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Discovery of Demurilactone A: A Specific Growth Inhibitor of L-Form Bacillus subtilis

  • Yousef Dashti*
    Yousef Dashti
    The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.
    *Email: [email protected], [email protected]
    More by Yousef Dashti
    Orcidhttps://orcid.org/0000-0002-8490-7763
  • , 
  • Fatemeh Mazraati Tajabadi
    Fatemeh Mazraati Tajabadi
    Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
    More by Fatemeh Mazraati Tajabadi
  • , 
  • Ling Juan Wu
    Ling Juan Wu
    The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.
    More by Ling Juan Wu
  • , 
  • Felaine Anne Sumang
    Felaine Anne Sumang
    The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.
    More by Felaine Anne Sumang
  • , 
  • Alexander Escasinas
    Alexander Escasinas
    Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
    More by Alexander Escasinas
  • , 
  • Nicholas Edward Ellis Allenby
    Nicholas Edward Ellis Allenby
    Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
    More by Nicholas Edward Ellis Allenby
  • , and 
  • Jeff Errington*
    Jeff Errington
    The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.
    Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
    *Email: [email protected]
    More by Jeff Errington
Cite this: ACS Infect. Dis. 2022, 8, 11, 2253–2258
Publication Date (Web):October 21, 2022
https://doi.org/10.1021/acsinfecdis.2c00220

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Abstract

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Metabolic profiling of the extracts from a library of actinobacteria led to the identification of a novel polyketide, demurilactone A, produced by Streptomyces strain DEM21308. The structure of the compound was assigned based on a detailed investigation of 1D/2D NMR spectra and HR-MS. Whole genome DNA sequencing, followed by bioinformatics analysis and insertional mutagenesis, identified type I polyketide synthases encoded by the dml gene cluster to direct the biosynthesis of this polyene macrolide. While the number of modules is consistent with the carbon backbone of the assigned structure, some discrepancies were identified in the domain organization of five modules. Close investigation of the amino acid sequences identified several mutations in the conserved motifs of nonfunctional domains. Furthermore, the absolute configuration of hydroxy-bearing stereocenters was proposed based on analyses of the ketoreductase domains. Remarkably, although demurilactone A has little detectable activity against normal-walled bacteria, it specifically inhibits the growth of cell wall-deficient “L-form” Bacillus subtilis at a minimum inhibitory concentration value of 16 μg/mL. Time-lapse microscopy analyses revealed that demurilactone affects membrane dynamics, probably by reducing membrane fluidity. This compound could be a powerful reagent for studying long-standing questions about the involvement of L-forms in recurrent infection.

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Introduction

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Specialized metabolites possess remarkable structural and chemical diversities and have been a reservoir of life-saving therapeutics for various diseases. (1) Modular polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), and their hybrids (PKS–NRPS) produce specialized metabolites with diverse structural features, which include some of the most important pharmaceuticals. Polyketide natural products made by type I modular PKS are the basis of about one-third of marketed medicines. (2) While polyketides have remarkably diverse structures, the molecular logic for their biosynthesis is simple. Modules of type I PKSs successively elongate the polyketide chain, process, and finally terminate the biosynthesis. (3) Each module consists of at least three domains, including acyltransferase (AT), an acyl carrier protein (ACP), and ketosynthase (KS) units, which collectively extend the polyketide chain by two carbon atoms. The AT domain loads the acyl units from acyl-coenzyme A (acyl-CoA) to the ACP, and KS domains are responsible for carbon–carbon bond formation between the acyl-ACP and the intermediate from the upstream module. In addition, PKS modules can also include ketoreductase (KR), dehydratase (DH), or enoyl reductase (ER) units, which successively reduce the β-keto group to a hydroxyl (KR), form a double bond via water removal (DH), and reduce the double bond to a single bond (ER). The final module contains a thioesterase (TE) that releases the polyketide chain through hydrolysis or cyclization. (3)
Peptidoglycan (PG) is the primary component of the bacterial cell wall, which is essential for the shape and structural integrity of the cell, and it creates a protective barrier against environmental factors. (4) Inhibition of bacterial cell wall biosynthesis by either β-lactam antibiotics or immune factors such as lysozyme can induce switching of bacterial cells into the cell-wall-deficient or L-form state. (5,6) L-forms are completely resistant to antibiotics targeting PG synthesis and are less susceptible to immune factors. (7,8) Upon termination of treatment with antibiotics, they can switch back into the walled state, and it has been suggested that the ability of the pathogenic bacteria to switch to the L-form and revert to the wall state might be involved in the recurrence of infection. (9−13) We have recently shown that L-form switching is a physiologically relevant phenomenon and possibly is responsible for recurrent urinary tract infections. (14) Thus, to avoid the recurrence of infection, it is crucial to have antibiotics targeting L-form bacteria during bacterial treatment with wall-targeting antibiotics. Herein, the structure of a novel growth inhibitor of L-form Bacillus subtilis, which we named demurilactone A, is described, together with its proposed biosynthetic assembly by type I PKS.

Results and Discussion

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Metabolic profiling of an ethyl acetate extract of Streptomyces strain DEM21308 cultured on a solid GYM medium using positive ion ESI-Q-TOF-MS showed a peak corresponding to the molecular formulas of C33H52O10 (calculated for C33H52NaO10+: 631.3456, found: 631.3451). A mass-directed purification approach on a semipreparative HPLC-Q-MS was used to isolate the compound from an ethyl acetate extract obtained from a 1 L culture of the strain. Comparison of the obtained molecular formulas and 1H NMR signals of the compound with chemical databases suggested that this compound was potentially a novel molecule; therefore, we proceeded to assign the planar structure using 1D/2D NMR experiments (Figure 1; Tables S1 and S2, and Figures S3–S14).

Figure 1

Figure 1. Structure of demurilactone A annotated with carbon numbers (left panel) and annotated with COSY and key HMBC correlations (right panel).

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The 1H NMR spectrum of the compound recorded in DMSO-d6 showed several overlapped proton signals. Shifting to a mixture of CD3OD/CDCl3 (3:1) helped resolve many of the peaks. Two spin systems were identified by correlation spectroscopy, HSQC, and HMBC correlations (Figure 1). HMBC correlations helped to link these fragments to each other. On the basis of observed 2J correlations of H14 (δH 1.98) and H16 (δH 1.65/1.73), as well as the 3J correlation of H17 (δH 4.19) to the quaternary carbon at δC 98.5, from one side, they are connected via C15. Similarly, the 3J correlation from H29 (δH 4.77), and the 2J/3J correlations of H2 (δH 5.83)/H3 (δH 7.26) to the carbonyl carbon at δC 169.3, established the existence of the ester bond that generates the 30-membered macrocycle ring. Based on its molecular formula, the compound has eight degrees of unsaturation, of which six are accounted for by five sets of olefins and one ester carbonyl. The remaining two degrees of unsaturation indicate the bicyclic nature of the molecule: one is the 30-membered macrocycle ring formed via the ester bond and the other is the hemiketal ring revealed by the 3J HMBC correlation of H11 (δH 4.02) to quaternary carbon C15 with the characteristic chemical shift at δC 98.5. All double bonds were assigned as E-configured, based on large coupling constants (J = ∼15) between the olefinic protons H2/H3, H4/H5, H6/H7, H8/H9, and H26/H27.
To identify the biosynthetic gene cluster (BGC) for demurilactone A, genomic DNA from the producer organism was isolated and sequenced using a combination of Nanopore and Illumina DNA sequencing methods. Bioinformatic analysis of assembled genome using antiSMASH (15) revealed six putative PKS BGCs in the genome. Analysis of the module organization and domain specificities of these PKSs identified a putative BGC, annotated as dml (GenBank accession number ON123837), which contains five genes (dmlE, dmlF, dmlG, dmlH, and dmlI) encoding a total of 15 type I PKS modules (Table 1 and Figure 3). The dml gene cluster contains nine other genes encoding the following proteins: a thioesterase (dmlJ), four regulators (dmlA, dmlC, dmlK, and dmlM), a transporter (dmlB), a putative dihydrodipicolinate synthase (dmlL), an aminoacyl-tRNA editor (dmlN), and one hypothetical protein (dmlD). Further details are summarized in Table 1.
Table 1. Predicted Functions of the Proteins Encoded by the dml BGC
protein Locus_tag predicted function
DmlA ctg4_73 transcriptional regulator-MarR family
DmlB ctg4_74 transporter-EamA family
DmlC ctg4_75 transcriptional regulator-MarR family
DmlD ctg4_76 hypothetical protein
DmlE ctg4_77 PKS
DmlF ctg4_78 PKS
DmlG ctg4_79 PKS
DmlH ctg4_80 PKS
DmlI ctg4_81 PKS
DmlJ ctg4_82 thioesterase
DmlK ctg4_83 LuxR family DNA-binding response regulator
DmlL ctg4_84 dihydrodipicolinate synthase family
DmlM ctg4_85 LysR family transcriptional regulator
DmlN ctg4_86 aminoacyl-tRNA editing domain
To confirm the involvement of the dml gene cluster in demurilactone A production, we inactivated dmlE via insertional mutagenesis. For this purpose, we created a new vector based on pSET152, in which phage integrase (ΦC31) and attP sites were replaced with a target nucleotide sequence from within dmlE. Liquid chromatography–mass spectrometry (LC–MS) analysis confirmed that the production of demurilactone A was abolished in the mutant strain (Figure 2).

Figure 2

Figure 2. Comparison of LC–MS profile of culture extracts from Streptomyces strain DEM21308 and Streptomyces strain DEM21308-ΩdmlE. Insertional mutagenesis in dmlE verified the involvement of the dml gene cluster in demurilactone A biosynthesis.

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The five modular PKS enzymes, DmlE–DmlI, are predicted to provide a loading module and 14 extender modules, which would generate the 30-carbon polyketide backbone via incorporation of 1 methyl malonyl-CoA and 13 malonyl-CoA extender units onto a priming isobutyl unit. This would be cyclized and released from the assembly line, catalyzed by the TE domain at the C-terminus of module 14 (Figure 3). Multiple sequence alignments were used to predict the substrate specificity of the AT domains. (16,17) With the exception of the loading and AT modules one and six, all modules showed a His-Ala-Phe-His motif that is specific for malonyl-CoA, (17) consistent with the assigned structure (Table S3). Module one is predicted to be specific for methyl malonyl-CoA due to the presence of a Tyr-Ala-Ser-His motif. The AT of module six has a Tyr-Ala-Pro-His motif which, based on the structure of demurilactone A, should be specific for malonyl-CoA. A comparison of the structure with the domain organization of the dml BGC suggested that the ER of module two and the DH of modules four, six, seven, and eight should be inactive (Figure 3).

