Post-translational control of vegetative cell separation enzymes through a direct interaction with specific inhibitor IseA in Bacillus subtilis

Three D,L-endopeptidases, LytE, LytF and CwlS, are involved in the vegetative cell separation in Bacillus subtilis. A novel cell surface protein, IseA, inhibits the cell wall lytic activities of these d,l-endopeptidases in vitro, and IseA negatively regulates the cell separation enzymes at the post-translational level. Immunofluorescence microscopy indicated that the IseA-3xFLAG fusion protein was specifically localized at cell separation sites and poles on the vegetative cell surface in a similar manner of the d,l-endopeptidases. Furthermore, pull-down assay showed that IseA binds to the catalytic domain of LytF, indicating that IseA is localized on the cell surface through the catalytic domain of LytF. Overexpression of IseA caused a long-chained cell morphology in the exponential growth phase, indicating that IseA inhibits the cell separation D,L-endopeptidases in vivo. Besides, overexpression of IseA in a cwlO disruptant affected cell growth, implying that IseA is also involved in the cell elongation event. However, although IseA inhibits the activities of LytE, LytF, CwlS and CwlO in vitro, it is unlikely to inhibit CwlS and CwlO in vivo. This is the first demonstration that the cell separation event is post-translationally controlled through a direct interaction between cell separation enzymes and a specific novel inhibitor in bacteria.     

A new D,L-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis

A new peptidoglycan hydrolase, Bacillus subtilis YojL (cell wall-lytic enzyme associated with cell separation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a d,l-endopeptidase. beta-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.     

The major and minor wall teichoic acids prevent the sidewall localization of vegetative DL-endopeptidase LytF in Bacillus subtilis

Cell separation in Bacillus subtilis depends on specific activities of DL-endopeptidases CwlS, LytF and LytE. Immunofluorescence microscopy (IFM) indicated that the localization of LytF depended on its N-terminal LysM domain. In addition, we revealed that the LysM domain efficiently binds to peptidoglycan (PG) prepared by chemically removing wall teichoic acids (WTAs) from the B. subtilis cell wall. Moreover, increasing amounts of the LysM domain bound to TagB- or TagO-depleted cell walls. These results strongly suggested that the LysM domain specifically binds to PG, and that the binding may be prevented by WTAs. IFM with TagB-, TagF- or TagO-reduced cells indicated that LytF-6xFLAG was observed not only at cell separation site and poles but also as a helical pattern along the sidewall. Moreover, we found that LytF was localizable on the whole cell surface in TagB-, TagF- or TagO-depleted cells. These results strongly suggest that WTAs inhibit the sidewall localization of LytF. Furthermore, the helical LytF localization was observed on the lateral cell surface in MreB-depleted cells, suggesting that cell wall modification by WTAs along the sidewall might be governed by an actin-like cytoskeleton homologue, MreB.     

Synthetic lethality of the lytE cwlO genotype in Bacillus subtilis is caused by lack of D,L-endopeptidase activity at the lateral cell wall

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two D,L-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the D,L-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these D,L-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of D,L-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.     

Solution Structure of IseA,an Inhibitor Protein of dl-Endopeptidases from Bacillus subtilis,Reveals a Novel Fold with a Characteristic Inhibitory Loop

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, dl-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the dl-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation dl-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3₁₀-helix, and eight β-strands, which is a novel fold like a “hacksaw.” Noteworthy is a dynamic loop between β4 and the 3₁₀-helix, which resembles a “blade.” The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4–3₁₀ loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4–3₁₀ loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.     

Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis.

Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 of Listeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-tagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by EsigmaD RNA polymerase. Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

The carboxy terminal domain of Epr, a minor extracellular serine protease, is essential for the swarming motility of Bacillus subtilis 168

In this study we have investigated the role of Epr, a minor extracellular serine protease, in the swarming motility of Bacillus subtilis 168. We identified that the protease activity of Epr was dispensable for swarming. Since the protease activity of Epr was confined to its N-terminal domain, we hypothesized instead that its C-terminal domain (CTD) could be critical for swarming. Our study showed that not only the expression of Epr-CTD was necessary, but also its secretion was crucial for the swarming motility of B. subtilis 168.     

Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole

The localization of penicillin-binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)-PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot-like pattern and also at mid-cell during cell division. PBP2 disappeared from mid-cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid-cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid-cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.     

Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1

The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.     

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis

We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis.     

Insertion and fate of the cell wall in Bacillus subtilis

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.     

