Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH

The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.     

Structural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.     

The requirement for pneumococcal MreC and MreD is relieved by inactivation of the gene encoding PBP1a

MreC and MreD, along with the actin homologue MreB, are required to maintain the shape of rod-shaped bacteria. The depletion of MreCD in rod-shaped bacteria leads to the formation of spherical cells and the accumulation of suppressor mutations. Ovococcus bacteria, such as Streptococcus pneumoniae, lack MreB homologues, and the functions of the S. pneumoniae MreCD (MreCD(Spn)) proteins are unknown. mreCD are located upstream from the pcsB cell division gene in most Streptococcus species, but we found that mreCD and pcsB are transcribed independently. Similarly to rod-shaped bacteria, we show that mreCD are essential in the virulent serotype 2 D39 strain of S. pneumoniae, and the depletion of MreCD results in cell rounding and lysis. In contrast, laboratory strain R6 contains suppressors that allow the growth of ΔmreCD mutants, and bypass suppressors accumulate in D39 ΔmreCD mutants. One class of suppressors eliminates the function of class A penicillin binding protein 1a (PBP1a). Unencapsulated Δpbp1a D39 mutants have smaller diameters than their pbp1a(+) parent or Δpbp2a and Δpbp1b mutants, which lack other class A PBPs and do not show the suppression of ΔmreCD mutations. Suppressed ΔmreCD Δpbp1a double mutants form aberrantly shaped cells, some with misplaced peptidoglycan (PG) biosynthesis compared to that of single Δpbp1a mutants. Quantitative Western blotting showed that MreC(Spn) is abundant (≈8,500 dimers per cell), and immunofluorescent microscopy (IFM) located MreCD(Spn) to the equators and septa of dividing cells, similarly to the PBPs and PG pentapeptides indicative of PG synthesis. These combined results are consistent with a model in which MreCD(Spn) direct peripheral PG synthesis and control PBP1a localization or activity.     

Penicillin-Binding Protein 2 Is Essential for the Integrity of Growing Cells of Escherichia coli ponB Strains

Analysis of Escherichia coli pbpA(Ts) or rodA(Ts) strains defective for penicillin-binding protein (PBP) 1A or PBP 1B indicated that the activity of PBP 2 is essential to prevent cell lysis in PBP 1B(-) strains and suggested that PBP 2 is active or activatable in rodA(Ts) mutants under restrictive conditions.     

Unstable Escherichia coli L forms revisited: growth requires peptidoglycan synthesis

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the beta-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the beta-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli-and possibly all L forms-have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.     

Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.     

Effects of Low PBP2b Levels on Cell Morphology and Peptidoglycan Composition in Streptococcus pneumoniae R6

Streptococcus pneumoniae produces two class B penicillin-binding proteins, PBP2x and PBP2b, both of which are essential. It is generally assumed that PBP2x is specifically involved in septum formation, while PBP2b is dedicated to peripheral cell wall synthesis. However, little experimental evidence exists to substantiate this belief. In the present study, we obtained evidence that strongly supports the view that PBP2b is essential for peripheral peptidoglycan synthesis. Depletion of PBP2b expression gave rise to long chains of cells in which individual cells were compressed in the direction of the long axis and looked lentil shaped. This morphological change is consistent with a role for pneumococcal PBP2b in the synthesis of the lateral cell wall. Depletion of PBP2x, on the other hand, resulted in lemon-shaped and some elongated cells with a thickened midcell region. Low PBP2b levels gave rise to changes in the peptidoglycan layer that made pneumococci sensitive to exogenously added LytA during logarithmic growth and refractory to chain dispersion upon addition of LytB. Interestingly, analysis of the cell wall composition of PBP2b-depleted pneumococci revealed that they had a larger proportion of branched stem peptides in their peptidoglycan than the corresponding undepleted cells. Furthermore, MurM-deficient mutants, i.e., mutants lacking the ability to synthesize branched muropeptides, were found to require much higher levels of PBP2b to sustain growth than those required by MurM-proficient strains. These findings might help to explain why increased incorporation of branched muropeptides is required for high-level beta-lactam resistance in S. pneumoniae.     

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi‐functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N‐deacetylase PgdA and the peptidoglycan O‐acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA‐dependent and OatA‐independent. Protein‐protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB‐like cell division protein and factors important for escape from the host immune system.     

