Most modern reviews about bacterial cell wall properties use the traditional classification of prokaryotic envelopes as either gram positive or gram negative (
27,
30). Mainly based on the variance of their peptidoglycan architecture, both envelope types show a characteristic difference when stained with crystal violet (Gram staining) (see reference
12). The multilayered peptidoglycan of gram-positive bacteria, with a thickness ranging from 20 to 40 nm, usually forms a physical barrier for the dye. In gram-negative bacteria, however, with their relatively thin sacculus of 2 to 6 nm, the stain can easily be washed out. Together with other characteristics, such as the occurrence of accessory cell wall polymers or the presence of an outer membrane, the peptidoglycan architecture largely determines the different properties of the two cell wall types in terms of mechanical stability, permeability, and resistance toward chemical substances (
106). Although this classification is generally useful, it oversimplifies the concept of bacterial cell walls. It also ignores, for example, that the largest and perhaps most diverse group of bacteria, the cyanobacteria, possess cell envelopes with a combination of these features.
Despite their overall gram-negative structure, the peptidoglycan layer found in cyanobacteria is considerably thicker than that of most gram-negative bacteria (Fig.
1). In unicellular strains such as
Synechococcus, its thickness is about 10 nm (
44), reaching 15 to 35 nm in filamentous species like
Phormidium uncinatum and more than 700 nm in large cyanobacteria like
Oscillatoria princeps(
56). Chemical analysis of cyanobacterial peptidoglycans has revealed further unusual characteristics. The degree of cross-linking between the peptidoglycan chains within the murein of
Synechocystis sp. strain PCC 6714 is far higher than the usual 20 to 33% found in most gram-negative bacterial peptidoglycans (
42), and in fact, the extent of cross-linking (56 to 63%) (
66) is more similar to the values reported for gram-positive bacteria (
76). On the other hand, most pentapeptides involved in cross-linkage contained only the typical gram-negative bacterial diamino acid
meso-diaminopimelic acid, in contrast to
l-diaminopimelic acid or
l-lysin in gram-positive peptidoglycan tetrapeptides (
107), although
l-lysin has been found in the pentapeptides of the cyanobacterium
Anabaena cylindrica(
75). Clearly, more studies are needed to establish the general composition of cyanobacterial peptidoglycans. Another typical constituent of gram-positive peptidoglycans, teichoic acid, is also missing in cyanobacterial cell walls. Nevertheless, the cyanobacterial peptidoglycan is complexed with specific polysaccharides (
62,
67) in a fashion very similar to that of gram-positive bacterial peptidoglycan; this feature is also found in the peptidoglycan of the closely related prochlorophytes (
64).
The unique mosaic of different bacterial cell wall features is also found in the cyanobacterial outer membranes: the lipopolysaccharides (LPS) of cyanobacteria not only contain small amounts of bound phosphate (
111) but also often lack ketodeoxyoctonate (
86,
127), a common LPS component of gram-negative bacterial outer membranes. On the other hand, the cyanobacterial O antigen is reminiscent of the
Escherichia coli O antigen (
69,
130) and is responsible for cyanophage adsorption (
108) or endotoxic activity in aquatic environments (
71); S. Fukuoka, Q. Adil, H. Kakita, and H. Obika, J. Endotoxin Res.
3:35, 1996). Furthermore, the outer membranes of cyanobacteria contain constituents not usually present in gram-negative bacteria. Among these components are carotenoids (
91,
102), unusual fatty acids, such as β-hydroxypalmitic acid which are found in the lipid A moiety (
112), or porins, which are anchored to the underlying peptidoglycan layer via bridge-like coiled-coil domains (
49). This list of cyanobacterium-specific cell envelope features is not complete but rather gives an idea how these cell walls might have changed as a consequence of specific adaptations during their (assumed) nearly 3.5 billion years of evolution (
25). Our minireview will therefore focus on some of these features and discuss them with respect to general concepts of transenvelope transport, cell wall design, motility, or evolution of this important group of bacteria. By necessity, we have selected a few aspects of the cyanobacterial envelopes, and we apologize that due to the limited space, certain topics such as the complex differentiation processes of cyanobacterial cell walls during heterocyst formation have been omitted.
