The putative Bacillus subtilis l,d-transpeptidase YciB is a lipoprotein that localizes to the cell poles in a divisome-dependent manner

Cell wall synthesis in bacteria is spatially organized by cytoskeletal structures. Common to all cell wall-bearing bacteria, the cytokinetic machinery localizes the cell wall synthesis to the site of septation. Recently, MinJ, a new component of the cytokinetic machinery, or divisome, of Bacillus subtilis has been described. MinJ is part of the division site selection system but also essential for correct assembly of the divisome. Here, I used the isolated PDZ domain of MinJ for co-elution experiments. One of the proteins that co-eluted was the so far uncharacterized, putative l,d-transpeptidase protein YciB. Evidence is shown that YciB localizes to the cell poles. YciB localization depends on the existence of a mature divisome, suggesting that l,d-transpeptidases are, like penicillin-binding proteins, part of the divisome.     

A synergistic role for two predicted inner membrane proteins of Escherichia coli in cell envelope integrity

The bacterial cytoplasmic membrane is a principal site of protein translocation, lipid and peptidoglycan biogenesis, signal transduction, transporters and energy generating components of the respiratory chain. Although 25–30% of bacterial proteomes consist of membrane proteins, a comprehensive understanding of their influence on fundamental cellular processes is incomplete. Here, we show that YciB and DcrB, two small cytoplasmic membrane proteins of previously unknown functions, play an essential synergistic role in maintaining cell envelope integrity of Escherichia coli. Lack of both YciB and DcrB results in pleiotropic cell defects including increased levels of lipopolysaccharide, membrane vesiculation, dynamic shrinking and extension of the cytoplasmic membrane accompanied by lysis and cell death. The stalling of an abundant outer membrane lipoprotein, Lpp, at the periplasmic face of the inner membrane leads to lethal inner membrane–peptidoglycan linkages. Additionally, the periplasmic chaperone Skp contributes to yciB dcrB mutant cell death by possibly mistargeting stalled porins into the inner membrane. Consistent with the idea of a compromised envelope in the yciB dcrB mutant, multiple envelope stress response systems are induced, with Cpx signal transduction being required for growth. Taken together, our results suggest a fundamental role for YciB and DcrB in cell envelope biogenesis.     

Division‐site localization of RodZ is required for efficient Z ring formation in Escherichia coli

Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ‐dependent manner. We also found that division‐site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division‐site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.     

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non‐canonical mechanisms may be employed. The rod‐shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998     

ULTRASTRUCTURAL ASPECTS OF EARLY STAGES IN COTTON FIBER ELONGATION

Fine structural alterations associated with early stages of cotton fiber elongation in Gossypium hirsutum L. var. dunn 56 C occur rapidly following anthesis and appear to be correlated with the formation of the central vacuole, plasma membrane, and primary cell wall as well as with increased protein synthesis necessary for cell elongation. Association of dilated cisternae of the endoplasmic reticulum with the tonoplast suggests that the endoplasmic reticulum is involved in the formation of the central vacuole. Dictyosome involvement in both plasma membrane and primary cell wall formation was suggested from observations of similarities between dictyosome associated vesicles, containing fibrils appearing similar in morphology to fibrils found in the primary cell wall, and plasma membrane associated vesicles. The single nucleolus found in cotton fibers enlarges following anthesis, shows segregation of granular and fibrillar components by 1 day postanthesis, develops a large “vacuole,” thus appearing ring-shaped, and occupies much of the nuclear volume by 2 days postanthesis. Prominent nucleoli were not observed in nuclei after 10 days postanthesis.     

