Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci.

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsiella pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42 degrees C were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.     

Bacterial morphogenesis: learning how cells make cells

Bacteria furnish tractable models for complex biological processes, and morphogenesis is now taking its turn. We can already explain in general terms how such elementary forms as rods and cocci are produced, and the shapes of several individual organisms are coming into focus. In most bacteria shape is maintained by the cell wall, specifically the peptidoglycan layer, which has the attributes of a strong stiff fabric. Compliance of that fabric with turgor pressure is an important aspect of morphogenesis. The shape of the wall sacculus is determined by the way it is deposited, which is controlled by a cytoskeleton made up of two molecular families. One, related to the eukaryotic tubulins, is responsible for the construction of the septum and the poles. The other, related to eukaryotic actins, localizes peptidoglycan synthesis in the lateral walls of rod-shaped cells. Just how the cytoskeleton itself is organized remains to be discovered, but it seems likely that, as in eukaryotes, the cytoskeleton is produced by self-organized assembly, guided by the fabric of the cell.     

Alterations in peptidoglycan chemical composition associated with rod-to-sphere transition in a conditional mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae Mir M7 is a spontaneous parentless morphology mutant which grows as cocci at pH 7 and as rods at pH 5.8. This strain has been characterized as defective in lateral wall formation (at pH7). Data suggest that the cell wall is mainly made up of poles of the rods (G. Satta, R. Fontana, P. Canepari, and G. Botta, J. Bacteriol. 137:727--734, 1979). In this work the isolation and the biochemical properties of the peptidoglycan of both Mir M7 rods and cocci and a nonconditional rod-shaped Mir M7 revertant (strain Mir A12) are described. The peptidoglycan of Mir M7 (both rods and cocci) and Mir A12 strains carried covalently bound proteins which could be easily removed by pronase treatment in Mir M7 rods and Mir A12 cells, but not in Mir M7 round cells. However, when the sodium dodecyl sulfate-insoluble residues of Mir M7 cocci were pretreated with ethylenediaminetetraacetic acid (EDTA), pronase digestion removed the covalently bound proteins, and pure peptidoglycan was obtained. EDTA treatment of the rigid layer of Mir M7 cocci removed amounts of Mg2+ and Ca2+, which were 10- and 50-fold higher, respectively, than the amount liberated from the rigid layer of Mir M7 rods and Mir A12 cells. Amino acid composition was qualitatively similar in both strains, but Mir M7 cocci contained a higher amount of alanine and glucosamine. Mir M7 cocci contained approximately 50% less peptidoglycan than rods. Under electron microscopy, the rigid layer of the Mir M7 rods and Mir A12 cells appeared to be rod-shaped and their shape remained unchanged after EDTA and pronase treatment. On the contrary, the Mir M7 cocci rigid layer appeared to be round, and after EDTA treatment it collapsed and lost any definite morphology. In spite of these alterations, the peptidoglycan of Mir M7 cocci still appeared able to determine the shape of the cell and protect it from osmotic shock and mechanical damages. The accumluation of divalent cations appeared necessary for the peptidoglycan to acquire sufficient rigidity for shape determination and cell protection. We concluded that the coccal shape in Mir M7 cells is not due to loss of cell wall rigidity but is a consequence of the formation of a round peptidoglycan molecule. The possibility that the alterations found in the Mir M7 cocci rigid layer may reflect natural differences in the biochemical composition of the septa and lateral wall of normally shaped bacteria is discussed.     

Rapid test for the serological separation of staphylococci from micrococci

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.     

Rapid test for the serological separation of staphylococci from micrococci.

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.     

Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins.

Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptidoglycan transpeptidases in these gram-positive cocci. Unlike Escherichia coli, in which the network structure of peptidoglycan is synthesized by multiple two-headed penicillin-binding proteins with both transpeptidase and transglycosylase activities, these gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase.     