CYANOBACTERIAL CELL WALLS AND ENDOSYMBIOSIS
Probably the most exciting recent results of cyanobacterial studies are hints indicating that properties once thought to be exclusive to higher plants have their roots back to cyanobacterial ancestors of today's chloroplasts. Originally, the concept of a cyanobacterium-chloroplast transition during evolution was based on the discovery of cyanelles, chloroplast-like cyanobacterial endosymbionts found in a number of taxonomically unrelated eukaryotes (
78). The cyanobacterial nature of these cyanelles was evident from their peptidoglycan-containing envelopes and their usage of phycobilisomes as light-harvesting complexes (
79). More direct evidence for the cyanobacterial nature of chloroplasts came later with the discovery of the prokaryote-type chloroplast genetic system and protein translation machinery, especially their 70S ribosomes, and their double-layered envelopes. The identification of further links accelerated with the availability of the first completely sequenced cyanobacterial genome (
87). Using the sequence of
Synechocystis sp. strain PCC 6803, it could be shown that plant phytochromes might have evolved from phytochrome-like molecules in cyanobacteria (
131; J. Hughes, T. Lamparter, F. Mittmann, E. Hartmann, W. Gartner, A. Wilde, and T. Borner, Letter, Nature 386:663, 1997; for a review, see reference
95) and that these proteins control similar light-dependent processes (
24,
70), despite their prokaryotic organization in two-component regulatory systems (
93).
Such links could also be established for the cyanobacterial cell walls. The analysis of the complete sequence of
Synechocystis sp. strain PCC 6803 allowed the identification of three open reading frames (ORFs), named synToc75, synToc34, and synTic55, with significant homology to components of the peptide-translocating system of the pea chloroplast (
16,
103). This peptide-translocating system is used by the chloroplast for the import of cytosolically synthesized proteins. This process became necessary as most of the genes of the prokaryotic chloroplast's ancestors were transferred after endosymbiosis to the cell nucleus. In pea chloroplasts, the protein translocation system is composed of two separate complexes, Toc and Tic, which are located in the outer (Toc) and inner (Tic) envelope of the chloroplast, respectively (
81). The Toc complex consists of at least three proteins designated Toc86, Toc75, and Toc34, but only the function of Toc75 has been elucidated in some detail. This protein forms a voltage-gated channel in lipid membranes in vitro (
52), a property which has also recently been demonstrated for the
Synechocystis homologue synToc75 (
51). The gating behavior of both channels changes dramatically in the presence of preproteins, suggesting that they form a major part of the protein translocation pore. No function is known for the second homologue, synToc34, which has the same distribution of GTP-binding sites and the same membrane anchor domain as its pea homologue.
Finally, the third ORF, synTic55, has a homologue in the chloroplast's inner envelope translocation complex, Tic, made up of the proteins Tic110, Tic55, Tic22, and Tic20. The structure and function of this translocon are less clear, as only a few components have been characterized so far. Nevertheless, synTic55 contains the same Rieske-type iron-sulfur cluster and mononuclear iron-binding site as the pea Tic55, indicating a similar function (
23). This function is, however, not yet known, but chemical modification with diethylpyrocarbonate arrests the translocation of the preprotein at the level of the inner membrane. As no other genes encoding potential homologues of the Toc and Tic machinery have yet been identified in
Synechocystis, we can only speculate about the origin of the other proteins. Altogether, these results suggest that the chloroplast protein import machinery was recruited from a preexisting transport system present in the cyanobacterial cell wall and thus support the hypothesis of a prokaryotic origin of the chloroplasts.