Figure 3

Figure 3. dml gene cluster and proposed biosynthetic pathway of demurilactone A. Abbreviations: AT; acyltransferase, KS; ketosynthase, KR; ketoreductase, DH; dehydratase, ER; enoyl reductase, TE; thioesterase. ACPs are shown in black circles. Inactive domains are shown with an asterisk in red.

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Multiple sequence alignments of the ER and several active ERs from well-characterized biosynthetic systems revealed a shorted sequence and multiple mutations in the NADPH binding motif. Thus, the conserved HAAAGGVGMA motif of functional ERs appears as HAPTGDVGAA in the DmlE ER protein (Table S4). It seems likely that these amino acid substitutions in the NADPH binding motif of the ER would disrupt the binding cleft and therefore impede accommodation of NADPH, and consequently make the ER nonfunctional. Donadio et al. (18) previously showed that mutagenesis of the coding region for the NADPH binding site in an ER could be used to generate an erythromycin analogue.
DH domains of PKS systems are recognizable by their signature HXXXGXXXXP motif near their N-terminus in which catalytic histidine is invariant. (19) Multiple sequence alignments of all active and inactive DH domains of dml BGC revealed that the histidine in the HXXXGXXXXP motif is replaced with tyrosine in DH modules six and eight (Table S5), which explains the nonfunctional nature of these two domains. DH modules four and seven both had substitutions in the conserved GYXYGPXF motif, in which the first glycine in the sequence is replaced with aspartic acid. In addition, module seven showed the replacement of phenylalanine in the expected LPFXW motif with valine. It has been hypothesized that this phenylalanine makes contact with the ACP. (19) Further experiments would be required to test whether these substitutions are sufficient to inactivate DH modules four and seven, but the deduced structure of demurilactone A strongly suggests that this is the case.
Subsequently, multiple sequence alignments of the KR domains were used to predict the absolute configuration of the hydroxy-bearing stereocenters of demurilactone A, based on the conserved regions of KRs that direct the stereochemistry. (20−24) This approach has been used to propose the absolute configuration of several macrolides including termidomycin A, (25) niphimycins, (26) brasilinolides, (27) caniferolides, (28) and quinolidomicin. (29) Based on the presence of the Leu-Asp-Asp (LDD) motif in the loop region and the absence of tryptophan (W) and the YXP motifs in the catalytic region, the KRs of modules 4, 6, 7, 9, 11, 12, 13, and 14 were assigned as B1 type (Table S6). Therefore, the chiral centers C-23, C-19, C-17, and C-13 are assigned as “S”. Furthermore, the B1 type assignment of modules 11–14 is consistent with trans-double bounds C-2/C-3, C-4/C-5, C-6/C-7, and C-8/C-9, as cis configured double bonds are only generated by a DH that operates on the product of an A-type KR. (21) Similarly, the KR domains of modules 1, 3, 5, and 10 were assigned as A1 type based on the absence of an LDD motif in the loop region and the presence of tryptophan (W) and lack of histidine (H), in the catalytic region (Table S6). This led us to propose the stereochemistry on C-29, C-25, C-21, and C-11 as “R”. Generation of the six-membered hemiketal ring of demurilactone A requires a carbonyl group on C15; therefore, the KR domain of module 8 must be inactive or skipped during the biosynthetic assembly of the core polyketide (Figure 3). Investigation of the active site of the KR of module 8, however, indicated that this domain harbors a functional active site. The process leading to the intramolecular cyclization for hemiketal formation remains to be investigated.
The similarity of demurilactone A to polyketide antifungal agents such as nystatin prompted us to test for possible antibiotic activity. The compound was tested against Candida albicans, Schizosaccharomyces pombe, Saccharomyces cerevisiae, methicillin-resistant Staphylococcus aureus, and B. subtilis, but no growth inhibitory effects were detected. Serendipitously, and unexpectedly, we found that the compound was highly active against L-form B. subtilis. Figure S16 shows a clear zone around the disc containing demurilactone A on a lawn of L-form B. subtilis. When tested in a liquid medium, demurilactone A had a minimum inhibitory concentration value of 16 μg/mL for L-form B. subtilis, while normal (walled) B. subtilis showed no growth inhibition even at 100 μg/mL, confirming that demurilactone A was differentially active against L-forms (Figure S17). Prolonged incubation of demurilactone A with L-forms did result in some growth at concentrations 16 and 20 μg/mL but not at 30 and 40 μg/mL, suggesting that the compound is bactericidal at higher concentrations (Figure S17).
To understand the mode of action of demurilactone A, we carried out time-lapse microscopy to observe the effects of the compound on L-form growth. Figure 4 shows the growth of L-form B. subtilis in the presence of DMSO (as a control) or demurilactone A. As expected, DMSO had no effect on growth. L-form cells continued to grow and divide after the addition of DMSO, and by 180 min, the field was already filled with cells. In the presence of demurilactone A, however, cells continued to grow larger but did not divide, such that 180 min after the addition of the compound, the cells had substantially enlarged without much of an increase in cell number. Division of L-form bacteria is independent of the FtsZ-based cell division machinery that is essential for walled bacteria. (7,8,13,30) Instead, they divide by a variety of processes involving membrane blebbing, tubulation, vesiculation, and fission, which is driven by excess membrane synthesis and is sensitive to membrane fluidity. (8,31) As the cells were still able to grow larger in the presence of demurilactone A, we assumed that excess membrane synthesis continued and that the inhibitory effect was mainly due to the loss of the dynamic biophysical properties of the membranes. Indeed, examination of cells very soon after the addition of demurilactone A revealed that within 2 min of the addition of the compound, cells started to become round and lost the dynamic shape changes that normally associate with actively dividing L-form cells (Figure S18). Taken together, these analyses suggest that demurilactone A inhibits the growth of L-form bacteria by reducing membrane dynamics, possibly by reducing membrane fluidity. Previous studies have shown that L-form B. subtilis cells are hypersensitive to peptide antibiotics nisin and daptomycin; (32) however, these two compounds also have lethal effects on walled B. subtilis. To our knowledge, this is the first natural product identified to inhibit the growth of L-form but not walled B. subtilis. Interestingly, the closely related polyketide antifungal antibiotic, nystatin, did not show growth inhibitory activity against L-form B. subtilis.

Figure 4

Figure 4. Inhibitory effect of demurilactone A on L-form B. subtilis proliferation (bottom panel) compared to the DMSO control (top panel). The first images shown were taken 20 min after the addition of DMSO (1 μL in 100 μL of cells) or demurilactone A (2 μL of demurilactone A at 1.6 μg/mL (diluted in NB/MSM) in 100 μL of cells), and the last images were taken at 180 min.

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Conclusions

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Evidence is growing for L-form bacteria as a source of antibiotic resistance, especially in recurrent infections. (9−14) Tackling this problem requires the use of combination chemotherapy including antibiotics targeting bacteria in the L-form state. The novel macrolide demurilactone A identified from a culture extract of Streptomyces DEM21308 is a potential starting point for novel drugs acting on L-form-dependent recurrent infection. Bioinformatics analysis of the sequenced genome of Streptomyces DEM21308 identified the putative dml BGC responsible for the assembly of demurilactone A, which was verified by insertional mutagenesis. Detailed investigation of the multiple sequence alignments revealed several inactive domains consistent with the structure assigned by HR-MS and NMR analysis.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.2c00220.

  • General experimental procedures; procedures for compound production and purification, genomic DNA sequencing and assembly, insertional mutagenesis, and antibacterial assays; NMR data and spectra; and multiple sequence alignments (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Yousef Dashti - The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.;  Present Address: Biomedical Building C81, Faculty of Medicine and Health, University of Sydney, Sydney NSW 2015, Australia;  Orcidhttps://orcid.org/0000-0002-8490-7763;  Email: [email protected] [email protected]
    • Jeff Errington - The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.;  Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.;  Present Address: Biomedical Building C81, Faculty of Medicine and Health, University of Sydney, Sydney NSW 2015, Australia;  Email: [email protected]
  • Authors
    • Fatemeh Mazraati Tajabadi - Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
    • Ling Juan Wu - The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.
    • Felaine Anne Sumang - The Centre for Bacterial Cell Biology, Biosciences Institute, Medical School, Newcastle University, Newcastle Upon Tyne NE2 4AX, U.K.
    • Alexander Escasinas - Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
    • Nicholas Edward Ellis Allenby - Odyssey Therapeutics Inc, The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, U.K.
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was funded by a Wellcome Investigator Award (209500) to J.E.