Covalent attachment of proteins to peptidoglycan

Bacterial surface proteins are key players in host-symbiont or host-pathogen interactions. How these proteins are targeted and displayed at the cell surface are challenging issues of both fundamental and clinical relevance. While surface proteins of Gram-negative bacteria are assembled in the outer membrane, Gram-positive bacteria predominantly utilize their thick cell wall as a platform to anchor their surface proteins. This surface display involves both covalent and noncovalent interactions with either the peptidoglycan or secondary wall polymers such as teichoic acid or lipoteichoic acid. This review focuses on the role of enzymes that covalently link surface proteins to the peptidoglycan, the well-known sortases in Gram-positive bacteria, and the recently characterized l,d-transpeptidases in Gram-negative bacteria.     

Peptidoglycan Synthesis in Cocci and Rods of a pH-Dependent, Morphologically Conditional Mutant of Klebsiella pneumoniae

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.     

Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell

Cell shape in most eubacteria is maintained by a tough external peptidoglycan cell wall. Recently, cell shape determining proteins of the MreB family were shown to form helical, actin-like cables in the cell. We used a fluorescent derivative of the antibiotic vancomycin as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria. In the rod-shaped bacterium B. subtilis, synthesis of the cylindrical part of the cell wall occurs in a helical pattern governed by an MreB homolog, Mbl. However, a few rod-shaped bacteria have no MreB system. Here, a rod-like shape can be achieved by a completely different mechanism based on use of polar growth zones derived from the division machinery. These results provide insights into the diverse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.     

A new morphogenesis pathway in bacteria: unbalanced activity of cell wall synthesis machineries leads to coccus-to-rod transition and filamentation in ovococci

Bacteria display a variety of shapes, which have biological relevance. In most eubacteria, cell shape is maintained by the tough peptidoglycan (PG) layer of the cell wall, the sacculus. The organization of PG synthesis machineries, orchestrated by different cytoskeletal elements, determines the specific shapes of sacculi. In rod-shaped bacteria, the actin-like (MreB) and the tubuline-like (FtsZ) cytoskeletons control synthesis of the sidewall (elongation) and the crosswall (septation) respectively. Much less is known concerning cell morphogenesis in cocci, which lack MreB proteins. While spherical cocci exclusively display septal growth, ovococci additionally display peripheral growth, which is responsible of the slight longitudinal expansion that generates their ovoid shape. Here, we report that the ovococcus Lactococcus lactis has the ability to become rod-shaped. L. lactis IL1403 wild-type cells form long aseptate filaments during both biofilm and planktonic growth in a synthetic medium. Nascent PG insertion and the division protein FtsK localize in multiple peripheral rings regularly spaced along the filaments. We show that filamentation results from septation inhibition, and that penicillin-binding proteins PBP2x and PBP2b play a direct role in this process. We propose a model for filament formation in L. lactis, and discuss the possible biological role of such morphological differentiation.     

Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus

Bacteria of the genus Staphylococcus are common pathogens responsible for a broad spectrum of human and animal infections and belong to the most important etiological factors causing food poisoning. Because of rapid increase in the prevalence of isolation of staphylococci resistant to many antibiotics, there is an urgent need for the development of new alternative chemotherapeutics. A number of studies have recently demonstrated the strong potential of peptidoglycan hydrolases (PHs) to control and treat infections caused by this group of bacteria. PHs cause rapid lysis and death of bacterial cells. The review concentrates on enzymes hydrolyzing peptidoglycan of staphylococci. Usually, they are characterized by high specificity to only Staphylococcus aureus cell wall components; however, some of them are also able to lyse cells of other staphylococci, e.g., Staphylococcus epidermidis-human pathogen of growing importance and also other groups of bacteria. Some PHs strengthen the bactericidal or bacteriostatic activity of common antibiotics, and as a result, they should be considered as component of combined therapy which could definitely reduced the development of bacterial resistance to both enzymes and antibiotics. The preliminary research revealed that most of these enzymes can be produced using heterologous, especially Escherichia coli expression systems; however, still much effort is required to develop more efficient and large-scale production technologies. This review discusses current state on knowledge with emphasis on the possibilities of application of PHs in the context of therapeutics for infections caused by staphylococci.     