Further relations between cyanobacteria and plant chloroplasts were demonstrated to be present in the cyanobacterial outer membrane. This outer membrane usually contains a species-specific repertoire of carotenoids, in addition to different lipids and proteins (
65,
91,
102). The fact that the chloroplast outer envelope membrane also contains such carotenoids (
28,
61) supports the speculation that this plastid envelope is another relic of the endocytic event that led to the development of this organelle (
29). It has been suggested that these carotenoids might protect the cyanobacterial cells from high light intensity, particularly in the UV range (
92), a hypothesis supported by the facts that the carotenoid synthesis in cyanobacteria is enhanced under these light qualities (
32) and that transcription of genes involved in the carotenoid synthesis such as
crtB (phytoene synthetase) and
crtP (phytoene desaturase) reaches its highest level under these light conditions (
34). However, carotenoids are not the only potential photoprotective substances found in cyanobacterial envelopes. In many species like
Nostoc commune, the carbohydrate sheaths (see “Cyanobacterial cell walls and external layers” below) contain aromatic pigments like scytonemin (
99) or oligosaccharide mycosporine amino acids (OS-MAAs) (
15,
110), which can effectively absorb UV light with their extended π electron systems (
25). In this organism, different qualities of UV light even lead to synthesis of different sunscreen pigments: UV-B (<315-nm wavelength) induced the formation of OS-MAAs, whereas UV-A (315 to 425 nm) induced almost exclusively scytonemin synthesis (
32). Most likely, these UV protection mechanisms have their origin in the conditions found on Earth 3.5 billion years ago, when the evolution of cyanobacteria began. The lack of an ozone layer at that time forced all organisms to adopt to extreme UV radiation. Even today, these mechanisms enable cyanobacteria to flourish in an extreme array of habitats, many of which, like tropical pools, rock surfaces, or terrestrial habitats of the Arctic and Antarctic, are characterized by intense solar radiation. However, some species like the antarctic cyanobacterium
Oscillatoria priestleyi try to avoid this radiation rather than tolerate it and simply escape from UV light by gliding motility (
100).
CYANOBACTERIAL CELL WALLS AND LOCOMOTION
Most bacteria that are able to move do so by means of flagella, small rotary motors (
83), which have never been found in cyanobacteria (
47). Nevertheless, many cyanobacteria are capable of a slow, surface-associated motility characterized as gliding (reviewed in reference
21). Although the precise mechanism of gliding is not understood, recent studies have revealed certain structural features invariably linked to this process. Among these features are distinct surface topographies, novel bacterial organelles (JCPs [discussed above]) and the continuous secretion of mucilage.
In most members of the family
Oscillatoriaceae, the surface topographies consist of parallel, helically arranged protein fibrils (
56), although another type of fibril has recently been described for some
Oscillatoria strains (
1). In all species studied so far, the arrangement of these surface fibrils correlates with the path of the filaments during locomotion. It has therefore been proposed that the surface fibrils serve as a screw thread guiding the rotation of the filaments, with the necessary thrust for locomotion being derived from the secretion of slime from the junctional pores (
58). In this model, the frequent reversal of movements of the filaments can be explained by the alternation of the set of junctional pores used. The importance of the protein fibrils and the secretion of slime for motility are substantiated by the observation that spontaneous mutants lacking these features are nonmotile (
57).
Only in
Phormidium have the protein fibrils been characterized at the molecular level (
57). They are formed by oscillin, a 646-aa glycoprotein (GenBank accession no. AF002131 ) which contains multiple repeats of a Ca
2+-binding nonapeptide (
3), a motif also found in SwmA (GenBank accession no. U48223 ), a surface protein of swimming
Synechococcus (
17,
18,
125), and HlyA, a partial ORF of
Anabaena (GenBank accession no. U13767 ) with unknown function. However, only for SwmA has a role in motility been established (
17). Compared with the role of oscillin in gliding, the role of SwmA in swimming is not so clear (
31,
96). The fact that a mutant lacking SwmA is still able to rotate indicates that the protein plays a role in the generation of thrust but not torque, but how the cells produce the thrust for their locomotion is not known.