Cell division and cell enlargement during potato tuber formation

Cell division and cell enlargement were studied to reveal the developmental mechanism of potato tuberization using both in vivo in vitro culture systems. Distribution of cells in S-phase was visualized by immunolabelling of incorporated bromodeoxyuridine (BrdU). Mitosis was detected in DAPI (4,6-di-amidino-2-phenylindole) or toluidine blue-stained sections. Timing and frequency of cell division were determined by daily cell counting, and cell enlargement was deduced from measurements of cell diameters.Under in vivo conditions, lateral underground buds developed into stolons due to transverse cell divisions and cell elongation in the apical region of the buds. At the onset of tuber formation, the elongation of stolons stopped and cells in pith and cortex enlarged and divided longitudinally, resulting in the swelling of the stolon tip. When tubers had a diameter of 0.8 cm, longitudinal divisions had stopped but randomly oriented division and cell enlargement occurred in the perimedullary region and continued until tubers reached their final diameter.In vitro tubers were formed by axillary buds on single node cuttings cultured under tuber-including conditions. They stopped growing at a diameter of 0.8 cm. Pith and cortex were involved in tuberization such as that found during the early stage of in vivo tuberization (<0.8 cm in diameter). The larger size of in vivo tubers is, however, due to further development of the perimedullary region, which is lacking in vitro conditions.Keywords: Cell division, cell enlargement, DNA synthesis, in vitro culture, potato, tuber formation.      

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.     

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation

A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In Escherichia coli, cell elongation requires crosslink cleavage by PG endopeptidases to make space for the incorporation of new PG material throughout the cell cylinder. Cell division, on the contrary, requires the localized synthesis and remodeling of new PG at midcell by the divisome. Little is known about the factors that modulate transitions between these two modes of PG biogenesis. In a transposon-insertion sequencing screen to identify mutants synthetically lethal with a defect in the division protein FtsP, we discovered that mutants impaired for cell division are sensitive to elevated activity of the endopeptidases. Increased endopeptidase activity in these cells was shown to interfere with the assembly of mature divisomes, and conversely, inactivation of MepS was found to suppress the lethality of mutations in essential division genes. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important point of regulation influencing the transition from elongation to the division mode of PG biogenesis.     

Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in Staphylococcus aureus

Lipoteichoic acid (LTA) is an important cell wall component of Gram‐positive bacteria. In Staphylococcus aureus it consists of a polyglycerolphosphate‐chain that is retained within the membrane via a glycolipid. Using an immunofluorescence approach, we show here that the LTA polymer is not surface exposed in S. aureus, as it can only be detected after digestion of the peptidoglycan layer. S. aureus mutants lacking LTA are enlarged and show aberrant positioning of septa, suggesting a link between LTA synthesis and the cell division process. Using a bacterial two‐hybrid approach, we show that the three key LTA synthesis proteins, YpfP and LtaA, involved in glycolipid production, and LtaS, required for LTA backbone synthesis, interact with one another. All three proteins also interacted with numerous cell division and peptidoglycan synthesis proteins, suggesting the formation of a multi‐enzyme complex and providing further evidence for the co‐ordination of these processes. When assessed by fluorescence microscopy, YpfP and LtaA fluorescent protein fusions localized to the membrane while the LtaS enzyme accumulated at the cell division site. These data support a model whereby LTA backbone synthesis proceeds in S. aureus at the division site in co‐ordination with cell division, while glycolipid synthesis takes place throughout the membrane.     

Cell division in a minimal bacterium in the absence of ftsZ

Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non‐essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.     

CtrA controls cell division and outer membrane composition of the pathogen Brucella abortus

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP‐seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.     

Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis

Although many membrane Ser/Thr‐kinases with PASTA motifs have been shown to control bacterial cell division and morphogenesis, inactivation of the Ser/Thr‐kinase PrkC does not impact Bacillus subtilis cell division. In this study, we show that PrkC localizes at the division septum. In addition, three proteins involved in cell division/elongation, GpsB, DivIVA and EzrA are required for stimulating PrkC activity in vivo. We show that GpsB interacts with the catalytic subunit of PrkC that, in turn, phosphorylates GpsB. These observations are not made with DivIVA and EzrA. Consistent with the phosphorylated residue previously detected for GpsB in a high‐throughput phosphoproteomic analysis of B. subtilis, we show that threonine 75 is the single PrkC‐mediated phosphorylation site in GpsB. Importantly, the substitution of this threonine by a phospho‐mimetic residue induces a loss of PrkC kinase activity in vivo and a reduced growth under high salt conditions as observed for gpsB and prkC null mutants. Conversely, substitution of threonine 75 by a phospho‐ablative residue does not induce such growth and PrkC kinase activity defects. Altogether, these data show that proteins of the divisome control PrkC activity and thereby phosphorylation of PrkC substrates through a negative feedback loop in B. subtilis.     

CDC25A pathway toward tumorigenesis: Molecular targets of CDC25A in cell-cycle regulation

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.     

Gibberellins and Subapical Cell Divisions in Relation to Bud Set and Bud Break in Salix pentandra

In young plants of Salix pentandra, a temperate zone deciduous woody species, elongation growth ceases and a terminal bud is formed at day lengths shorter than a critical length. This is the first step in dormancy development, making survival under harsh winter conditions possible. Early studies strongly indicate that gibberellin is involved in the photoperiodic control of bud set and bud break. GA₁ action was studied by application under short days to plants where cessation of shoot elongation had occurred, followed by subsequent anatomic investigations of shoot tips. Under short days the frequency of cell division decreased rapidly along with the earlier observed decrease in GA₁ levels. Application of GA₁ to short-day–induced terminal buds rapidly stimulated cell division in apices several days before visible shoot elongation in response to this treatment was observed. One day after GA₁ application a fourfold increase in cell division frequency in apices was observed, increasing to a maximum of sevenfold 2 days after application. Long-day treatment leading to induction of bud break after about 4–6 days was followed by slowly increasing frequency of cell divisions. In earlier studies of this species, short days and gibberellins had no effect on cell elongation. These data show that increased GA₁ content, by application or long-day treatment, results in increased frequency of mitosis. This strongly indicates that GA₁ affects stem elongation in connection with bud set and bud break primarily by affecting cell divisions in subapical tissues. Received February 26, 1999; accepted October 8, 1999     

Megacell phenotype and its relation to metabolic alterations in transketolase deficient strain of Bacillus pumilus

Fermentation with transketolase (tkt) deficient strain of Bacillus is the only reported industrially viable process for production of D ‐ribose, a commercially important pentose sugar. In addition to direct effects of tkt deficiency, the mutation in non‐oxidative part of pentose phosphate pathway (PPP) is known to display several unexpected physiological characteristics such as decreased ability to utilize D ‐glucose, altered carbon catabolite repression, lack of motility, etc. Here we demonstrate the morphological plasticity of tkt deficient strain of Bacillus pumilus ATCC 21951 and its possible relation with D ‐ribose productivity, a measure of carbon flux through PPP. The bacilli divide normally in nutrient rich media such as Luria–Bertani (LB) broth while showing cell elongation of up to 20‐fold without a visible septum accompanied by moderate to high extracellular D ‐ribose accumulation in glucose‐rich media. The cells stained with DAPI (4′‐6‐diamidino‐2‐phenylindole) and anti FtsZ antibody showed nucleoid separation and Z‐ring formation in LB broth but not in glucose‐rich media. FtsZ protein is known to localize at the future division site forming a ring, called Z‐ring, at an early stage in cytokinesis. The strain experiences inhibition or delay in Z‐ring formation resulting in cell elongation, possibly due to its altered cell membrane composition resulting from tkt deficiency. We hypothesize that the lack of PPP intermediates may have two effects on the strain: (i) altered the cell membrane leading to delay in Z‐ring formation and cell elongation; and (ii) induction of genes of the oxidative part of PPP resulting in D ‐ribose accumulation. Nutrient rich media such as LB broth may alleviate these metabolite deficiencies thereby restoring normal cell division and inhibiting excessive D ‐ribose accumulation. The D ‐ribose productivity and cell elongation may therefore be co‐morbid. The results have implications in designing optimal media and monitoring strategy based on morphological analysis. Biotechnol. Bioeng. 2009;102: 1387–1397. © 2008 Wiley Periodicals, Inc.     

The Arabidopsis KNOLLE Protein Is a Cytokinesis-specific Syntaxin

In higher plant cytokinesis, plasma membrane and cell wall originate by vesicle fusion in the plane of cell division. The Arabidopsis KNOLLE gene, which is required for cytokinesis, encodes a protein related to vesicle-docking syntaxins. We have raised specific rabbit antiserum against purified recombinant KNOLLE protein to show biochemically and by immunoelectron microscopy that KNOLLE protein is membrane associated. Using immunofluorescence microscopy, KNOLLE protein was found to be specifically expressed during mitosis and, unlike the plasma membrane H⁺-ATPase, to localize to the plane of division during cytokinesis. Arabidopsis dynamin-like protein ADL1 accumulates at the plane of cell plate formation in knolle mutant cells as in wild-type cells, suggesting that cytokinetic vesicle traffic is not affected. Furthermore, electron microscopic analysis indicates that vesicle fusion is impaired. KNOLLE protein was detected in mitotically dividing cells of various parts of the developing plant, including seedling root, inflorescence meristem, floral meristems and ovules, and the cellularizing endosperm, but not during cytokinesis after the male second meiotic division. Thus, KNOLLE is the first syntaxin-like protein that appears to be involved specifically in cytokinetic vesicle fusion.     

A new factor stimulating peptidoglycan hydrolysis to separate daughter cells in Caulobacter crescentus

Cell division in Gram‐negative bacteria involves the co‐ordinated invagination of the three cell envelope layers to form two new daughter cell poles. This complex process starts with the polymerization of the tubulin‐like protein FtsZ into a Z‐ring at mid‐cell, which drives cytokinesis and recruits numerous other proteins to the division site. These proteins are involved in Z‐ring constriction, inner‐ and outer‐membrane invagination, peptidoglycan remodelling and daughter cell separation. Three papers in this issue of Molecular Microbiology, from the teams of Lucy Shapiro, Martin Thanbichler and Christine Jacobs‐Wagner, describe a novel protein, called DipM for Division Involved Protein with LysM domains, that is required for cell division in Caulobacter crescentus. DipM localizes to the mid‐cell during cell division, where it is necessary for the hydrolysis of the septal peptidoglycan to remodel the cell wall. Loss of DipM results in severe defects in cell envelope constriction, which is deleterious under fast‐growth conditions. State‐of‐the‐art microscopy experiments reveal that the peptidoglycan is thicker and that the cell wall is incorrectly organized in DipM‐depleted cells compared with wild‐type cells, demonstrating that DipM is essential for reorganizing the cell wall at the division site, for envelope invagination and cell separation in Caulobacter.     

Molecular cloning and expression of a MAP kinase homologue from pea

The cdc2 kinases are important cell cycle regulators in all eukaryotes. MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants. We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases. One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae. This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library. The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases. An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs). Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues.     

The lipid II flippase RodA determines morphology and growth in Corynebacterium glutamicum

Lipid II flippases play an essential role in cell growth and the maintenance of cell shape in many rod‐shaped bacteria. The putative lipid II flippase RodA is an integral membrane protein and member of the SEDS (shape, elongation, division and sporulation) protein family. In contrast to its homologues in Escherichia coli and Bacillus subtilis little is known about the role of RodA in actinobacteria. In this study, we describe the localization and function of RodA in Corynebacterium glutamicum, a rod‐shaped, apically growing actinobacterium. RodA‐GFP localizes exclusively at the cell poles. Surprisingly, time‐lapse microscopy revealed that apical cell growth is sustained in a rodA deletion strain. However, growth rates and antibiotic susceptibility are altered. In the absence of RodA, it appears that apical growth is driven by lateral diffusion of lipid II that is likely flipped by the septal flippase, FtsW. Furthermore, we applied a previously described synthetic in vivo system in combination with FRET to identify an interaction between C. glutamicum RodA and the polar growth organizing protein DivIVA.