Mutations in cell elongation genes mreB,mrdA and mrdB suppress the shape defect of RodZ‐deficient cells

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process.     

The LpxL acyltransferase is required for normal growth and penta‐acylation of lipid A in Burkholderia cenocepacia

Lipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa‐acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra‐ and penta‐acylated lipid A. “Late” acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2′ position of lipid A resulting in penta‐acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary β ‐hydroxymyristate (3‐OH‐C14:0) chain at the 3 position, leading to tetra‐acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta‐acylated lipid A, suggesting lipid A penta‐acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle.     

The life-cycle proteins RodA of Escherichia coli and SpoVE of Bacillus subtilis have very similar primary structures

Comparison of the predicted amino acid sequence of the cell-cycle RodA protein with the National Research Foundation protein sequence database shows that the 370-amino-acid RodA, a protein that is essential for wall elongation in Escherichia coli and maintenance of the rod shape of the cell, is highly analogous, in terms of primary structure, with the Bacillus subtilis SpoVE protein involved in stage V of sporulation.     

Determination of bacterial rod shape by a novel cytoskeletal membrane protein

Cell shape is critical for growth, and some genes are involved in bacterial cell morphogenesis. Here, we report a novel gene, rodZ, required for the determination of rod shape in Escherichia coli. Cells lacking rodZ no longer had rod shape but rather were round or oval. These round cells were smaller than known round mutant cells, including mreB and pbpA mutants; both are known to lose rod shape. Morphogenesis from rod cells to round cells and vice versa, caused by depletion and overproduction of RodZ, respectively, revealed that RodZ could regulate the length of the long axis of the cell. RodZ is a membrane protein with bitopic topology such that the N‐terminal region including a helix‐turn‐helix motif is in the cytoplasm, whereas the C‐terminal region is exposed in the periplasm. GFP–RodZ forms spirals along the lateral axis of the cell beneath the cell membrane, similar to the MreB bacterial actin. Thus, RodZ may mediate spatial information from cytoskeletal proteins in the cytoplasm to a peptidoglycan synthesis machinery in the periplasm.     

The three‐component system EsrISR regulates a cell envelope stress response in Corynebacterium glutamicum

When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two‐component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino‐terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two‐component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non‐stress conditions. The resulting EsrISR three‐component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI‐esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane‐associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322‐3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI‐esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress‐responsive system whose components are highly conserved in the phylum Actinobacteria.     

RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli

The bacterial MreB actin cytoskeleton is required for cell shape maintenance in most non‐spherical organisms. In rod‐shaped cells such as Escherichia coli, it typically assembles along the long axis in a spiral‐like configuration just underneath the cytoplasmic membrane. How this configuration is controlled and how it helps dictate cell shape is unclear. In a new genetic screen for cell shape mutants, we identified RodZ (YfgA) as an important transmembrane component of the cytoskeleton. Loss of RodZ leads to misassembly of MreB into non‐spiral structures, and a consequent loss of cell shape. A juxta‐membrane domain of RodZ is essential to maintain rod shape, whereas other domains on either side of the membrane have critical, but partially redundant, functions. Though one of these domains resembles a DNA‐binding motif, our evidence indicates that it is primarily responsible for association of RodZ with the cytoskeleton.     

Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39

In ellipsoid‐shaped ovococcus bacteria, such as the pathogen Streptococcus pneumoniae (pneumococcus), side‐wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin‐binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG‐domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. The ‘synthetic viable’ genetic relationship between Δpbp1a and ΔmltG mutations extends to essential ΔmreCD and ΔrodZ mutations that misregulate peripheral PG synthesis. Remarkably, the single MltG(Y488D) change suppresses the requirement for PBP2b, MreCD, RodZ and RodA. Structural modeling and comparisons, catalytic‐site changes and an interspecies chimera indicate that pneumococcal MltG is the functional homologue of the recently reported MltG endo‐lytic transglycosylase of Escherichia coli. Depletion of pneumococcal MltG or mltG(Y488D) increases sphericity of cells, and MltG localizes with peripheral PG synthesis proteins during division. Finally, growth of Δpbp1a ΔmltG or mltG(Y488D) mutants depends on induction of expression of the WalRK TCS regulon of PG hydrolases. These results fit a model in which MltG releases anchored PG glycan strands synthesized by PBP1a for crosslinking by a PBP2b:RodA complex in peripheral PG synthesis.     

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod‐shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re‐assemble, and MreB‐free zones were subsequently observed in the cytoplasmic membrane. These MreB‐free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y‐shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.     

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.     

Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non‐medial septa

Mycobacterium spp., rod‐shaped cells belonging to the phylum Actinomycetes, lack the Min‐ and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid‐cell. We show that the position for establishment of the FtsZ‐ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non‐medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane‐specific dye FM4‐64 and fluorescent antibiotic vancomycin (FL‐Vanco), respectively, showed that many division sites were off‐centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time‐lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post‐septal DNA transport and unequal polar growth may compensate for the non‐medial division site placement in Mycobacterium spp.     

De novo morphogenesis in L‐forms via geometric control of cell growth

In virtually all bacteria, the cell wall is crucial for mechanical integrity and for determining cell shape. Escherichia coli's rod‐like shape is maintained via the spatiotemporal patterning of cell‐wall synthesis by the actin homologue MreB. Here, we transiently inhibited cell‐wall synthesis in E. coli to generate cell‐wall‐deficient, spherical L‐forms, and found that they robustly reverted to a rod‐like shape within several generations after inhibition cessation. The chemical composition of the cell wall remained essentially unchanged during this process, as indicated by liquid chromatography. Throughout reversion, MreB localized to inwardly curved regions of the cell, and fluorescent cell wall labelling revealed that MreB targets synthesis to those regions. When exposed to the MreB inhibitor A22, reverting cells regrew a cell wall but failed to recover a rod‐like shape. Our results suggest that MreB provides the geometric measure that allows E. coli to actively establish and regulate its morphology.     

