Lytic response of Escherichia coli cells to inhibitors of penicillin-binding proteins 1a and 1b as a timed event related to cell division.

In growing cultures of Escherichia coli, simultaneous inhibition of penicillin-binding proteins 1a and 1b (PBPs 1) by a beta-lactam efficiently induces cell lysis. However, the lytic behavior of cultures initiating growth in the presence of beta-lactams specifically inhibiting PBPs 1 suggested that the triggering of cell lysis was a cell division-related event, at least in the first cell cycle after the resumption of growth (F. Garcia del Portillo, A. G. Pisabarro, E. J. de la Rosa, and M. A. de Pedro, J. Bacteriol. 169:2410-2416, 1987). To investigate whether this apparent correlation would hold true in actively growing cells, we studied the lytic behavior of cultures of E. coli aligned for cell division which were challenged with beta-lactams at different times after alignment. Cell division was aligned either by nutritional shift up or by chromosome replication alignment. Specific inhibition of PBPs 1 with the beta-lactam cefsulodin resulted in a delayed onset of lysis which was coincident in time with the resumption of cell division. The apparent correlation between the initiation of lysis and cell division was abolished when cefsulodin was used in combination with the PBP 2-specific inhibitor mecillinam, leading to the onset of lysis at a constant time after the addition of the beta-lactams. The results presented clearly argue in favor of the hypothesis that the triggering of cell lysis after inhibition of PBPs 1 is a cell division-correlated event dependent on the activity of PBP 2.     

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.     

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.     

The SUN41 and SUN42 genes are essential for cell separation in Candida albicans

Completion of the yeast cell cycle involves extensive remodelling of the cell wall upon separation of mother and daughter cells. We have studied two members of the ascomycete-specific SUN gene family in Candida albicans. Inactivation of SUN41 yields defects in cell separation and hyphal elongation while inactivation of SUN42 results in minor phenotypic alterations. Simultaneous inactivation of SUN41 and SUN42 is synthetically lethal due to lysis of mother cells after septation. Electronic microscopy reveals cell wall defects mainly localized in the region surrounding the septa. This phenotype is osmoremediable and the conditional double mutants show increased sensitivity to cell wall or cell membrane perturbing agents. The essential function shared by Sun41p and Sun42p is conserved among yeasts because UTH1, a Saccharomyces cerevisiae SUN gene, suppresses the lethality of SUN41 and SUN42 conditional mutants. Investigation of functional genomic data obtained in S. cerevisiae reveals links between members of the SUN gene family and the RAM pathway regulating cell wall-degrading enzymes specifically involved during cell separation. Thus, the main function of ascomycetous Sun proteins appears linked to cell wall remodelling, with a probable role in counter-balancing cell wall degradation to avoid cell lysis upon cell separation.     

Cell elongation and septation are two mutually exclusive processes in Escherichia coli

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a β-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length. Received: 23 January 1997 / Accepted: 2 May 1997     

Self-cycling operation increases productivity of recombinant protein in Escherichia coli

Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express β-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF.     

A novel suppressor of ras1 in fission yeast, byr4, is a dosage- dependent inhibitor of cytokinesis

A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the ras1 signaling pathways. Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation. The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2- phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis. Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells. When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with ras1. The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of ras1 and scd1, suggesting a link between pathways needed for cell polarity and cytokinesis. Overexpression of byr4 also partially bypasses the need for ras1 for sporulation. The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products. A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdc15, which fail to form a medial F-actin ring in mitosis. A slower migrating byr4 protein was found in cells with a mutation in the beta-tubulin gene, which arrests cells at the metaphase-anaphase transition.     

Differential effect of mutational impairment of penicillin-binding proteins 1A and 1B on Escherichia coli strains harboring thermosensitive mutations in the cell division genes ftsA, ftsQ, ftsZ, and pbpB.