The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia coli.     

femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

femA is a chromosomally encoded factor, occurring naturally in Staphylococcus aureus, which is essential for the expression of high-level methicillin resistance in this organism. The production of a low-affinity penicillin-binding protein, PBP2a or PBP2', which is intimately involved with methicillin resistance in S. aureus, is not influenced by femA. To elucidate a possible physiological function of the 48-kDa protein encoded by femA, several related methicillin-resistant, methicillin-susceptible, and Tn551 insertionally inactivated femA mutants were analyzed for possible changes in cell wall structure and metabolism. Independent of the presence of mec, the methicillin resistance determinant, all femA mutants had a reduced peptidoglycan (PG) glycine content (up to 60% in the molar ratio of glycine/glutamic acid) compared to that of related femA+ parent strains. Additional effects of femA inactivation and the subsequent decrease in PG-associated glycine were (i) reduced digestion of PG by recombinant lysostaphin, (ii) unaltered digestion of PG by Chalaropsis B-muramidase, (iii) reduced cell wall turnover, (iv) reduced whole-cell autolysis, and (v) increased sensitivity towards beta-lactam antibiotics. Also, the PG-associated glycine content of a femA::Tn551 methicillin-susceptible strain was restored concomitantly with the methicillin resistance to a level almost equal to that of its femA+ methicillin-resistant parent strain by introduction of plasmid pBBB31, encoding femA.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.     

Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins.

The penicillin-binding proteins (PBPs) of 209 cell division (or growth) temperature-sensitive mutants of Streptococcus faecium were analyzed in this study. A total of nine strains showed either constitutive or temperature-sensitive conditional damage in the PBPs. Analysis of these nine strains yielded the following results: one carried a PBP 1 constitutively showing a lower molecular weight; one constitutively lacked PBP 2; two lacked PBP 3 at 42 degrees C, but not at 30 degrees C; one was normal at 30 degrees C but at 42 degrees C lacked PBP 3 and overproduced PBP 5; two were normal at 42 degrees C and lacked PBP 5 at 30 degrees C; one constitutively lacked PBP 5; and one carried a PBP 6 constitutively split in two bands. The mutant lacking PBP 3 and overproducing PBP 5 continued to grow at 42 degrees C for 150 min and then lysed. Revertants selected for growth capability at 42 degrees C from the mutants altered in PBPs 5 and 6 maintained the same PBP alterations, while those isolated from the strains with altered PBP 1 or lacking PBP 2 or PBP 3 showed a normal PBP pattern. Penicillin-resistant derivatives were isolated at 30 degrees C from the mutants lacking PBP 2 and from that lacking PBP 3. All these derivatives continued to show the same PBP damage as the parents, but overproduced PBP 5 and grew at 42 degrees C. These findings indicate that high-molecular-weight, but not low-molecular-weight, PBPs are essential for cell growth in S. faecium. This is in complete agreement with previous findings obtained with a different experimental system. On the basis of both previous and present data it is suggested that PBPs 1, 2, and 3 appear necessary for cell growth at optimal temperature (and at maximal rate), but not for cell growth at a submaximal one (or at a reduced rate), and an overproduced PBP 5 is capable of taking over the function of PBPs 1, 2, and 3.     

Differential methicillin susceptibilities of peptidoglycan syntheses in methicillin-resistant Staphylococcus aureus.

The mechanism of staphylococcal resistance to methicillin is unknown. Peptidoglycan synthesis was studied in a methicillin-resistant and a derived methicillin-sensitive Staphylococcus aureus strain. Although the methicillin minimum inhibitory concentration for growth of the methicillin-resistant strain was 1,600 micrograms/ml, peptidoglycan synthesis by the organism incubated in a wall synthesis solution was inhibited about 90% by 5 micrograms of methicillin per ml. In contrast, high concentrations of methicillin added to actively growing cultures of the methicillin-resistant strain had little effect on growth or peptidoglycan synthesis. Peptidoglycan synthesis in chloramphenicol-treated cultures was more susceptible to methicillin than it was in actively growing cultures of the methicillin-resistant strain. It is proposed that in this strain cell wall thickening peptidoglycan synthesis which predominates in cell wall synthesis solution and chloramphenicol-treated cultures is methicillin sensitive, whereas peptidoglycan synthesis involved in cell division, primarily in the region of the septum, which predominates in actively growing cultures is methicillin resistant. Both cell wall thickening and septal peptidoglycan syntheses are methicillin sensitive in the methicillin-sensitive strain.     

Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates

Staphylococcus aureus penicillin-binding protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the division site but its recruitment to the forming division septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d-cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the division site by binding to its substrate, which is localized at that place. In methicillin-resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.     

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.     

Control of cell shape and elongation by the rodA gene in Bacillus subtilis

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli , the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA , encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG-inducible P_spac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.     

Peptidoglycan synthesis and turnover in cell division mutants of Agmenellum.

The synthesis and turnover of peptidoglycan in Agmenellum quadruplicatum was investigated using D-[U-14C]alanine followed by proteolytic digestion. The rate of turnover of alanine in the peptide portion of the peptidoglycan was measured in strain BG-1 and in two division mutants of this strain: one was blocked in cell separation; and the other was a low-temperature, conditional cell division mutant. The peptide portion of peptidoglycan turned over in all three strains tested, but no correlation was observed between septum formation or cell separation and the rate of turnover. Peptidoglycan synthesis was measured during induced division in snake forms of strain SN-29. A stimulation of peptidoglycan synthesis was observed during the period of cross-wall formation, even in the absence of new protein synthesis. Thus in A. quadruplicatum, cross-wall synthesis is accompanied by a stimulation of peptidoglycan synthesis.