Morphogenesis of rod-shaped sacculi

For growth and division of rod-shaped bacteria, the cylindrical part of the sacculus has to be elongated and two new cell poles have to be synthesized. The elongation is performed by a protein complex, the elongase that inserts disaccharidepentapeptide units at a limited number of discrete sites while using the cytoskeletal MreB helix as a tracking device. Upon initiation of cell division by positioning of the cytoskeletal Z-ring at mid cell, a switch from dispersed to concentrated local peptidoglycan-synthesis occurs. From this point on, peptidoglycan synthesis is for a large part redirected from elongating activity to synthesis of new cell poles by the divisome. The divisome might be envisioned as an extended elongase because apart from its basic peptidoglycan synthesizing activity, specific functions have to be added. These are conversion from a cylinder to a sphere, invagination of the outer membrane and addition of hydrolases that allow separation of the daughter cells. The elongase and the divisome are dynamic hyperstructures that probably share part of their proteins. Although this multifunctionality and flexibility form a barrier to the functional elucidation of its individual subunits, it helps the cells to survive a variety of emergency situations and to proliferate securely.     

Attachment of the chromosome to the cell poles: the strategy for the growth of bacteria in two and three dimensions.

Bacteria such as Staphylococcus, Lampropedia, and Sarcina develop in characteristic two-or three-dimensional groups of cells. We propose here a model of how bacteria may generate such groupings by an extension of an earlier model for rod-shaped bacteria. No other mechanism for forming two- or three-dimensional structures of groups of cells has been proposed. Our earlier model for division of rod-shaped bacteria into nearly equal-sized daughters assumed that the origin and terminus DNA were attached at a critical time to polar wall sites. While such binding was speculative 20 years ago, it has now been established that the DNA for the origin of replication, at least during some part of the cell cycle is located in the pole for several different bacteria. Evidence is also building showing that the terminus DNA region is sometimes located at a position in the cell that will develop into two new poles. Here, a new extension of the concept that polar sites bind specifically origin and terminus DNA of the chromosome is presented that can explain how division takes place in one and then in another dimension to form two-dimensional tablets of four cells or large planar arrays. A further possible extension to three dimensions to generate octets of cells is proposed.     

The PXL1 gene of Saccharomyces cerevisiae encodes a paxillin-like protein functioning in polarized cell growth

The Saccharomyces cerevisiae open reading frame YKR090w encodes a predicted protein displaying similarity in organization to paxillin, a scaffolding protein that organizes signaling and actin cytoskeletal regulating activities in many higher eucaryotic cell types. We found that YKR090w functions in a manner analogous to paxillin as a mediator of polarized cell growth; thus, we have named this gene PXL1 (Paxillin-like protein 1). Analyses of pxl1Delta strains show that PXL1 is required for the selection and maintenance of polarized growth sites during vegetative growth and mating. Genetic analyses of strains lacking both PXL1 and the Rho GAP BEM2 demonstrate that such cells display pronounced growth defects in response to different conditions causing Rho1 pathway activation. PXL1 also displays genetic interactions with the Rho1 effector FKS1. Pxl1p may therefore function as a modulator of Rho-GTPase signaling. A GFP::Pxl1 fusion protein localizes to sites of polarized cell growth. Experiments mapping the localization determinants of Pxl1p demonstrate the existence of localization mechanisms conserved between paxillin and Pxl1p and indicate an evolutionarily ancient and conserved role for LIM domain proteins in acting to modulate cell signaling and cytoskeletal organization during polarized growth.     

Fission yeast cytoskeletons and cell polarity factors: connecting at the cortex

Cell polarity is a fundamental property of cells from unicellular to multicellular organisms. Most of the time, it is essential so that the cells can achieve their function. The fission yeast Schizosaccharomyces pombe is a powerful genetic model organism for studying the molecular mechanisms of the cell polarity process. Indeed, S. pombe cells are rod-shaped and cell growth is restricted at the poles. The accurate localization of the cell growth machinery at the cell cortex, which involves the actin cytoskeleton, depends on cell polarity pathways that are temporally and spatially regulated. The importance of interphase microtubules and cell polarity factors acting at the cortex of cell ends in this process has been shown. Here, we review recent advances in knowledge of molecular pathways leading to the establishment of a cellular axis in fission yeast. We also describe the role of cortical proteins and mitotic cytoskeletal rearrangements that control the symmetry of cell division.     

Generation of buds, swellings, and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti.

Inhibition of cell division in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis results in elongation into long filaments many times the length of dividing cells. As a first step in characterizing the Rhizobium meliloti cell division machinery, we tested whether R. meliloti cells could also form long filaments after cell division was blocked. Unexpectedly, DNA-damaging agents, such as mitomycin C and nalidixic acid, caused only limited elongation. Instead, mitomycin C in particular induced a significant proportion of the cells to branch at the poles. Moreover, methods used to inhibit septation, such as FtsZ overproduction and cephalexin treatment, induced growing cells to swell, bud, or branch while increasing in mass, whereas filamentation was not observed. Overproduction of E. coli FtsZ in R. meliloti resulted in the same branched morphology, as did overproduction of R. meliloti FtsZ in Agrobacterium tumefaciens. These results suggest that in these normally rod-shaped species and perhaps others, branching and swelling are default pathways for increasing mass when cell division is blocked.     

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.