References

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    The induction of L-forms of Bacillus subtilis from protoplasts is described. The method involved the frequent subculture of the unstable L-form on a growth medium supplemented with lysozyme and horse serum. A stable culture, which did not revert when lysozyme and horse serum were omitted from the medium, was obtained after 13 subcultures. This culture could be grown on solid and in liquid medium by routine microbiological methods. Long-term storage of these cells was achieved by freeze drying and maintenance in glycerol at -70 degrees C. The cultural adaptability of the L-form is described and discussed with respect to methods of cultivation and growth.
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  6. 6
    Kawai, Y.; Mickiewicz, K.; Errington, J. Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria. Cell 2018, 172, 1038– 1049,  DOI: 10.1016/j.cell.2018.01.021
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    Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria
    Kawai, Yoshikazu; Mickiewicz, Katarzyna; Errington, Jeff
    Cell (Cambridge, MA, United States) (2018), 172 (5), 1038-1049.e10CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    β-Lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiol. culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiol., osmoprotective conditions, for various Gram-pos. bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also obsd. in macrophages and in an animal model, presumably due to the prodn. of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.
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    Leaver, M.; Domínguez-Cuevas, P.; Coxhead, J. M.; Daniel, R. A.; Errington, J. Life without a wall or division machine in Bacillus subtilis. Nature 2009, 457, 849– 853,  DOI: 10.1038/nature07742
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    Life without a wall or division machine in Bacillus subtilis
    Leaver, M.; Dominguez-Cuevas, P.; Coxhead, J. M.; Daniel, R. A.; Errington, J.
    Nature (London, United Kingdom) (2009), 457 (7231), 849-853CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    A review and discussion. The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivs. of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biol. or their means of propagation. The authors have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Using genome sequencing, they have identified a single point mutation that predisposes cells to grow without a wall. Propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resoln. mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.
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  8. 8
    Mercier, R.; Kawai, Y.; Errington, J. Excess membrane synthesis drives a primitive mode of cell proliferation. Cell 2013, 152, 997– 1007,  DOI: 10.1016/j.cell.2013.01.043
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    Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation
    Mercier, Romain; Kawai, Yoshikazu; Errington, Jeff
    Cell (Cambridge, MA, United States) (2013), 152 (5), 997-1007CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The peptidoglycan cell wall is a hallmark of the bacterial subkingdom. Surprisingly, many modern bacteria retain the ability to switch into a wall-free state called the L-form. L-form proliferation is remarkable in being independent of the normally essential FtsZ-based division machinery and in occurring by membrane blebbing and tubulation. We show that mutations leading to excess membrane synthesis are sufficient to drive L-form division in Bacillus subtilis. Artificially increasing the cell surface area to vol. ratio in wild-type protoplasts generates similar shape changes and cell division. Our findings show that simple biophys. processes could have supported efficient cell proliferation during the evolution of early cells and provide an extant biol. model for studying this problem.
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    Clasener, H. Pathogenicity of the L-Phase of Bacteria. Annu. Rev. Microbiol. 1972, 26, 55– 84,  DOI: 10.1146/annurev.mi.26.100172.000415
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    Pathogenicity of the L-phase of bacteria
    Clasener H
    Annual review of microbiology (1972), 26 (), 55-84 ISSN:0066-4227.
    There is no expanded citation for this reference.
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  10. 10
    Domingue, G. J.; Woody, H. B. Bacterial persistence and expression of disease. Clin. Microbiol. Rev. 1997, 10, 320– 344,  DOI: 10.1128/cmr.10.2.320
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    Bacterial persistence and expression of disease
    Domingue G J Sr; Woody H B
    Clinical microbiology reviews (1997), 10 (2), 320-44 ISSN:0893-8512.
    A considerable body of experimental and clinical evidence supports the concept that difficult-to-culture and dormant bacteria are involved in latency of infection and that these persistent bacteria may be pathogenic. This review includes details on the diverse forms and functions of individual bacteria and attempts to make this information relevant to the care of patients. A series of experimental studies involving host-bacterium interactions illustrates the probability that most bacteria exposed to a deleterious host environment can assume a form quite different from that of a free-living bacterium. A hypothesis is offered for a kind of reproductive cycle of morphologically aberrant bacteria as a means to relate their diverse tissue forms to each other. Data on the basic biology of persistent bacteria are correlated with expression of disease and particularly the mechanisms of both latency and chronicity that typify certain infections. For example, in certain streptococcal and nocardial infections, it has been clearly established that wall-defective forms can be induced in a suitable host. These organisms can survive and persist in a latent state within the host, and they can cause pathologic responses compatible with disease. A series of cases illustrating idiopathic conditions in which cryptic bacteria have been implicated in the expression of disease is presented. These conditions include nephritis, rheumatic fever, aphthous stomatitis, idiopathic hematuria, Crohn's disease, and mycobacterial infections. By utilizing PCR, previously nonculturable bacilli have been identified in patients with Whipple's disease and bacillary angiomatosis. Koch's postulates may have to be redefined in terms of molecular data when dormant and nonculturable bacteria are implicated as causative agents of mysterious diseases.
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  11. 11
    Onwuamaegbu, M.; Belcher, R.; Soare, C. Cell wall-deficient bacteria as a cause of infections: A review of the clinical significance. J. Int. Med. Res. 2005, 33, 1– 20,  DOI: 10.1177/147323000503300101
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    Cell wall-deficient bacteria as a cause of infections: a review of the clinical significance
    Onwuamaegbu M E; Belcher R A; Soare C
    The Journal of international medical research (2005), 33 (1), 1-20 ISSN:0300-0605.
    Cell wall-deficient bacteria (CWDB) are pleomorphic bacterial forms. These atypical organisms may occur naturally or they can be induced in the laboratory. Their presence has been known about for over a century, but a definite link to clinical disease outcomes has not been demonstrated. A number of case reports and laboratory studies suggest some disease associations, however. Considerable controversy surrounds the true relevance of CWDB to disease; there is a widespread belief that they may represent a response by the walled organism to adverse extracellular conditions like antibiotic pressure. This review looks at studies published between 1934 and 2003, which were identified by Dialog DataStar using the key words 'cell wall deficient bacteria and clinical significance and infections' and by further scanning the reference list at the end of the papers retrieved. We conclude that the evidence for the clinical significance of CWDB in disease is not compelling.
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    Allan, E. J.; Hoischen, C.; Gumpert, J., Chapter 1 Bacterial L-Forms. In Advances in Applied Microbiology; Academic Press, 2009; Vol. 68, pp 1– 39.
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    Errington, J.; Mickiewicz, K.; Kawai, Y.; Wu, L. J. L-form bacteria, chronic diseases and the origins of life. Philos. Trans. R. Soc. London, Ser. B 2016, 371, 20150494,  DOI: 10.1098/rstb.2015.0494
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    Mickiewicz, K. M.; Kawai, Y.; Drage, L.; Gomes, M. C.; Davison, F.; Pickard, R.; Hall, J.; Mostowy, S.; Aldridge, P. D.; Errington, J. Possible role of L-form switching in recurrent urinary tract infection. Nat. Commun. 2019, 10, 4379,  DOI: 10.1038/s41467-019-12359-3
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    14
    Possible role of L-form switching in recurrent urinary tract infection
    Mickiewicz Katarzyna M; Kawai Yoshikazu; Drage Lauren; Davison Frances; Aldridge Phillip D; Errington Jeff; Gomes Margarida C; Mostowy Serge; Pickard Robert; Hall Judith
    Nature communications (2019), 10 (1), 4379 ISSN:.
    Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E. coli strains from patient samples readily transition from the walled state to L-form during challenge with a cell wall targeting antibiotic. Following antibiotic withdrawal, they then efficiently transition back to the walled state. E. coli switches between walled and L-form states in a zebrafish larva infection model. The results suggest that L-form switching is a physiologically relevant phenomenon that may contribute to the recurrence of infection in older patients with rUTI, and potentially other infections.
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  15. 15
    Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H. U.; Bruccoleri, R.; Lee, S. Y.; Fischbach, M. A.; Müller, R.; Wohlleben, W.; Breitling, R.; Takano, E.; Medema, M. H. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015, 43, W237– W243,  DOI: 10.1093/nar/gkv437
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    antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters
    Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A.; Muller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H.
    Nucleic Acids Research (2015), 43 (W1), W237-W243CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Microbial secondary metab. constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chems. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodol. for novel compd. discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and anal. of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission nos. are assigned to functionally classify all enzyme-coding genes. Addnl., chem. structure prediction has been improved by incorporating polyketide redn. states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.
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  16. 16
    Haydock, S. F.; Aparicio, J. F.; Molnár, I.; Schwecke, T.; Khaw, L. E.; König, A.; Marsden, A. F.; Galloway, I. S.; Staunton, J.; Leadlay, P. F. Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases. FEBS Lett. 1995, 374, 246– 248,  DOI: 10.1016/0014-5793(95)01119-y
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    16
    Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases
    Haydock, Stephen F.; Aparicio, Jesus F.; Molnar, Istvan; Schwecke, Torsten; Khaw, Lake Ee; Koenig, Ariane; Marsden, Andrew F. A.; Galloway, Ian S.; Staunton, James; et al.
    FEBS Letters (1995), 374 (2), 246-8CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
    The amino acid sequences of a large no. of polyketide synthase domains that catalyze the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) were compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences were sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-detd. structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, although not close enough to make direct contact with bound substrate.
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  17. 17
    Del Vecchio, F.; Petkovic, H.; Kendrew, S. G.; Low, L.; Wilkinson, B.; Lill, R.; Cortés, J.; Rudd, B. A. M.; Staunton, J.; Leadlay, P. F. Active-site residue, domain and module swaps in modular polyketide synthases. J. Ind. Microbiol. Biotechnol. 2003, 30, 489– 494,  DOI: 10.1007/s10295-003-0062-0
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    Active-site residue, domain and module swaps in modular polyketide synthases
    Del Vecchio, Francesca; Petkovic, Hrvoje; Kendrew, Steven G.; Low, Lindsey; Wilkinson, Barrie; Lill, Rachel; Cortes, Jesus; Rudd, Brian A. M.; Staunton, Jim; Leadlay, Peter F.
    Journal of Industrial Microbiology & Biotechnology (2003), 30 (8), 489-494CODEN: JIMBFL; ISSN:1367-5435. (Springer-Verlag)
    Sequence comparisons of multiple acyltransferase (AT) domains from modular polyketide synthases (PKSs) have highlighted a correlation between a short sequence motif and the nature of the extender unit selected. When this motif was specifically altered in the bimodular model PKS DEBS1-TE of Saccharopolyspora erythraea, the products included triketide lactones in which acetate extension units had been incorporated instead of propionate units at the predicted positions. We also describe a cassette system for convenient construction of hybrid modular PKSs based on the tylosin PKS in Streptomyces fradiae and demonstrate its use in domain and module swaps.
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    Donadio, S.; McAlpine, J. B.; Sheldon, P. J.; Jackson, M.; Katz, L. An erythromycin analog produced by reprogramming of polyketide synthesis. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7119– 7123,  DOI: 10.1073/pnas.90.15.7119
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    18
    An erythromycin analog produced by reprogramming of polyketide synthesis
    Donadio, Stefano; McAlpine, James B.; Sheldon, Paul J.; Jackson, Marianna; Katz, Leonard
    Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (15), 7119-23CODEN: PNASA6; ISSN:0027-8424.
    The polyketide-derived macrolactone of the antibiotic erythromycin is made through successive condensation and processing of 7 C3 units. The 4th cycle involves complete processing of the newly formed β-keto group (β-keto redn., dehydration, and enoyl redn.) to yield the methylene that will appear at C-7 of the lactone ring. Synthesis of this mol. in Saccharopolyspora erythraea is detd. by the 3 large eryA genes, organized in 6 modules, each governing 1 condensation cycle. Two amino acid substitutions were introduced in the putative NAD(P)H-binding motif in the proposed enoyl reductase domain encoded by eryAII. The metabolite produced by the resulting strain was identified as Δ6,7-anhydroerythromycin C resulting from failure of enoyl redn. during the 4th cycle of synthesis of the macrolactone. This result demonstrates the involvement of at least the enoyl reductase from the 4th module in the 4th cycle and indicates that a virtually complete macrolide can be produced through reprogramming of polyketide synthesis.
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    Keatinge-Clay, A. Crystal structure of the erythromycin polyketide synthase dehydratase. J. Mol. Biol. 2008, 384, 941– 953,  DOI: 10.1016/j.jmb.2008.09.084
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    Crystal Structure of the Erythromycin Polyketide Synthase Dehydratase
    Keatinge-Clay, Adrian
    Journal of Molecular Biology (2008), 384 (4), 941-953CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    The dehydratases (DHs) of modular polyketide synthases (PKSs) catalyze dehydrations that occur frequently in the biosynthesis of complex polyketides, yet little is known about them structurally or mechanistically. Here, the structure of a DH domain, isolated from the fourth module of the erythromycin PKS, is presented at 1.85 Å resoln. As with the DH of the highly related animalian fatty acid synthase, the DH monomer possesses a double-hotdog fold. Two symmetry mates within the crystal lattice make a contact that likely represents the DH dimerization interface within an intact PKS. Conserved hydrophobic residues on the DH surface indicate potential interfaces with two other PKS domains, the ketoreductase and the acyl carrier protein. Mutation of an invariant arginine at the hypothesized acyl carrier protein docking site in the context of the erythromycin PKS resulted in decreased prodn. of the erythromycin precursor 6-deoxyerythronolide B. The structure elucidates how the α-hydrogen and β-hydroxyl group of a polyketide substrate interact with the catalytic histidine and aspartic acid in the DH active site prior to dehydration.
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    Zheng, J.; Taylor, C. A.; Piasecki, S. K.; Keatinge-Clay, A. T. Structural and functional analysis of A-type ketoreductases from the amphotericin modular polyketide synthase. Structure 2010, 18, 913– 922,  DOI: 10.1016/j.str.2010.04.015
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    20
    Structural and Functional Analysis of A-Type Ketoreductases from the Amphotericin Modular Polyketide Synthase
    Zheng, Jian-Ting; Taylor, Clint A.; Piasecki, Shawn K.; Keatinge-Clay, Adrian T.
    Structure (Cambridge, MA, United States) (2010), 18 (8), 913-922CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    Complex polyketides are characterized by multiple chiral centers harboring hydroxyl and alkyl substituents. To investigate the mechanisms by which these stereocenters are set, several high-resoln. structures of the ketoreductase (KR) domain from the second module of the amphotericin modular polyketide synthase (PKS) were solved. This first structural anal. of an A-type KR helps reveal how these KRs direct polyketide intermediates into their active sites from the side opposite that used by B-type KRs, resulting in a β-hydroxyl group of opposite stereochem. A comparison of structures obtained in the absence and presence of ligands reveals an induced fit mechanism that is important for catalysis. Activity assays of mutants of KRs from the first and second modules of the amphotericin PKS reveal the relative contributions of several active site residues toward catalysis and stereocontrol. Together, these results highlight the possibility of region-specific modification of polyketides through active site engineering of KRs.
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    Keatinge-Clay, A. T. Stereocontrol within polyketide assembly lines. Nat. Prod. Rep. 2016, 33, 141– 149,  DOI: 10.1039/c5np00092k
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    21
    Stereocontrol within polyketide assembly lines
    Keatinge-Clay, Adrian T.
    Natural Product Reports (2016), 33 (2), 141-149CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
    A review. Most of the stereocenters of polyketide natural products are established during assembly line biosynthesis. The body of knowledge for how stereocenters are set is now large enough to begin constructing phys. models of key reactions. Interactions between stereocenter-forming enzymes and polyketide intermediates are examd. here at at. resoln., drawing from the most current structural and functional information of ketosynthases (KSs), ketoreductases (KRs), dehydratases (DHs), enoylreductases (ERs), and related enzymes. While many details remain to be exptl. detd., our understanding of the chem. and phys. mechanisms utilized by the chirality-molding enzymes of modular PKSs is rapidly advancing.
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    Caffrey, P. Conserved amino acid residues correlating with ketoreductase stereospecificity in modular polyketide synthases. ChemBioChem 2003, 4, 654– 657,  DOI: 10.1002/cbic.200300581
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    22
    Conserved amino acid residues correlating with ketoreductase stereospecificity in modular polyketide synthases
    Caffrey, Patrick
    ChemBioChem (2003), 4 (7), 654-657CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    An anal. of ketoreductase (KR) sequences was carried out to better understand how modular polyketide synthases (PKSs) control alc. stereochem. in nascent oligoketide chains. Two types of alc. stereochem., designated A and B, were considered. KR domains were divided into groups according to whether they generate A- or B-type alc. stereochem. A- and B-type KR sequences were aligned to allow identification of residues characteristic of each group. Very few consistent differences were found between A- and B-type KRs. The LDD motif and residues P144 and N148 correlate well with the formation of B-type alc. stereochem. The absence of these residues and the presence of W141 correlate with A-type alc. stereochem. The PKSs that were studied also contain KRs for which the stereochem. outcome of the ketoredn. is hidden by subsequent dehydration, or dehydration and enoyl redn. These domains have the LDD motif and PN motifs characteristic of B-type KRs. This suggests that the preferred substrate for a PKS dehydratase domain is a B-type 3-hydroxyacyl chain, as is present in fatty acid biosynthesis.
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    Khosla, C.; Gokhale, R. S.; Jacobsen, J. R.; Cane, D. E. Tolerance and specificity of polyketide synthases. Annu. Rev. Biochem. 1999, 68, 219– 253,  DOI: 10.1146/annurev.biochem.68.1.219
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    Tolerance and specificity of polyketide synthases
    Khosla, Chaitan; Gokhale, Rajesh S.; Jacobsen, John R.; Cane, David E.
    Annual Review of Biochemistry (1999), 68 (), 219-253CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
    A review with 89 refs. Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Addnl. complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the mol. recognition features of polyketide synthases. Within this decade, a variety of genetic, biochem., and chem. investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review.
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    Böhm, I.; Holzbaur, I. E.; Hanefeld, U.; Cortési, J.; Staunton, J.; Leadlay, P. F. Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension. Chem. Biol. 1998, 5, 407– 412,  DOI: 10.1016/S1074-5521(98)90157-0
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    Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension
    Bohm, Ines; Hlazbaur, Ines E.; Hanefeld, Ulf; Cortes, Jesus; Staunton, Jim; Leadlay, Peter F.
    Chemistry & Biology (1998), 5 (8), 407-412CODEN: CBOLE2; ISSN:1074-5521. (Current Biology Ltd.)