The fine structure of Micrococcus radiophilus and Micrococcus radioproteolyticus

The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freeze-etching techniques and the two species were found to be similar in the fine structure. The only significant difference was in the appearance of the surfaces of the cell walls in freeze-etched preparations.Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different from that of other micrococci and Gram-positive cocci, it is recommended that they be reclassified into a new genus.     

A model for the three-dimensional structure of peptidoglycan in staphylococci

Although the monomeric units of peptidoglycan in Staphylococcus aureus and other staphylococci are well known, the complete structure of the peptidoglycan has not been elucidated. The peptidoglycan monomeric unit may be divided into three parts: (1) glycan chain piece, consisting of N-acetylglucosaminyl-N-acetylmuramic acid; (2) connecting peptide extending from L-alanine to the alpha-amino group of L-lysine; (3) peptide chain piece, consisting of D-alanine, the remainder of L-lysine not included in the connecting peptide, and pentaglycine (S. aureus) or mixed glycine and serine residues (other staphylococci) attached to the epsilon amino group of lysine. The deformation of cross wall into hemisphere in the course of cell division, the distensibility of peptidoglycan, and the appearance of circular (? spiral) lines in the cross wall and on the surface of the newly-formed hemisphere are clues to the structure of peptidoglycan. In the proposed model, cross wall is formed as a linear spiral with 20 turns extending in a plane from periphery to center of the cell. During cell division, the cross wall is bisected. The cross wall spiral becomes a spiral forming the peripheral wall of a new hemisphere. The width of the spiral on the cell surface is maintained by rigid glycan chains and by covalent bonds linking turns of the spiral. The length of the spiral is about 30 times the diameter of the cell. Flexible polypeptide sheets consisting of parallel polypeptide chains run along the length of the spiral. Individual polypeptides contain an average of ten peptide chain pieces. The glycan chain is a helix with two disaccharide residues per turn; consequently consecutive connecting peptides project in opposite directions and are perpendicular both to the glycan chain and to the peptide chain. In cross wall, hydrogen bonding between polypeptide chains enables the polypeptide sheet to transmit changes in tension. The deformation of cross wall into peripheral wall requires doubling of the external surface area of the peptidoglycan. A change in the angle of the glycan chain with respect to the peptide chain results in an increase of the distance between peptide chains, causing the doubling of surface area. Implications of the model include explanations for the initiation of cell division and for the existence of osmotically growth-dependent staphylococci.     

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Mechanisms for maintaining cell shape in rod-shaped Gram-negative bacteria

For the rod-shaped Gram-negative bacterium Escherichia coli, changes in cell shape have critical consequences for motility, immune system evasion, proliferation and adhesion. For most bacteria, the peptidoglycan cell wall is both necessary and sufficient to determine cell shape. However, how the synthesis machinery assembles a peptidoglycan network with a robustly maintained micron-scale shape has remained elusive. To explore shape maintenance, we have quantified the robustness of cell shape in three Gram-negative bacteria in different genetic backgrounds and in the presence of an antibiotic that inhibits division. Building on previous modelling suggesting a prominent role for mechanical forces in shape regulation, we introduce a biophysical model for the growth dynamics of rod-shaped cells to investigate the roles of spatial regulation of peptidoglycan synthesis, glycan-strand biochemistry and mechanical stretching during insertion. Our studies reveal that rod-shape maintenance requires insertion to be insensitive to fluctuations in cell-wall density and stress, and even a simple helical pattern of insertion is sufficient for over sixfold elongation without significant loss in shape. In addition, we demonstrate that both the length and pre-stretching of newly inserted strands regulate cell width. In sum, we show that simple physical rules can allow bacteria to achieve robust, shape-preserving cell-wall growth.     

Bacterial cell wall synthesis: new insights from localization studies.

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Cell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicum

Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.     

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.     

Cell wall structure and function in lactic acid bacteria

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.