To study the functional differences between penicillin-binding proteins (PBPs) 1A and 1B, as well as their recently postulated involvement in the septation process (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989), a series of isogenic strains with mutations in the genes coding for PBP 1A (ponA) or PBP 1B (ponB) or in the cell division-specific genes ftsA, ftsQ, pbpB, and ftsZ was constructed and used as the start point to produce double mutants combining the ponA or ponB characters with mutations in cell division genes. PBP 1A seemed to be unable to preserve cell integrity by itself, requiring the additional activities of PBP 2, PBP 3, and FtsQ. PBP 1B was apparently endowed with a more versatile biosynthetic potential that permitted a substantial enlargement of PBP 1A-deficient cells when PBP 2 or 3 was inhibited or when FtsQ was inactive. beta-Lactams binding to PBP 2 (mecillinam) or 3 (furazlocillin) caused rapid lysis in a ponB background. The lytic effect of furazlocillin to ponB cell division double mutants was suppressed at the restrictive temperature irrespective of the identity of the mutated cell division gene. These results indicate that PBPs 1A and 1B play distinct roles in cell wall synthesis and support the idea of a relevant involvement of PBP 1B in peptidoglycan synthesis at the time of septation.     

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.     

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by sertoli cells of the rat

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.     

Autocides produced by Myxococcus xanthus.

Ethanol extracts of Myxococcus xanthus contained several substances, referred to as autocides, which were bactericidal to the producing strain but showed no activity against other bacteria. The autocides were produced by growing cells and remained largely cell bound throughout the growth cycle; ca. 5% of the autocidal activity was found in the supernatant fluid at the time cell lysis began. The autocides were separated by sequential-column and thin-layer chromatography into five active fractions (AM I through AM V). Each of the fractions was at least 20 times more active against M. xanthus than against the other gram-negative or gram-positive bacteria tested. AM I, AM IV, and AM V were inactive against yeasts, whereas a mixture of fractions AM II and AM III was active against Rhodotorula sp. At low concentrations, AM I reversibly inhibited the growth of M. xanthus; at higher concentrations of AM I, the cells lysed within 1 h. The lowest concentration of AM IV that showed any activity caused rapid cell death and lysis. The mode of action of the major autocide, AM V, was different from that of AM I and AM IV. During the initial 2 h of treatment, the viable count of M. xanthus cells remained constant; during the next few hours killing occurred without lysis; within 24 h lysis was complete. The autocidal activity of each of the fractions was expressed when the cells were suspended in buffer, as well as in growth medium. The possible role of autocides in developmental lysis of M. xanthus is discussed.     

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways.

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, could be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VI. THE BEHAVIOR OF INDIVIDUAL CELLS ON NUTRIENT AGAR

Freely suspended cells of a long continuously cultured strain of carrot were dispersed in nutrient agar medium in Petri dishes. After inoculation, cells in randomly chosen fields were photographed at low magnification. The same cells were photographed at intervals over the next 23 days. The fate of 678 units, 24% of which grew visibly, was determined from the photographic record. Cells and units displayed a marked degree of individuality. In some units, there was a progressive increase in cell number; in others, after a few divisions, growth ceased; in still other units, cells enlarged without dividing. Although the evidence of cell division in free cells is conclusive, its incidence and maintenance increased with the size of the initial unit. In general, growth started after an “induction period” of about 5 days, but some cells remained apparently unchanged considerably longer after inoculation, before they started to grow. Although single cells of all sizes were observed to grow by one means or another, it was generally the shorter ones that continued to grow into viable colonies. Elongated cells predominated in the carrot strains used; these grew into colonies first by septation, with little or no over-all increase in size, followed by a stage of cell enlargement. Growth by both cell enlargement and division then ensued. During growth by septation within single cells, some derived cells were seen to burst and die. The most frequent divisions were observed during septation when the average cell generation time was 24 hr. Lacking the controls that normally operate in the intact plant body, there was much variation in the forms assumed during growth, a number of which are illustrated. The importance of epigenetic factors, or external controls, that determine the growth of cells in the plant body is stressed.     

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.     

Cell shape and division in Escherichia coli: experiments with shape and division mutants.