    Polyketides are a large and structurally diverse group of natural products that include antibiotics, antifungal agents and immunosuppressant compds. Polyketides are biosynthesized in filamentous bacteria on modular polyketide synthases (PKSs) in which each cycle of chain extension requires a different 'module' of enzymic activities. The recently proposed dimeric model for modular PKSs predicts that even a single-module PKS should be catalytically active in the absence of other PKS components. Researchers are also interested in manipulating the stereochem. outcome of polyketide chain extension using genetic engineering of domains within each module. We have constructed a minimal modular PKS from the erythromycin-producing PKS (DEBS) of Saccharopolyspora erythraea. The diketide synthase (DKS1-2) consists of a single chimeric extension module, derived from the DEBS module 1 ketoacyl-ACP synthase (KS), sandwiched between a loading module and a chain-terminating thioesterase. When DKS1-2 was expressed in S. erythraea, the strain preferentially accumulated the diketide (2R,3S)-2-methyl-3-hydroxy pentanoic acid. These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2-methyl-3-hydroxy intermediate by epimerizing the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The obsd. formation of the alternative (2R)-2-methyl-3-hydroxy product catalyzed by DKS1-2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS.
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    Um, S.; Guo, H.; Thiengmag, S.; Benndorf, R.; Murphy, R.; Rischer, M.; Braga, D.; Poulsen, M.; de Beer, Z. W.; Lackner, G.; Beemelmanns, C. Comparative genomic and metabolic analysis of Streptomyces sp. RB110 morphotypes illuminates genomic rearrangements and formation of a new 46-membered antimicrobial macrolide. ACS Chem. Biol. 2021, 16, 1482– 1492,  DOI: 10.1021/acschembio.1c00357
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    Comparative Genomic and Metabolic Analysis of Streptomyces sp. RB110 Morphotypes Illuminates Genomic Rearrangements and Formation of a New 46-Membered Antimicrobial Macrolide
    Um, Soohyun; Guo, Huijuan; Thiengmag, Sirinthra; Benndorf, Rene; Murphy, Robert; Rischer, Maja; Braga, Daniel; Poulsen, Michael; de Beer, Z. Wilhelm; Lackner, Gerald; Beemelmanns, Christine
    ACS Chemical Biology (2021), 16 (8), 1482-1492CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Morphotype switches frequently occur in Actinobacteria and are often assocd. with disparate natural product prodn. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clin. important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic anal. of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.
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    Hu, Y.; Wang, M.; Wu, C.; Tan, Y.; Li, J.; Hao, X.; Duan, Y.; Guan, Y.; Shang, X.; Wang, Y.; Xiao, C.; Gan, M. Identification and Proposed Relative and Absolute Configurations of Niphimycins C-E from the Marine-Derived Streptomyces sp. IMB7-145 by Genomic Analysis. J. Nat. Prod. 2018, 81, 178– 187,  DOI: 10.1021/acs.jnatprod.7b00859
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    Identification and Proposed Relative and Absolute Configurations of Niphimycins C-E from the Marine-Derived Streptomyces sp. IMB7-145 by Genomic Analysis
    Hu, Yuanyuan; Wang, Mian; Wu, Chunyan; Tan, Yi; Li, Jiao; Hao, Xiaomeng; Duan, Yanbo; Guan, Yan; Shang, Xiaoya; Wang, Yiguang; Xiao, Chunling; Gan, Maoluo
    Journal of Natural Products (2018), 81 (1), 178-187CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society-American Society of Pharmacognosy)
    Anal. of the whole genome sequence of Streptomyces sp. IMB7-145 revealed the presence of seven type I polyketide synthase biosynthetic gene clusters, one of which was highly homologous to the biosynthetic gene cluster of azalomycin F. Detailed bioinformatic anal. of the modular organization of the PKS gene suggested that this gene is responsible for niphimycin biosynthesis. Guided by genomic anal., a large-scale cultivation ultimately led to the discovery and characterization of four new niphimycin congeners, namely niphimycins C-E (1-3) and 17-O-methylniphimycin (4). The configurations of most stereocenters of niphimycins have not been detd. to date. Inthe present study, the relative configurations were elucidated by spectroscopic anal., including J-based anal. and the CNMR database method. Further, the full abs. configurations of niphimycins were completely proposed for the first time based on biosynthetic gene cluster anal. of the ketoreductase and enoylreductase domains for hydroxy- and methyl-bearing stereocenters. Compds. 1, 3, 4, and niphimycin Iα (5) showed antimicrobial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC: 8-64 μg/mL), as well as cytotoxicity against the human HeLa cancer cell line (IC50: 3.0-9.0 μM). In addn., compds. 1 and 5 displayed significant activity against several Mycobacterium tuberculosis clin. isolates (MIC: 4-32 μg/mL).
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    Chiu, H.-T.; Weng, C.-P.; Lin, Y.-C.; Chen, K.-H. Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes. Org. Biomol. Chem. 2016, 14, 1988– 2006,  DOI: 10.1039/c5ob02292d
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    Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-L-fucose biosynthetic enzymes
    Chiu, Hsien-Tai; Weng, Chien-Pao; Lin, Yu-Chin; Chen, Kuan-Hung
    Organic & Biomolecular Chemistry (2016), 14 (6), 1988-2006CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-L-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match expts. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and mol. levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
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    Pérez-Victoria, I.; Oves-Costales, D.; Lacret, R.; Martín, J.; Sánchez-Hidalgo, M.; Díaz, C.; Cautain, B.; Vicente, F.; Genilloud, O.; Reyes, F. Structure elucidation and biosynthetic gene cluster analysis of caniferolides A–D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066. Org. Biomol. Chem. 2019, 17, 2954– 2971,  DOI: 10.1039/C8OB03115K
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    Structure elucidation and biosynthetic gene cluster analysis of caniferolides A-D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066
    Perez-Victoria, Ignacio; Oves-Costales, Daniel; Lacret, Rodney; Martin, Jesus; Sanchez-Hidalgo, Marina; Diaz, Caridad; Cautain, Bastien; Vicente, Francisca; Genilloud, Olga; Reyes, Fernando
    Organic & Biomolecular Chemistry (2019), 17 (11), 2954-2971CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D. Their connectivity was detd. by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochem. of each stereocluster in these compds. was established using NOE anal., the universal database method and J-based configuration anal., further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics anal. led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochem. outcome of their biosynthesis, confirming the relative stereochem. of each stereocluster already detd. by NMR and establishing their stereochem. relationship, ultimately rendering the abs. configuration of all chiral centers. Furthermore, based on our results and already published data, it has been possible to derive the complete abs. configuration of the related macrolides PM100117 and PM100118, astolides A and B, and deplelides A and B. Caniferolides A-D showed pronounced antifungal activity against Candida albicans and Aspergillus fumigatus alongside antiproliferative activity against 5 human tumoral cell lines.
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    Hashimoto, T.; Hashimoto, J.; Kozone, I.; Amagai, K.; Kawahara, T.; Takahashi, S.; Ikeda, H.; Shin-ya, K. Biosynthesis of quinolidomicin, the largest known macrolide of terrestrial origin: Identification and heterologous expression of a biosynthetic gene cluster over 200 kb. Org. Lett. 2018, 20, 7996– 7999,  DOI: 10.1021/acs.orglett.8b03570
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    Biosynthesis of quinolidomicin, the largest known macrolide of terrestrial origin: Identification and heterologous expression of a biosynthetic gene cluster over 200 kb
    Hashimoto, Takuya; Hashimoto, Junko; Kozone, Ikuko; Amagai, Keita; Kawahara, Teppei; Takahashi, Shunji; Ikeda, Haruo; Shin-ya, Kazuo
    Organic Letters (2018), 20 (24), 7996-7999CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    Quinolidomicin A1 is the largest macrolide compd. from terrestrial sources, consisting of a 60-membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.
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    Studer, P.; Staubli, T.; Wieser, N.; Wolf, P.; Schuppler, M.; Loessner, M. J. Proliferation of Listeria monocytogenes L-form cells by formation of internal and external vesicles. Nat. Commun. 2016, 7, 13631,  DOI: 10.1038/ncomms13631
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    Proliferation of Listeria monocytogenes L-form cells by formation of internal and external vesicles
    Studer, Patrick; Staubli, Titu; Wieser, Noemi; Wolf, Patrick; Schuppler, Markus; Loessner, Martin J.
    Nature Communications (2016), 7 (), 13631CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    L-Forms are cell wall-deficient bacteria that divide through unusual mechanisms, involving dynamic perturbations of the cellular shape and generation of vesicles, independently of the cell-division protein FtsZ. Here we describe FtsZ-independent mechanisms, involving internal and external vesicles, by which Listeria monocytogenes L-forms proliferate. Using micromanipulation of single cells and vesicles, we show that small vesicles are formed by invagination within larger intracellular vesicles, receive cytoplasmic content, and represent viable progeny. In addn., the L-forms can reproduce by pearling, i.e., generation of extracellular vesicles that remain transiently linked to their mother cell via elastic membranous tubes. Using photobleaching and fluorescence recovery, we demonstrate cytoplasmic continuity and transfer through these membranous tubes. Our findings indicate that L-forms' polyploidy and extended interconnectivity through membranous tubes contribute to the generation of viable progeny independently of dedicated division machinery, and further support L-forms as models for studies of potential multiplication mechanisms of hypothetical primitive cells.
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    Mercier, R.; Domínguez-Cuevas, P.; Errington, J. Crucial role for membrane fluidity in proliferation of primitive cells. Cell Rep. 2012, 1, 417– 423,  DOI: 10.1016/j.celrep.2012.03.008
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    Crucial role for membrane fluidity in proliferation of primitive cells
    Mercier, Romain; Dominguez-Cuevas, Patricia; Errington, Jeff
    Cell Reports (2012), 1 (5), 417-423CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell wall-deficient "L-form" state, requiring remarkable physiol. and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ-based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, the authors uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the sepn. of progeny cells. The failure in scission is assocd. with a redn. in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophys. processes may have been sufficient for proliferation of primitive cells.
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    Wolf, D.; Domínguez-Cuevas, P.; Daniel, R. A.; Mascher, T. Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis. Antimicrob. Agents Chemother. 2012, 56, 5907– 5915,  DOI: 10.1128/aac.00770-12
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    Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis
    Wolf, Diana; Dominguez-Cuevas, Patricia; Daniel, Richard A.; Mascher, Thorsten
    Antimicrobial Agents and Chemotherapy (2012), 56 (11), 5907-5915CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
    L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-pos. model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to β-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here the authors studied the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. The authors' results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, the authors established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).
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  • Abstract

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    Figure 1

    Figure 1. Structure of demurilactone A annotated with carbon numbers (left panel) and annotated with COSY and key HMBC correlations (right panel).

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    Figure 2

    Figure 2. Comparison of LC–MS profile of culture extracts from Streptomyces strain DEM21308 and Streptomyces strain DEM21308-ΩdmlE. Insertional mutagenesis in dmlE verified the involvement of the dml gene cluster in demurilactone A biosynthesis.

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    Figure 3

    Figure 3. dml gene cluster and proposed biosynthetic pathway of demurilactone A. Abbreviations: AT; acyltransferase, KS; ketosynthase, KR; ketoreductase, DH; dehydratase, ER; enoyl reductase, TE; thioesterase. ACPs are shown in black circles. Inactive domains are shown with an asterisk in red.

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    Figure 4

    Figure 4. Inhibitory effect of demurilactone A on L-form B. subtilis proliferation (bottom panel) compared to the DMSO control (top panel). The first images shown were taken 20 min after the addition of DMSO (1 μL in 100 μL of cells) or demurilactone A (2 μL of demurilactone A at 1.6 μg/mL (diluted in NB/MSM) in 100 μL of cells), and the last images were taken at 180 min.

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    This article references 32 other publications.

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      Natural Products as Sources of New Drugs from 1981 to 2014
      Newman, David J.; Cragg, Gordon M.
      Journal of Natural Products (2016), 79 (3), 629-661CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society-American Society of Pharmacognosy)
      This contribution is a completely updated and expanded version of the four prior analogous reviews that were published in this journal in 1997, 2003, 2007, and 2012. In the case of all approved therapeutic agents, the time frame has been extended to cover the 34 years from Jan. 1, 1981, to Dec. 31, 2014, for all diseases worldwide, and from 1950 (earliest so far identified) to Dec. 2014 for all approved antitumor drugs worldwide. As mentioned in the 2012 review, we have continued to utilize our secondary subdivision of a "natural product mimic", or "NM", to join the original primary divisions and the designation "natural product botanical", or "NB", to cover those botanical "defined mixts." now recognized as drug entities by the U.S. FDA (and similar organizations). From the data presented in this review, the utilization of natural products and/or their novel structures, in order to discover and develop the final drug entity, is still alive and well. For example, in the area of cancer, over the time frame from around the 1940s to the end of 2014, of the 175 small mols. approved, 131, or 75%, are other than "S" (synthetic), with 85, or 49%, actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the anti-infective area being dependent on natural products and their structures. We wish to draw the attention of readers to the rapidly evolving recognition that a significant no. of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore it is considered that this area of natural product research should be expanded significantly.
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      Newman, D. J.; Cragg, G. M. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. 2012, 75, 311– 335,  DOI: 10.1021/np200906s
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      Natural Products As Sources of New Drugs over the 30 Years from 1981 to 2010
      Newman, David J.; Cragg, Gordon M.
      Journal of Natural Products (2012), 75 (3), 311-335CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society-American Society of Pharmacognosy)
      This review is an updated and expanded version of the three prior reviews that were published in this journal in 1997, 2003, and 2007. In the case of all approved therapeutic agents, the time frame has been extended to cover the 30 years from Jan. 1, 1981, to Dec. 31, 2010, for all diseases worldwide, and from 1950 (earliest so far identified) to Dec. 2010 for all approved antitumor drugs worldwide. We have continued to utilize our secondary subdivision of a "natural product mimic" or "NM" to join the original primary divisions and have added a new designation, "natural product botanical" or "NB", to cover those botanical "defined mixts." that have now been recognized as drug entities by the FDA and similar organizations. From the data presented, the utility of natural products as sources of novel structures, but not necessarily the final drug entity, is still alive and well. Thus, in the area of cancer, over the time frame from around the 1940s to date, of the 175 small mols., 131, or 74.8%, are other than "S" (synthetic), with 85, or 48.6%, actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the anti-infective area being dependent on natural products and their structures. Although combinatorial chem. techniques have succeeded as methods of optimizing structures and have been used very successfully in the optimization of many recently approved agents, we are able to identify only one de novo combinatorial compd. approved as a drug in this 30-yr time frame. We wish to draw the attention of readers to the rapidly evolving recognition that a significant no. of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore we consider that this area of natural product research should be expanded significantly.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XitVeku78%253D&md5=395ac7378f07d122a5789d7b440f858d
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      Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms
      Fischbach, Michael A.; Walsh, Christopher T.
      Chemical Reviews (Washington, DC, United States) (2006), 106 (8), 3468-3496CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review on the logic, machinery, and mechanisms of the polyketide synthase (PKS) and nonribosomal peptide synthetase enzymic assembly lines. This basic knowledge of PKS and NRPS systems provides context for more efficient efforts in combinatorial biosynthesis to create collections of natural product variants with novel structure and function.
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      Egan, A. J. F.; Errington, J.; Vollmer, W. Regulation of peptidoglycan synthesis and remodelling. Nat. Rev. Microbiol. 2020, 18, 446– 460,  DOI: 10.1038/s41579-020-0366-3
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      Regulation of peptidoglycan synthesis and remodelling
      Egan, Alexander J. F.; Errington, Jeff; Vollmer, Waldemar
      Nature Reviews Microbiology (2020), 18 (8), 446-460CODEN: NRMACK; ISSN:1740-1526. (Nature Research)
      Abstr.: Bacteria surround their cell membrane with a net-like peptidoglycan layer, called sacculus, to protect the cell from bursting and maintain its cell shape. Sacculus growth during elongation and cell division is mediated by dynamic and transient multiprotein complexes, the elongasome and divisome, resp. In this Review we present our current understanding of how peptidoglycan synthases are regulated by multiple and specific interactions with cell morphogenesis proteins that are linked to a dynamic cytoskeletal protein, either the actin-like MreB or the tubulin-like FtsZ. Several peptidoglycan synthases and hydrolases require activation by outer-membrane-anchored lipoproteins. We also discuss how bacteria achieve robust cell wall growth under different conditions and stresses by maintaining multiple peptidoglycan enzymes and regulators as well as different peptidoglycan growth mechanisms, and we present the emerging role of LD-transpeptidases in peptidoglycan remodelling.
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      Induction and cultivation of a stable L-form of Bacillus subtilis
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      The Journal of applied bacteriology (1991), 70 (4), 339-43 ISSN:0021-8847.
      The induction of L-forms of Bacillus subtilis from protoplasts is described. The method involved the frequent subculture of the unstable L-form on a growth medium supplemented with lysozyme and horse serum. A stable culture, which did not revert when lysozyme and horse serum were omitted from the medium, was obtained after 13 subcultures. This culture could be grown on solid and in liquid medium by routine microbiological methods. Long-term storage of these cells was achieved by freeze drying and maintenance in glycerol at -70 degrees C. The cultural adaptability of the L-form is described and discussed with respect to methods of cultivation and growth.
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    6. 6
      Kawai, Y.; Mickiewicz, K.; Errington, J. Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria. Cell 2018, 172, 1038– 1049,  DOI: 10.1016/j.cell.2018.01.021
      6
      Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria
      Kawai, Yoshikazu; Mickiewicz, Katarzyna; Errington, Jeff
      Cell (Cambridge, MA, United States) (2018), 172 (5), 1038-1049.e10CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      β-Lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiol. culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiol., osmoprotective conditions, for various Gram-pos. bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also obsd. in macrophages and in an animal model, presumably due to the prodn. of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.
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    7. 7
      Leaver, M.; Domínguez-Cuevas, P.; Coxhead, J. M.; Daniel, R. A.; Errington, J. Life without a wall or division machine in Bacillus subtilis. Nature 2009, 457, 849– 853,  DOI: 10.1038/nature07742
      7
      Life without a wall or division machine in Bacillus subtilis
      Leaver, M.; Dominguez-Cuevas, P.; Coxhead, J. M.; Daniel, R. A.; Errington, J.
      Nature (London, United Kingdom) (2009), 457 (7231), 849-853CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review and discussion. The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivs. of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biol. or their means of propagation. The authors have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Using genome sequencing, they have identified a single point mutation that predisposes cells to grow without a wall. Propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resoln. mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.
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    8. 8
      Mercier, R.; Kawai, Y.; Errington, J. Excess membrane synthesis drives a primitive mode of cell proliferation. Cell 2013, 152, 997– 1007,  DOI: 10.1016/j.cell.2013.01.043
      8
      Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation
      Mercier, Romain; Kawai, Yoshikazu; Errington, Jeff
      Cell (Cambridge, MA, United States) (2013), 152 (5), 997-1007CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The peptidoglycan cell wall is a hallmark of the bacterial subkingdom. Surprisingly, many modern bacteria retain the ability to switch into a wall-free state called the L-form. L-form proliferation is remarkable in being independent of the normally essential FtsZ-based division machinery and in occurring by membrane blebbing and tubulation. We show that mutations leading to excess membrane synthesis are sufficient to drive L-form division in Bacillus subtilis. Artificially increasing the cell surface area to vol. ratio in wild-type protoplasts generates similar shape changes and cell division. Our findings show that simple biophys. processes could have supported efficient cell proliferation during the evolution of early cells and provide an extant biol. model for studying this problem.
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    9. 9
      Clasener, H. Pathogenicity of the L-Phase of Bacteria. Annu. Rev. Microbiol. 1972, 26, 55– 84,  DOI: 10.1146/annurev.mi.26.100172.000415
      9
      Pathogenicity of the L-phase of bacteria
      Clasener H
      Annual review of microbiology (1972), 26 (), 55-84 ISSN:0066-4227.
      There is no expanded citation for this reference.
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    10. 10
      Domingue, G. J.; Woody, H. B. Bacterial persistence and expression of disease. Clin. Microbiol. Rev. 1997, 10, 320– 344,  DOI: 10.1128/cmr.10.2.320
      10
      Bacterial persistence and expression of disease
      Domingue G J Sr; Woody H B
      Clinical microbiology reviews (1997), 10 (2), 320-44 ISSN:0893-8512.
      A considerable body of experimental and clinical evidence supports the concept that difficult-to-culture and dormant bacteria are involved in latency of infection and that these persistent bacteria may be pathogenic. This review includes details on the diverse forms and functions of individual bacteria and attempts to make this information relevant to the care of patients. A series of experimental studies involving host-bacterium interactions illustrates the probability that most bacteria exposed to a deleterious host environment can assume a form quite different from that of a free-living bacterium. A hypothesis is offered for a kind of reproductive cycle of morphologically aberrant bacteria as a means to relate their diverse tissue forms to each other. Data on the basic biology of persistent bacteria are correlated with expression of disease and particularly the mechanisms of both latency and chronicity that typify certain infections. For example, in certain streptococcal and nocardial infections, it has been clearly established that wall-defective forms can be induced in a suitable host. These organisms can survive and persist in a latent state within the host, and they can cause pathologic responses compatible with disease. A series of cases illustrating idiopathic conditions in which cryptic bacteria have been implicated in the expression of disease is presented. These conditions include nephritis, rheumatic fever, aphthous stomatitis, idiopathic hematuria, Crohn's disease, and mycobacterial infections. By utilizing PCR, previously nonculturable bacilli have been identified in patients with Whipple's disease and bacillary angiomatosis. Koch's postulates may have to be redefined in terms of molecular data when dormant and nonculturable bacteria are implicated as causative agents of mysterious diseases.
