Long-Chain Polyphosphate Causes Cell Lysis and Inhibits Bacillus cereus Septum Formation, Which Is Dependent on Divalent Cations

We investigated the cellular mechanisms that led to growth inhibition, morphological changes, and lysis of Bacillus cereus WSBC 10030 when it was challenged with a long-chain polyphosphate (polyP). At a concentration of 0.1% or higher, polyP had a bacteriocidal effect on log-phase cells, in which it induced rapid lysis and reductions in viable cell counts of up to 3 log units. The cellular debris consisted of empty cell wall cylinders and polar caps, suggesting that polyP-induced lysis was spatially specific. This activity was strictly dependent on active growth and cell division, since polyP failed to induce lysis in cells treated with chloramphenicol and in stationary-phase cells, which were, however, bacteriostatically inhibited by polyP. Similar observations were made with B. cereus spores; 0.1% polyP inhibited spore germination and outgrowth, and a higher concentration (1.0%) was even sporocidal. Supplemental divalent metal ions (Mg²⁺ and Ca²⁺) could almost completely block and reverse the antimicrobial activity of polyP; i.e., they could immediately stop lysis and reinitiate rapid cell division and multiplication. Interestingly, a sublethal polyP concentration (0.05%) led to the formation of elongated cells (average length, 70 μm) after 4 h of incubation. While DNA replication and chromosome segregation were undisturbed, electron microscopy revealed a complete lack of septum formation within the filaments. Exposure to divalent cations resulted in instantaneous formation and growth of ring-shaped edges of invaginating septal walls. After approximately 30 min, septation was complete, and cell division resumed. We frequently observed a minicell-like phenotype and other septation defects, which were probably due to hyperdivision activity after cation supplementation. We propose that polyP may have an effect on the ubiquitous bacterial cell division protein FtsZ, whose GTPase activity is known to be strictly dependent on divalent metal ions. It is tempting to speculate that polyP, because of its metal ion-chelating nature, indirectly blocks the dynamic formation (polymerization) of the Z ring, which would explain the aseptate phenotype.     

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.     

Cell cycle-independent lysis of Escherichia coli by cefsulodin, an inhibitor of penicillin-binding proteins 1a and 1b.

Cefsulodin lyses actively growing Escherichia coli by binding specifically to penicillin-binding proteins (PBPs) 1a and 1b. Recent findings (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989) have linked cefsulodin-induced lysis to septation during the first division cycle after a nutritional shift-up or chromosome replication realignment. We synchronized cells by membrane filtration to determine whether cefsulodin-induced lysis depended on septation in normally growing cells. Populations of newly divided cells were allowed to grow for variable lengths of time. Cefsulodin was added to these synchronous cultures, which represented points in two to three rounds of the cell cycle. Since the cell numbers were small, a new lysis assay was developed that was based on the release of DNA measured by fluorometry. Lysis occurred at a constant time after addition of the antibiotic, regardless of the time in the cell cycle at which the addition was made. Thus, cefsulodin-induced lysis is not linked to septation or to any other cell cycle-related event.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

Relationship between the DNA content and mesosome number in cells of Bacillus.

In cells of Bacillus there is evidence that deoxyribonucleic acid forms an association with some membranous structure within the cell, possibly mesosomes. Cells of varieties of Bacillus cereus and Bacillus subtilis were examined to see if any quantitative relationship existed between the numbers of mesosomes and DNA content. No direct relationship could be domonstrated. However, cells of Bacillus cereus var. alesti A(-) maintained a characteristic and constant DNA content and number of mesosomes regardless of growth conditions. During sporulation, a variant of A(-), termed A(-)3, SEQUESTERS ITS DNA at both ends of the cell, leaving a small amount of DNA but no mesosomes in the center compartment. Since this center compartment is capableof growth and division upon replacement in fresh medium (rejuventation) it was examinedfor mesosome content as DNA synthesis and division were initiated. In most cells, acentral mesosome was formed at the site of cell septum formation; however, the presenceof a mesosome was not an absolute prerequisite for cell division. We propose that atthe onset of cell growth, mesosomes primarily function in the process of cell septum formation. As growth and division proceed, mesosomes are produced in characteristicnumbers and may act as the site of DNA synthesis and (or) segregation.     

Production and release of polyphosphate by a genetically engineered strain of Escherichia coli.

A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium. PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis. PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium. When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed. Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C. These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E. coli recombinants. High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E. coli recombinant. The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium. Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.     

Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.     

Dual Role for the O-Acetyltransferase OatA in Peptidoglycan Modification and Control of Cell Septation in Lactobacillus plantarum

Until now, peptidoglycan O-acetyl transferases (Oat) were only described for their peptidoglycan O-acetylating activity and for their implication in the control of peptidoglycan hydrolases. In this study, we show that a Lactobacillus plantarum mutant lacking OatA is unable to uncouple cell elongation and septation. Wild-type cells showed an elongation arrest during septation while oatA mutant cells continued to elongate at a constant rate without any observable pause during the cell division process. Remarkably, this defect does not result from a default in peptidoglycan O-acetylation, since it can be rescued by wild-type OatA as well as by a catalytic mutant or a truncated variant containing only the transmembrane domain of the protein. Consistent with a potential involvement in division, OatA preferentially localizes at mid-cell before membrane invagination and remains at this position until the end of septation. Overexpression of oatA or its inactive variants induces septation-specific aberrations, including asymmetrical and dual septum formation. Overproduction of the division inhibitors, MinC or MinD, leads to cell filamentation in the wild type while curved and branched cells are observed in the oatA mutant, suggesting that the Min system acts differently on the division process in the absence of OatA. Altogether, the results suggest that OatA plays a key role in the spatio-temporal control of septation, irrespective of its catalytic activity.     