Double mutants which carry mutations in genes (rodA, pbpA) required for cell elongation (i.e., maintenance of rod shape) in combination with mutations in genes (ftsA, ftsI, ftsQ, or ftsZ) required for septation were constructed. Such mutants were able to grow for about two mass doublings at a normal rate at the restrictive temperature (42 degrees C). The morphology of the cells formed under these conditions was interpreted by assuming the existence of a generalized system for peptidoglycan growth together with two additional systems which modify the shape of the growing peptidoglycan layer. The results also showed that different fts genes probably control different stages in septation. ftsZ (sulB or sfiB) appears to be required for the earliest step in septation, ftsQ and ftsI (pbpB or sep) are required for a later step or steps, and ftsA is required only for the latest stages in septation.     

The cell cycle block and lysis of an activated T cell hybridoma are distinct processes with different Ca2+ requirements and sensitivity to cyclosporine A

Stimulation of transformed T cells leads to both lymphokine secretion and inhibition of spontaneous growth. Studies performed with an Ag-specific T cell hybridoma demonstrated that growth inhibition is an early (within 1 h) manifestation of activation. Experiment in which extracellular Ca2+ was chelated or in which cyclosporine A was included indicated that activation-associated growth inhibition is a two-step process. The first phase is the establishment of a G1/S cell cycle block; it does not require extracellular Ca2+ and is not prevented by the addition of cyclosporine A. The second phase is cell lysis. It can be detected 4 to 6 h after activation, requires the presence of extracellular Ca2+, and is prevented when stimulation occurs in the presence of cyclosporine A. The observation that both Ca2+ depletion and cyclosporine A prevented IL-2 secretion at all time points indicates that the pathways leading to lymphokine secretion and the G1/S block diverge early in the course of the cellular response, and establish the cell cycle block as a distinct activation event with unique characteristics.     

Effects of etoposide on the proliferation of hexaploid H1 (ES) cells

Etoposide is a specific inhibitor of topoisomerase II, which is an enzyme that enables double-stranded DNA to pass through another double-stranded DNA. Topoisomerase II is a major constituent of chromosome scaffold, existing at appreciable amounts in cells. To examine the effects of etoposide on the cell cycle, hexaploid H1 (ES) cells (6H1 cells) were used with diploid H1 (ES) cells (2H1 cells) as a control. Exponentially growing 2H1 and 6H1 cells were exposed to etoposide at various concentrations, and cultured for about 60 days in L15F10 medium with leukemia inhibitory factor. With a high concentration of etoposide (1 μM), the DNA histograms showed G(2)/M accumulation, suggesting that etoposide arrested the cell cycle at the G(2)/M phase. With a low concentration of etoposide (50 nM), the cell proliferation was suppressed with a doubling time of 98.4 h for 2H1 cells and 51.6 h for 6H1 cells, and without significant alteration in DNA histograms. Time-lapse videography revealed that 6H1 cells survived in the medium containing 50 nM etoposide had a cell cycle time of 18.8 h, which was equivalent to 19.2 h of the doubling time for the 6H1 cell population in drug-free medium, suggesting that a part of the cell population died and was excluded from the cell system. It was concluded that etoposide affected the cell cycle at a wide range of concentrations.     

cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.     

The RSF1 gene regulates septum formation in Saccharomyces cerevisiae.

Septum formation in the mitotic cell cycle of the budding yeast Saccharomyces cerevisiae occurs by conversion of the chitin ring, laid down at bud formation, into the primary septum. We show here that under certain conditions this septation is dependent on the newly identified RSF1 gene. However, cells harboring the rsf1-1 mutation accumulated in a postcytokinesis state, with delayed conversion of the chitin-rich annulus into the primary septum. This rsf1-1-mediated inhibition of septum formation only occurred under conditions of biosynthetic stress and was correlated with biosynthetically mediated inhibition of the cell-cycle regulatory step START. The RSF1 gene is distinct from the CHS2 chitin synthase gene that is responsible for septation, and thus RSF1 most likely encodes a regulator of chitin synthesis. We hypothesize that RSF1 activity facilitates septum formation during times of biosynthetic stress, to allow efficient septation even under these conditions.