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    11. 11
      Onwuamaegbu, M.; Belcher, R.; Soare, C. Cell wall-deficient bacteria as a cause of infections: A review of the clinical significance. J. Int. Med. Res. 2005, 33, 1– 20,  DOI: 10.1177/147323000503300101
      11
      Cell wall-deficient bacteria as a cause of infections: a review of the clinical significance
      Onwuamaegbu M E; Belcher R A; Soare C
      The Journal of international medical research (2005), 33 (1), 1-20 ISSN:0300-0605.
      Cell wall-deficient bacteria (CWDB) are pleomorphic bacterial forms. These atypical organisms may occur naturally or they can be induced in the laboratory. Their presence has been known about for over a century, but a definite link to clinical disease outcomes has not been demonstrated. A number of case reports and laboratory studies suggest some disease associations, however. Considerable controversy surrounds the true relevance of CWDB to disease; there is a widespread belief that they may represent a response by the walled organism to adverse extracellular conditions like antibiotic pressure. This review looks at studies published between 1934 and 2003, which were identified by Dialog DataStar using the key words 'cell wall deficient bacteria and clinical significance and infections' and by further scanning the reference list at the end of the papers retrieved. We conclude that the evidence for the clinical significance of CWDB in disease is not compelling.
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    12. 12
      Allan, E. J.; Hoischen, C.; Gumpert, J., Chapter 1 Bacterial L-Forms. In Advances in Applied Microbiology; Academic Press, 2009; Vol. 68, pp 1– 39.
      There is no corresponding record for this reference.
    13. 13
      Errington, J.; Mickiewicz, K.; Kawai, Y.; Wu, L. J. L-form bacteria, chronic diseases and the origins of life. Philos. Trans. R. Soc. London, Ser. B 2016, 371, 20150494,  DOI: 10.1098/rstb.2015.0494
      There is no corresponding record for this reference.
    14. 14
      Mickiewicz, K. M.; Kawai, Y.; Drage, L.; Gomes, M. C.; Davison, F.; Pickard, R.; Hall, J.; Mostowy, S.; Aldridge, P. D.; Errington, J. Possible role of L-form switching in recurrent urinary tract infection. Nat. Commun. 2019, 10, 4379,  DOI: 10.1038/s41467-019-12359-3
      14
      Possible role of L-form switching in recurrent urinary tract infection
      Mickiewicz Katarzyna M; Kawai Yoshikazu; Drage Lauren; Davison Frances; Aldridge Phillip D; Errington Jeff; Gomes Margarida C; Mostowy Serge; Pickard Robert; Hall Judith
      Nature communications (2019), 10 (1), 4379 ISSN:.
      Recurrent urinary tract infection (rUTI) is a major medical problem, especially in the elderly and infirm, but the nature of the reservoir of organisms responsible for survival and recolonisation after antibiotic treatment in humans is unclear. Here, we demonstrate the presence of cell-wall deficient (L-form) bacteria in fresh urine from 29 out of 30 older patients with rUTI. In urine, E. coli strains from patient samples readily transition from the walled state to L-form during challenge with a cell wall targeting antibiotic. Following antibiotic withdrawal, they then efficiently transition back to the walled state. E. coli switches between walled and L-form states in a zebrafish larva infection model. The results suggest that L-form switching is a physiologically relevant phenomenon that may contribute to the recurrence of infection in older patients with rUTI, and potentially other infections.
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    15. 15
      Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H. U.; Bruccoleri, R.; Lee, S. Y.; Fischbach, M. A.; Müller, R.; Wohlleben, W.; Breitling, R.; Takano, E.; Medema, M. H. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015, 43, W237– W243,  DOI: 10.1093/nar/gkv437
      15
      antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters
      Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A.; Muller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H.
      Nucleic Acids Research (2015), 43 (W1), W237-W243CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Microbial secondary metab. constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chems. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodol. for novel compd. discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and anal. of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission nos. are assigned to functionally classify all enzyme-coding genes. Addnl., chem. structure prediction has been improved by incorporating polyketide redn. states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.
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    16. 16
      Haydock, S. F.; Aparicio, J. F.; Molnár, I.; Schwecke, T.; Khaw, L. E.; König, A.; Marsden, A. F.; Galloway, I. S.; Staunton, J.; Leadlay, P. F. Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases. FEBS Lett. 1995, 374, 246– 248,  DOI: 10.1016/0014-5793(95)01119-y
      16
      Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases
      Haydock, Stephen F.; Aparicio, Jesus F.; Molnar, Istvan; Schwecke, Torsten; Khaw, Lake Ee; Koenig, Ariane; Marsden, Andrew F. A.; Galloway, Ian S.; Staunton, James; et al.
      FEBS Letters (1995), 374 (2), 246-8CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
      The amino acid sequences of a large no. of polyketide synthase domains that catalyze the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) were compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences were sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-detd. structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, although not close enough to make direct contact with bound substrate.
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    17. 17
      Del Vecchio, F.; Petkovic, H.; Kendrew, S. G.; Low, L.; Wilkinson, B.; Lill, R.; Cortés, J.; Rudd, B. A. M.; Staunton, J.; Leadlay, P. F. Active-site residue, domain and module swaps in modular polyketide synthases. J. Ind. Microbiol. Biotechnol. 2003, 30, 489– 494,  DOI: 10.1007/s10295-003-0062-0
      17
      Active-site residue, domain and module swaps in modular polyketide synthases
      Del Vecchio, Francesca; Petkovic, Hrvoje; Kendrew, Steven G.; Low, Lindsey; Wilkinson, Barrie; Lill, Rachel; Cortes, Jesus; Rudd, Brian A. M.; Staunton, Jim; Leadlay, Peter F.
      Journal of Industrial Microbiology & Biotechnology (2003), 30 (8), 489-494CODEN: JIMBFL; ISSN:1367-5435. (Springer-Verlag)
      Sequence comparisons of multiple acyltransferase (AT) domains from modular polyketide synthases (PKSs) have highlighted a correlation between a short sequence motif and the nature of the extender unit selected. When this motif was specifically altered in the bimodular model PKS DEBS1-TE of Saccharopolyspora erythraea, the products included triketide lactones in which acetate extension units had been incorporated instead of propionate units at the predicted positions. We also describe a cassette system for convenient construction of hybrid modular PKSs based on the tylosin PKS in Streptomyces fradiae and demonstrate its use in domain and module swaps.
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    18. 18
      Donadio, S.; McAlpine, J. B.; Sheldon, P. J.; Jackson, M.; Katz, L. An erythromycin analog produced by reprogramming of polyketide synthesis. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7119– 7123,  DOI: 10.1073/pnas.90.15.7119
      18
      An erythromycin analog produced by reprogramming of polyketide synthesis
      Donadio, Stefano; McAlpine, James B.; Sheldon, Paul J.; Jackson, Marianna; Katz, Leonard
      Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (15), 7119-23CODEN: PNASA6; ISSN:0027-8424.
      The polyketide-derived macrolactone of the antibiotic erythromycin is made through successive condensation and processing of 7 C3 units. The 4th cycle involves complete processing of the newly formed β-keto group (β-keto redn., dehydration, and enoyl redn.) to yield the methylene that will appear at C-7 of the lactone ring. Synthesis of this mol. in Saccharopolyspora erythraea is detd. by the 3 large eryA genes, organized in 6 modules, each governing 1 condensation cycle. Two amino acid substitutions were introduced in the putative NAD(P)H-binding motif in the proposed enoyl reductase domain encoded by eryAII. The metabolite produced by the resulting strain was identified as Δ6,7-anhydroerythromycin C resulting from failure of enoyl redn. during the 4th cycle of synthesis of the macrolactone. This result demonstrates the involvement of at least the enoyl reductase from the 4th module in the 4th cycle and indicates that a virtually complete macrolide can be produced through reprogramming of polyketide synthesis.
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    19. 19
      Keatinge-Clay, A. Crystal structure of the erythromycin polyketide synthase dehydratase. J. Mol. Biol. 2008, 384, 941– 953,  DOI: 10.1016/j.jmb.2008.09.084
      19
      Crystal Structure of the Erythromycin Polyketide Synthase Dehydratase
      Keatinge-Clay, Adrian
      Journal of Molecular Biology (2008), 384 (4), 941-953CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      The dehydratases (DHs) of modular polyketide synthases (PKSs) catalyze dehydrations that occur frequently in the biosynthesis of complex polyketides, yet little is known about them structurally or mechanistically. Here, the structure of a DH domain, isolated from the fourth module of the erythromycin PKS, is presented at 1.85 Å resoln. As with the DH of the highly related animalian fatty acid synthase, the DH monomer possesses a double-hotdog fold. Two symmetry mates within the crystal lattice make a contact that likely represents the DH dimerization interface within an intact PKS. Conserved hydrophobic residues on the DH surface indicate potential interfaces with two other PKS domains, the ketoreductase and the acyl carrier protein. Mutation of an invariant arginine at the hypothesized acyl carrier protein docking site in the context of the erythromycin PKS resulted in decreased prodn. of the erythromycin precursor 6-deoxyerythronolide B. The structure elucidates how the α-hydrogen and β-hydroxyl group of a polyketide substrate interact with the catalytic histidine and aspartic acid in the DH active site prior to dehydration.
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    20. 20
      Zheng, J.; Taylor, C. A.; Piasecki, S. K.; Keatinge-Clay, A. T. Structural and functional analysis of A-type ketoreductases from the amphotericin modular polyketide synthase. Structure 2010, 18, 913– 922,  DOI: 10.1016/j.str.2010.04.015
      20
      Structural and Functional Analysis of A-Type Ketoreductases from the Amphotericin Modular Polyketide Synthase
      Zheng, Jian-Ting; Taylor, Clint A.; Piasecki, Shawn K.; Keatinge-Clay, Adrian T.
      Structure (Cambridge, MA, United States) (2010), 18 (8), 913-922CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Complex polyketides are characterized by multiple chiral centers harboring hydroxyl and alkyl substituents. To investigate the mechanisms by which these stereocenters are set, several high-resoln. structures of the ketoreductase (KR) domain from the second module of the amphotericin modular polyketide synthase (PKS) were solved. This first structural anal. of an A-type KR helps reveal how these KRs direct polyketide intermediates into their active sites from the side opposite that used by B-type KRs, resulting in a β-hydroxyl group of opposite stereochem. A comparison of structures obtained in the absence and presence of ligands reveals an induced fit mechanism that is important for catalysis. Activity assays of mutants of KRs from the first and second modules of the amphotericin PKS reveal the relative contributions of several active site residues toward catalysis and stereocontrol. Together, these results highlight the possibility of region-specific modification of polyketides through active site engineering of KRs.
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    21. 21
      Keatinge-Clay, A. T. Stereocontrol within polyketide assembly lines. Nat. Prod. Rep. 2016, 33, 141– 149,  DOI: 10.1039/c5np00092k
      21
      Stereocontrol within polyketide assembly lines
      Keatinge-Clay, Adrian T.
      Natural Product Reports (2016), 33 (2), 141-149CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. Most of the stereocenters of polyketide natural products are established during assembly line biosynthesis. The body of knowledge for how stereocenters are set is now large enough to begin constructing phys. models of key reactions. Interactions between stereocenter-forming enzymes and polyketide intermediates are examd. here at at. resoln., drawing from the most current structural and functional information of ketosynthases (KSs), ketoreductases (KRs), dehydratases (DHs), enoylreductases (ERs), and related enzymes. While many details remain to be exptl. detd., our understanding of the chem. and phys. mechanisms utilized by the chirality-molding enzymes of modular PKSs is rapidly advancing.
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    22. 22
      Caffrey, P. Conserved amino acid residues correlating with ketoreductase stereospecificity in modular polyketide synthases. ChemBioChem 2003, 4, 654– 657,  DOI: 10.1002/cbic.200300581
      22
      Conserved amino acid residues correlating with ketoreductase stereospecificity in modular polyketide synthases
      Caffrey, Patrick
      ChemBioChem (2003), 4 (7), 654-657CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      An anal. of ketoreductase (KR) sequences was carried out to better understand how modular polyketide synthases (PKSs) control alc. stereochem. in nascent oligoketide chains. Two types of alc. stereochem., designated A and B, were considered. KR domains were divided into groups according to whether they generate A- or B-type alc. stereochem. A- and B-type KR sequences were aligned to allow identification of residues characteristic of each group. Very few consistent differences were found between A- and B-type KRs. The LDD motif and residues P144 and N148 correlate well with the formation of B-type alc. stereochem. The absence of these residues and the presence of W141 correlate with A-type alc. stereochem. The PKSs that were studied also contain KRs for which the stereochem. outcome of the ketoredn. is hidden by subsequent dehydration, or dehydration and enoyl redn. These domains have the LDD motif and PN motifs characteristic of B-type KRs. This suggests that the preferred substrate for a PKS dehydratase domain is a B-type 3-hydroxyacyl chain, as is present in fatty acid biosynthesis.
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    23. 23
      Khosla, C.; Gokhale, R. S.; Jacobsen, J. R.; Cane, D. E. Tolerance and specificity of polyketide synthases. Annu. Rev. Biochem. 1999, 68, 219– 253,  DOI: 10.1146/annurev.biochem.68.1.219
      23
      Tolerance and specificity of polyketide synthases
      Khosla, Chaitan; Gokhale, Rajesh S.; Jacobsen, John R.; Cane, David E.
      Annual Review of Biochemistry (1999), 68 (), 219-253CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review with 89 refs. Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Addnl. complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the mol. recognition features of polyketide synthases. Within this decade, a variety of genetic, biochem., and chem. investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review.
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    24. 24
      Böhm, I.; Holzbaur, I. E.; Hanefeld, U.; Cortési, J.; Staunton, J.; Leadlay, P. F. Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension. Chem. Biol. 1998, 5, 407– 412,  DOI: 10.1016/S1074-5521(98)90157-0
      24
      Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension
      Bohm, Ines; Hlazbaur, Ines E.; Hanefeld, Ulf; Cortes, Jesus; Staunton, Jim; Leadlay, Peter F.
      Chemistry & Biology (1998), 5 (8), 407-412CODEN: CBOLE2; ISSN:1074-5521. (Current Biology Ltd.)
      Polyketides are a large and structurally diverse group of natural products that include antibiotics, antifungal agents and immunosuppressant compds. Polyketides are biosynthesized in filamentous bacteria on modular polyketide synthases (PKSs) in which each cycle of chain extension requires a different 'module' of enzymic activities. The recently proposed dimeric model for modular PKSs predicts that even a single-module PKS should be catalytically active in the absence of other PKS components. Researchers are also interested in manipulating the stereochem. outcome of polyketide chain extension using genetic engineering of domains within each module. We have constructed a minimal modular PKS from the erythromycin-producing PKS (DEBS) of Saccharopolyspora erythraea. The diketide synthase (DKS1-2) consists of a single chimeric extension module, derived from the DEBS module 1 ketoacyl-ACP synthase (KS), sandwiched between a loading module and a chain-terminating thioesterase. When DKS1-2 was expressed in S. erythraea, the strain preferentially accumulated the diketide (2R,3S)-2-methyl-3-hydroxy pentanoic acid. These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2-methyl-3-hydroxy intermediate by epimerizing the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The obsd. formation of the alternative (2R)-2-methyl-3-hydroxy product catalyzed by DKS1-2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS.
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    25. 25
      Um, S.; Guo, H.; Thiengmag, S.; Benndorf, R.; Murphy, R.; Rischer, M.; Braga, D.; Poulsen, M.; de Beer, Z. W.; Lackner, G.; Beemelmanns, C. Comparative genomic and metabolic analysis of Streptomyces sp. RB110 morphotypes illuminates genomic rearrangements and formation of a new 46-membered antimicrobial macrolide. ACS Chem. Biol. 2021, 16, 1482– 1492,  DOI: 10.1021/acschembio.1c00357
      25
      Comparative Genomic and Metabolic Analysis of Streptomyces sp. RB110 Morphotypes Illuminates Genomic Rearrangements and Formation of a New 46-Membered Antimicrobial Macrolide
      Um, Soohyun; Guo, Huijuan; Thiengmag, Sirinthra; Benndorf, Rene; Murphy, Robert; Rischer, Maja; Braga, Daniel; Poulsen, Michael; de Beer, Z. Wilhelm; Lackner, Gerald; Beemelmanns, Christine
      ACS Chemical Biology (2021), 16 (8), 1482-1492CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Morphotype switches frequently occur in Actinobacteria and are often assocd. with disparate natural product prodn. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clin. important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic anal. of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.
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    26. 26
      Hu, Y.; Wang, M.; Wu, C.; Tan, Y.; Li, J.; Hao, X.; Duan, Y.; Guan, Y.; Shang, X.; Wang, Y.; Xiao, C.; Gan, M. Identification and Proposed Relative and Absolute Configurations of Niphimycins C-E from the Marine-Derived Streptomyces sp. IMB7-145 by Genomic Analysis. J. Nat. Prod. 2018, 81, 178– 187,  DOI: 10.1021/acs.jnatprod.7b00859
      26
      Identification and Proposed Relative and Absolute Configurations of Niphimycins C-E from the Marine-Derived Streptomyces sp. IMB7-145 by Genomic Analysis
      Hu, Yuanyuan; Wang, Mian; Wu, Chunyan; Tan, Yi; Li, Jiao; Hao, Xiaomeng; Duan, Yanbo; Guan, Yan; Shang, Xiaoya; Wang, Yiguang; Xiao, Chunling; Gan, Maoluo
      Journal of Natural Products (2018), 81 (1), 178-187CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society-American Society of Pharmacognosy)
      Anal. of the whole genome sequence of Streptomyces sp. IMB7-145 revealed the presence of seven type I polyketide synthase biosynthetic gene clusters, one of which was highly homologous to the biosynthetic gene cluster of azalomycin F. Detailed bioinformatic anal. of the modular organization of the PKS gene suggested that this gene is responsible for niphimycin biosynthesis. Guided by genomic anal., a large-scale cultivation ultimately led to the discovery and characterization of four new niphimycin congeners, namely niphimycins C-E (1-3) and 17-O-methylniphimycin (4). The configurations of most stereocenters of niphimycins have not been detd. to date. Inthe present study, the relative configurations were elucidated by spectroscopic anal., including J-based anal. and the CNMR database method. Further, the full abs. configurations of niphimycins were completely proposed for the first time based on biosynthetic gene cluster anal. of the ketoreductase and enoylreductase domains for hydroxy- and methyl-bearing stereocenters. Compds. 1, 3, 4, and niphimycin Iα (5) showed antimicrobial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC: 8-64 μg/mL), as well as cytotoxicity against the human HeLa cancer cell line (IC50: 3.0-9.0 μM). In addn., compds. 1 and 5 displayed significant activity against several Mycobacterium tuberculosis clin. isolates (MIC: 4-32 μg/mL).
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    27. 27
      Chiu, H.-T.; Weng, C.-P.; Lin, Y.-C.; Chen, K.-H. Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes. Org. Biomol. Chem. 2016, 14, 1988– 2006,  DOI: 10.1039/c5ob02292d
      27
      Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-L-fucose biosynthetic enzymes
      Chiu, Hsien-Tai; Weng, Chien-Pao; Lin, Yu-Chin; Chen, Kuan-Hung
      Organic & Biomolecular Chemistry (2016), 14 (6), 1988-2006CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-L-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match expts. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and mol. levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
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    28. 28
      Pérez-Victoria, I.; Oves-Costales, D.; Lacret, R.; Martín, J.; Sánchez-Hidalgo, M.; Díaz, C.; Cautain, B.; Vicente, F.; Genilloud, O.; Reyes, F. Structure elucidation and biosynthetic gene cluster analysis of caniferolides A–D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066. Org. Biomol. Chem. 2019, 17, 2954– 2971,  DOI: 10.1039/C8OB03115K
      28
      Structure elucidation and biosynthetic gene cluster analysis of caniferolides A-D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066
      Perez-Victoria, Ignacio; Oves-Costales, Daniel; Lacret, Rodney; Martin, Jesus; Sanchez-Hidalgo, Marina; Diaz, Caridad; Cautain, Bastien; Vicente, Francisca; Genilloud, Olga; Reyes, Fernando
      Organic & Biomolecular Chemistry (2019), 17 (11), 2954-2971CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D. Their connectivity was detd. by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochem. of each stereocluster in these compds. was established using NOE anal., the universal database method and J-based configuration anal., further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics anal. led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochem. outcome of their biosynthesis, confirming the relative stereochem. of each stereocluster already detd. by NMR and establishing their stereochem. relationship, ultimately rendering the abs. configuration of all chiral centers. Furthermore, based on our results and already published data, it has been possible to derive the complete abs. configuration of the related macrolides PM100117 and PM100118, astolides A and B, and deplelides A and B. Caniferolides A-D showed pronounced antifungal activity against Candida albicans and Aspergillus fumigatus alongside antiproliferative activity against 5 human tumoral cell lines.
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    29. 29
      Hashimoto, T.; Hashimoto, J.; Kozone, I.; Amagai, K.; Kawahara, T.; Takahashi, S.; Ikeda, H.; Shin-ya, K. Biosynthesis of quinolidomicin, the largest known macrolide of terrestrial origin: Identification and heterologous expression of a biosynthetic gene cluster over 200 kb. Org. Lett. 2018, 20, 7996– 7999,  DOI: 10.1021/acs.orglett.8b03570
      29
      Biosynthesis of quinolidomicin, the largest known macrolide of terrestrial origin: Identification and heterologous expression of a biosynthetic gene cluster over 200 kb
      Hashimoto, Takuya; Hashimoto, Junko; Kozone, Ikuko; Amagai, Keita; Kawahara, Teppei; Takahashi, Shunji; Ikeda, Haruo; Shin-ya, Kazuo
      Organic Letters (2018), 20 (24), 7996-7999CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      Quinolidomicin A1 is the largest macrolide compd. from terrestrial sources, consisting of a 60-membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.
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    30. 30
      Studer, P.; Staubli, T.; Wieser, N.; Wolf, P.; Schuppler, M.; Loessner, M. J. Proliferation of Listeria monocytogenes L-form cells by formation of internal and external vesicles. Nat. Commun. 2016, 7, 13631,  DOI: 10.1038/ncomms13631
      30
      Proliferation of Listeria monocytogenes L-form cells by formation of internal and external vesicles
      Studer, Patrick; Staubli, Titu; Wieser, Noemi; Wolf, Patrick; Schuppler, Markus; Loessner, Martin J.
      Nature Communications (2016), 7 (), 13631CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      L-Forms are cell wall-deficient bacteria that divide through unusual mechanisms, involving dynamic perturbations of the cellular shape and generation of vesicles, independently of the cell-division protein FtsZ. Here we describe FtsZ-independent mechanisms, involving internal and external vesicles, by which Listeria monocytogenes L-forms proliferate. Using micromanipulation of single cells and vesicles, we show that small vesicles are formed by invagination within larger intracellular vesicles, receive cytoplasmic content, and represent viable progeny. In addn., the L-forms can reproduce by pearling, i.e., generation of extracellular vesicles that remain transiently linked to their mother cell via elastic membranous tubes. Using photobleaching and fluorescence recovery, we demonstrate cytoplasmic continuity and transfer through these membranous tubes. Our findings indicate that L-forms' polyploidy and extended interconnectivity through membranous tubes contribute to the generation of viable progeny independently of dedicated division machinery, and further support L-forms as models for studies of potential multiplication mechanisms of hypothetical primitive cells.
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    31. 31
      Mercier, R.; Domínguez-Cuevas, P.; Errington, J. Crucial role for membrane fluidity in proliferation of primitive cells. Cell Rep. 2012, 1, 417– 423,  DOI: 10.1016/j.celrep.2012.03.008
      31
      Crucial role for membrane fluidity in proliferation of primitive cells
      Mercier, Romain; Dominguez-Cuevas, Patricia; Errington, Jeff
      Cell Reports (2012), 1 (5), 417-423CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell wall-deficient "L-form" state, requiring remarkable physiol. and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ-based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, the authors uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the sepn. of progeny cells. The failure in scission is assocd. with a redn. in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophys. processes may have been sufficient for proliferation of primitive cells.
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    32. 32
      Wolf, D.; Domínguez-Cuevas, P.; Daniel, R. A.; Mascher, T. Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis. Antimicrob. Agents Chemother. 2012, 56, 5907– 5915,  DOI: 10.1128/aac.00770-12
      32
      Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis
      Wolf, Diana; Dominguez-Cuevas, Patricia; Daniel, Richard A.; Mascher, Thorsten
      Antimicrobial Agents and Chemotherapy (2012), 56 (11), 5907-5915CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
      L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-pos. model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to β-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here the authors studied the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. The authors' results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, the authors established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).
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