Role of SpoVG in Asymmetric Septation in Bacillus subtilis

Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and other spoVG loss-of-function mutations enabled citC mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor sigmaF. However, little induction of mother cell-specific, sigmaE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed sigmaF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Accumulation of Polyphosphates and Expression of High Molecular Weight Exopolyphosphatase in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of cultivation time and concentration of inorganic phosphate (P(i)) in the culture medium on the accumulation of polyphosphates (polyP) and the activity of two cytosolic exopolyphosphatases of the yeast Saccharomyces cerevisiae was studied: an exopolyphosphatase of 40 kD encoded by PPX1 and a high molecular weight exopolyphosphatase encoded by another gene. Depletion of polyP in the cells on P(i) starvation is a signal factor for the accumulation of polyP after the subsequent addition of 5-20 mM P(i) and glucose to the cells or spheroplasts. A high activity of both exopolyphosphatases does not prevent the accumulation of polyP. The expression of the high molecular weight exopolyphosphatase is due to the acceleration of metabolism in cells that have reached the stage of growth deceleration on the addition of P(i) and glucose or complete culture medium. This process may occur independently from the accumulation of polyP. The activity of exopolyphosphatase PPX1 depends less on the mentioned factors, decreasing 10-fold only under conditions of phosphate surplus at the stationary growth stage.     

Periodic accumulation of cdc15 mRNA is not necessary for septation in Schizosaccharomyces pombe

Analysis of Schizosaccharomyces pombe mutants that are defective in septum formation and cytokinesis has identified the product of the cdc15 gene as a key element in formation of a division septum. S. pombe cells lacking cdc15p function cannot assemble a functional medial ring, and do not make a division septum. cdc15 mRNA accumulates periodically during the cell cycle, peaking after entry into mitosis, and increased expression of the gene in G2-arrested cells can promote F-actin ring formation. Here, we have investigated the effects of mutations that block cell division upon the expression of cdc15 in synchronised cell populations, and analysed the expression of cdc15 when septum formation is induced by ectopic activation of the septation signalling network. We concluded the following: (i) the septation signalling network genes are not required for periodic accumulation of cdc15 mRNA; (ii) induction of septum formation in G2-arrested cells by activation of the septation signalling network does not result in accumulation of cdc15 mRNA, which is therefore not a prerequisite for septum formation; (iii) failure to turn off septum formation at the end of mitosis results in continued expression of cdc15; and (iv) periodic accumulation of cdc15 mRNA is mediated by a 97 bp region 5' to the mRNA start site.     

Septal localization of penicillin-binding protein 1 in Bacillus subtilis.

Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.     

Purification and Properties of Exopolyphosphatase from the Cytosol of Saccharomyces cerevisiae Not Encoded by the PPX1 Gene

A novel exopolyphosphatase has been isolated from the cytosol of Saccharomyces cerevisiae grown to the stationary phase after its transfer from phosphate-deficient to complete medium. The PPX1 gene responsible for 40-kD exopolyphosphatase of the cytosol does not encode it. Specific activity of the preparation is 150 U/mg, purification degree is 319, and the yield is 16.9%. The minimal molecular mass of the active but unstable enzyme complex is approximately 125 kD. A stable enzyme complex with a molecular mass of approximately 500 kD is composed of two polypeptides of approximately 32 and 35 kD and apparently polyphosphates (polyP). Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with polyP3, not inhibited by antibodies suppressing the activity of 40-kD exopolyphosphatase, inhibited by EDTA, and stimulated by divalent cations to a lesser extent. The high-molecular-mass exopolyphosphatase hydrolyzes polyP with an average chain length of 208 to 15 phosphate residues to the same extent, but is inactive with ATP, PPi, and p-nitrophenyl phosphate. The activity with polyP3 is 13% of that with polyP208. The Km values for polyP208, polyP15, and polyP3 hydrolysis are 3.5, 75, and 1100 microM, respectively. The enzyme is most active at pH approximately 7. Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of 1 mM enhances it 2-fold. The enzyme under study is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles.     

Escherichia coli strains blocked in Tat-dependent protein export exhibit pleiotropic defects in the cell envelope

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.     

Cross Talk between NDR Kinase Pathways Coordinates Cytokinesis with Cell Separation in Schizosaccharomyces pombe

NDR (nuclear Dbf-2-related) kinases constitute key regulatory nodes in signaling networks that control multiple biological processes such as growth, proliferation, mitotic exit, morphogenesis, and apoptosis. Two NDR pathways called the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR) exist in the fission yeast Schizosaccharomyces pombe. The SIN promotes cytokinesis, and the MOR drives cell separation at the end of cytokinesis and polarized growth during interphase. We showed previously that cross talk exists between these two pathways, with the SIN inhibiting the MOR during cytokinesis through phosphorylation of the MOR component Nak1 by the SIN Sid2 kinase. The reason for this inhibition remained uncertain. We show here that failure to inhibit MOR signaling during cytokinesis results in cell lysis at the site of septum formation. Time-lapse analysis revealed that MOR signaling during cytokinesis causes cells to prematurely initiate septum degradation/cell separation. The cell lysis phenotype is due to premature initiation of cell separation because it can be rescued by mutations in genes required for cell separation/septum degradation. We also shed further light on how the SIN inhibits the MOR. Sid2 phosphorylation of the MOR proteins Sog2 and Nak1 is required to prevent cell lysis during cytokinesis. Together, these results show that SIN inhibition of the MOR enforces proper temporal ordering of cytokinetic events.