CYANOBACTERIAL CELL WALLS AND EXTERNAL LAYERS
Frequently, bacterial cell envelopes are covered by external surface layers such as S-layers and carbohydrate structures (
13,
126). S-layers are two-dimensional crystalline arrays formed by a single species of (glyco)protein which covers the entire surface of a cell (
117). They function as protective coats or molecular sieves or are involved in cell adhesion and recognition (
14,
118). The first observation of S-layers in cyanobacteria was reported about 30 years ago (
60), but there have been only a few detailed studies since then (
68,
73,
80). As can be seen in Fig.
3, the structures of S-layers can be surprisingly well resolved using electron microscopy in conjunction with image analysis techniques (
4). In addition to
Phormidium (
56), S-layers have been identified on more than 20 strains of
Synechococcus(
124),
Synechocystis and
Microcystisspp. (
119),
Gloecapsa alpicola(
60),
Cyanothece minerva (
45),
Aphanothece halophytica(
116), and on a
Chroococcidiopsis strain (
20). It has also been shown that cyanobacterial S-layers can participate in the formation of fine-grain minerals (
114), a process involved in the formation of stromatolites (
122).
In response to various environmental factors, many cyanobacteria produce diverse external carbohydrate structures (Fig.
4) (
126) which sometimes contain pigments such as scytonemin (
32) and OS-MAAs (
15). Some, like the slime used for gliding motility are readily secreted (discussed above), whereas others such as the sheath of the same species are more firmly attached to the cell (
55). Only a few cyanobacterial exopolysaccharides have been defined structurally, although some details of their composition are known (
126). The sheaths of
P. uncinatum and
Nostoc commune contain cellulose-like homoglucan fibrils which are cross-linked by minor monosaccharides (
39,
55).
Microcystis flos-aquae synthesizes an exopolysaccharide with a composition similar to that of pectin, containing up to 83% galacturonic acid (
121). Information about whether its structure also resembles that of pectin is not yet available.
Anabaena flos-aquae synthesizes two different polysaccharides: a xyloglucan containing glucose and xylose in a molar ratio of 8:1 and a more complex polysaccharide containing uronic acid, glucose, xylose, and ribose in the molar ratio of 10:6:1:1 (
121). This list is far from complete but should highlight the similarity between some cyanobacterial and plant carbohydrates. This observation provokes an exciting speculation. According to the so-called endosymbiont hypothesis, early plant cells started out as amoeboid anaerobic cells without photosynthesis. Only after the establishment of a stable endosymbiosis with a photosynthetic (cyano)bacterium could the early plant cells subvert the endosymbiont's ability to produce carbohydrate for their own use. Today's plant carbohydrates might, therefore, be a consequence of this event and might have evolved from common ancient exopolysaccharides in cyanobacteria. This leads to the question what are the main functions of extracellular carbohydrates in cyanobacteria? Besides being another protective coat for the cells and the photoprotective function discussed earlier, these carbohydrates embed the cells in an extremely hydrated gel-like matrix, which can retain humidity effectively even under water stress. In doing so, the carbohydrates might even prevent dehydration of the cells during temporary dryness. If, however, the unfavorable conditions continue, the delayed desiccation of the carbohydrate matrix gives the cells enough time to activate more specific protection mechanisms. Among these mechanisms are the synthesis of membrane-protective sugars like trehalose (
101) or the production of specific stress proteins (
109) which help the cells to survive desiccation and rehydration processes. The resulting extraordinary drought resistance of cyanobacteria (reviewed in reference
98) explains their predominance in many desert and tropical soils, terrestrial and subaerial habitats, where an increased tolerance of desiccation is the only way to survive.