Apical assemblies of intermediate filament‐like protein FilP are highly dynamic and affect polar growth determinant DivIVA in Streptomyces venezuelae

Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled‐coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament‐like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP‐mCherry fusion protein, we show that FilP accumulates in gradient‐like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.     

Bacterial cell division: regulating Z-ring formation

The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site. Z rings appear to be synthesized in a bi‐directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane. It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur. However, a Z ring can be made to form at positions other than at the division site. How does a cell regulate utilization of a NS at the correct location and at the right time? In rod‐shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion. It is suggested that in B. subtilis, the main role of the Min proteins is to inhibit division at the nucleoid‐free cell poles. In E. coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid‐cell or whether its main role is to ensure that division inhibition occurs away from mid‐cell, a role analogous to that in B. subtilis. While the nucleoid negatively influences Z‐ring formation in its vicinity in these rod‐shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid‐cell Z ring is unresolved. Recent evidence suggests that in B. subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co‐ordination between chromosome replication and cell division.     

A M23B family metallopeptidase of Helicobacter pylori required for cell shape,pole formation and virulence

The molecular basis of the regulation of specific shapes and their role for the bacterial fitness remain largely unknown. We focused in this study on the Gram‐negative and spiral‐shaped Helicobacter pylori. To colonize its unique niche, H. pylori needs to reach quickly the human gastric mucosa, by swimming to and through the mucus layer. For that reason, the specific shape of H. pylori is predicted to be necessary for optimal motility in vivo, and consequently for its colonization ability. Here, we describe the involvement of a PG‐modifying enzyme, HdpA (HP0506), in the mouse colonization ability of this bacterium, by regulating its shape. Indeed, the inactivation of the hp0506 gene led to a stocky and branched phenotype, affecting H. pylori colonization capacity despite a normal motility phenotype in vitro. In contrast, the overexpression of the hp0506 gene induced the transformation of H. pylori from rod to dividing cocci shaped bacteria. Furthermore, we demonstrated by PG analysis and enzymology, that HdpA carried both d ,d ‐carboxypeptidase and d ,d ‐endopeptidase activities. Thus, HdpA is the first enzyme belonging to the M23‐peptidase family able to perform the d ,d ‐carboxypeptidation and regulate cell shape.     

Induction of only limited elongation instead of filamentation by inhibition of cell division in Corynebacterium glutamicum

In order to characterize the cell-division mechanism of coryneform bacteria, we tried to isolate cell-division mutants from Corynebacterium glutamicum after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, such as Escherichia coli fts mutants, which form long filaments at the restrictive temperatures. At the non-permissive temperature, most of the mutants formed club-shaped or dumbbell-shaped, elongated rod cells, but no filament formers were isolated. Then we examined the effects of cell division inhibitors on this organism. Cephalexin and sparfloxacin, which are the inhibitors of septation and DNA synthesis respectively, and are known to cause cell filamentation in E. coli, did not cause filamentation in C. glutamicum but induced morphological changes that were similar to those observed with the temperature-sensitive ts mutants of C.␣glutamicum. These results suggest that C. glutamicum has a unique regulation mechanism, that is, the inhibition of cell division leads to cessation of cell elongation. Received: 5 February 1998 / Received revision: 6 April 1998 / Accepted: 27 April 1998     

Kin1 is a plasma membrane‐associated kinase that regulates the cell surface in fission yeast

Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1‐GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1Δ null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue‐sensitive allele kin1‐as1. Kin1 inhibition primarily promotes delocalization of plasma membrane‐associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen‐activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen‐activated protein kinase components reduces kin1Δ hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall.     

Structure and chemistry of the Dufour gland in Pristomyrmex ants (Hymenoptera, Formicidae)

Billen, J., Ito, F., Tsuji, K., Schoeters, E., Maile, R. and Morgan, E. D. 2000. Structure and chemistry of the Dufour gland in Pristomyrmex ants (Hymenoptera, Formicidae). —Acta Zoologica (Stockholm) 81 : 159–166 All individuals of the three species of Pristomyrmex studied have a Dufour gland with a conspicuous hammer‐shaped distal part, that is connected to a thin‐walled proximal reservoir through a very narrow stalk. The secretory distal part is formed by high columnar cells that are characterized by a highly folded apical wall. The lateral cell junctions apically correspond with the crenel tops, which gives individual cells a peculiar shape with a deep apical depression. The cytoplasm of the secretory cells contains a very well developed smooth endoplasmic reticulum and Golgi apparatus, numerous mitochondria and lysosomes. Histochemical analysis indicates a positive reaction for the presence of a lipid secretion in the hammer‐like part. Gas chromatographic analysis of glands of P. pungens workers reveals the secretion to be formed of a mixture of simple volatile monoterpene hydrocarbons; α–pinene, β–pinene, limonene and camphene. Similar, but species characteristic mixtures of four monoterpenes were found in Pristomyrmex brevispinosus and Pristomyrmex sp.1. Behavioural experiments did not allow a conclusive determination of the function of the gland.     

Microbial production of L -glutamate and L -glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb

Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum.