Quantal Behavior of a Diffusible Factor Which Initiates Septum Formation at Potential Division Sites in Escherichia coli

Analysis of nucleated cell size in a minicell-producing strain of Escherichia coli and in its parental strain shows that the two distributions are considerably different. A model is proposed to account for this difference. The model states that: (i) in the mutant population, the cell poles are available as potential division sites in addition to the normally located division sites; (ii) the probability of a division occurring at any of the potential division sites is equal; and (iii) only enough "division factor" arises at each unit cell doubling to permit a single division. This factor is utilized entirely in the formation of a single septum. Thus, the occurrence of a polar division with the production of an anucleate minicell (which occurs only in the mutant strain) prevents the occurrence of a non-polar division, with the result that the average nucleated cell length is increased in minicell-producing strains. The model has been used to construct a theoretical population, and a number of parameters of the real and theoretical populations have been compared. The two populations are very similar in all of the parameters measured.     

Cell division suppression in the Bacillus subtilis div IC-A1 minicell-producing mutant.

Growth and division patterns of Bacillus subtilis wild-type (div IV-A1+) and minicell-producing mutant (div IV-A1) clones were studied after spore germination during microcolony development in chambers that facilitate continuous observation with a phase contrast microscope. Data obtained from 13 div IV-A1+ clones were used to derive the equation DE equals [(mum minus 17.6)/8.8], which expresses the relationship of cell divisions present in clones of various lengths. This equation was used to determine the number of divisions expected in div IV-A1 clones if the mutant clones were able to divide as often as wild-type clones. The observed number of divisions present in mutant clones was found to be only 25.27% of the number expected on the basis of this equation. Although individual div IV-A1 clones varied in the percentage of division equivalents expressed, there appeared to be no correlation between the overall clone growth rate and the number of divisions expressed. Culturing div IV-A1+ and div IV-A1 clones together in the same growth chamber revealed that there were no diffusible interactions influencing the division phenotypes of either mutant or wild-type cells. At later stages of growth, mixed microcolonies containing cells of both genotypes were formed. A length analysis of individual cells in these populations indicated that the relative division suppression of mutant compared with wild-type cells characteristic of the initial stages of clone development was maintained. It is likely, therefore, that the excessive length of minicell-producing cells (div IV-A1) is a reflection primarily of division suppression in the mutant and not simply of mislocation of division along cell length.     

Altered membrane proteins in a minicell-producing mutant of Bacillus subtilis.

The Bacillus subtilis minicell-producing mutant divIV-B1 has a membrane protein profile that is strikingly different from that of the other minicell-producing mutant, divIV-A1, or that of wild-type strain CU403.     

Clonal Analysis of Cell Division in the Bacillus subtilis div IV-B1 Minicell-Producing Mutant

Spores of the Bacillus subtilis minicell-producing mutant div IV-B1 were germinated and grown to microcolonies in chambers which facilitate continuous observation of the developing clones with a phase-contrast microscope. Time lapse photographs were taken of 46 clones, covering the period from the beginning of outgrowth until at least two rounds of cell division had been completed. Cell lineages were constructed from contour length measurements of the photographs. These data include cell lengths, division site locations, and cell numbers in clones of various ages. From these data we have determined that the probability of a minicell being produced at any division by the div IV-B1 mutant is 0.31. The location of the abnormal division site which generates the first minicell produced in the outgrowing clone appears to be random with respect to the existing cell poles. In contrast, the location of the second abnormal division site, and hence the second minicell, is not random but rather occurs preferentially in proximity to the first minicell. This clustering of abnormal events suggests that division site location is related to pole age (generations), although other influences on minicell clustering cannot be ruled out at present.     

Dependency of size of Saccharomyces cerevisiae cells on growth rate.

The mean size and percentage of budded cells of a wild-type haploid strain of Saccharomyces cerevisiae grown in batch culture over a wide range of doubling times (tau) have been measured using microscopic measurements and a particle size analyzer. Mean size increased over a 2.5-fold range with increasing growth rate (from tau = 450 min to tau = 75 min). Mean size is principally a function of growth rate and not of a particular carbon source. The duration of the budded phase increased at slow growth rates according to the empirical equation, budded phase = 0.5 tau + 27 (all in minutes). Using a recent model of the cell cycle in which division is thought to be asymmetric, equations have been derived for mean cell age and mean cell volume. The data are consistent with the notion that initiation of the cell cycle occurs at "start" after attainment of a critical cell size, and this size is dependent on growth rate, being, at slow growth rates, 63% of the volume of fast growth rates. Previous reports are reanalyzed in the light of the unequal division model and associated population equations.     

Minicell Yield and Cell Division Suppression in Bacillus subtilis Mutants

Minicell yield is determined by the probability of a minicell-producing division and the relationship of growth to division in Bacillus subtilis mutants.     

A Novel Protein,RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ΔrafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ΔrafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ΔrafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ΔrafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ΔrafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.     

Identification of Salmonella functions critical for bacterial cell division within eukaryotic cells

Salmonella typhimurium multiplication inside eukaryotic host cells is critical for virulence. Salmonella typhimurium strain SL1344 appears as filaments upon growth in macrophages and MelJuSo cells, a human melanoma cell line, indicating a specific blockage in the bacterial cell division process. Several studies have investigated the host cell response impairing bacterial division. However, none looked at the bacterial factors involved in inhibition of Salmonella division inside eukaryotic cells. We show here that blockage in the bacterial division process is sulA-independent and takes place after FtsZ-ring assembly. Salmonella typhimurium genes in which mutations lead to filamentous growth within host cells were identified by a large scale mutagenesis approach on strain 12023, revealing bacterial functions crucial for cell division within eukaryotic cells. We finally demonstrate that SL1344 filamentation is a result of hisG mutation, requires the activity of an enzyme of the histidine biosynthetic pathway HisFH and is specific for the vacuolar environment.     

Synthesis of peptidoglycan and membrane during the division cycle of rod-shaped, gram-negative bacteria.

A modified procedure for determining the pattern of peptidoglycan synthesis during the division cycle has allowed the measurement of the rate of side wall synthesis during the division cycle without the contribution due to pole formation. As predicted by a model proposing that the surface growth of the cell is regulated by mass increase, we find a decrease in side wall synthesis in the latter half of the division cycle. This supports the proposal that, upon invagination, pole growth accommodates a significant proportion of the increasing cell mass and that residual side wall growth occurs in response to the residual mass increase not accommodated by pole volume. The observed side wall synthesis patterns support the proposal that mass increase is a major, and possibly sole, regulator of bacterial surface increase. Membrane synthesis during the division cycle of the gram-negative, rod-shaped bacteria Escherichia coli and Salmonella typhimurium has also been measured with similar methods. The rate of membrane synthesis--measured by incorporation of radioactive glycerol or palmitate relative to simultaneous labeling with radioactive leucine--exhibits the same pattern as peptidoglycan synthesis. The results are compatible with a model of cell surface growth containing the following elements. (i) During the period of the division cycle prior to invagination, growth of the cell occurs predominantly in the side wall and the cell grows only in length. (ii) When invagination begins, pole growth accommodates some cytoplasmic increase, leading to a concomitant decrease in side wall synthesis. (iii) Surface synthesis increases relative to mass synthesis during the last part of the division cycle because of pole formation. It is proposed here that membrane synthesis passively follows the pattern of peptidoglycan synthesis during the division cycle.     

Growth of the surface of Corynebacterium diphtheriae

Surface structure and growth of the surface of Corynebacterium diphtheriae mitis strain were investigated by scanning electron microscopy and the immunofluorescence technique. The surface of the cell revealed by the scanning electron microscope showed a few elevated circular zones which encompassed the cell. The cell diameter increased at this zone and this gave the club-shaped appearance to this species. The cell surface labeled with specific antibodies against the whole bacterial cell and tagged with ferritin remained at a constant length during cell division cycles and the new cell surface emerged from the polar ends of the cell. This new wall surface was completely devoid of the ferritin particles indicating that the cell wall component on the old preexistent wall was completely conserved. A similar finding was obtained by immunofluorescence microscopy. C. diphtheriae, unlike Bacillus spp., showed apical growth as has been observed in fungal cells.     

Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division

In the fission yeast Schizosaccharomyces pombe, nutritional reduction of growth rate by supplying poor nitrogen, carbon or phosphate sources causes a decrease in cell size. The effect on cell division following three different nutritional shifts-up has been investigated. In all cases, about 20% of the cells divide at the original cell length, and then cell division stops for a period. Cell division then resumes at the new faster rate, cell length at division being characteristic of the new medium. Further investigation reveals that the first effect of the shift is to inhibit nuclear division rapidly and completely. These results are strongly suggestive of the operation of a cell size requirement for entry into nuclear division. The cell size necessary for nuclear division is set, or modulated, by the prevailing growth conditions. This model is confirmed by a nutritional shift-down, where nuclear division and cell division are stimulated after the shift. Cell length at division falls rapidly until the new shorter length is attained, when a new steady state is assumed at a slower growth rate. The control system is compared with that in bacteria, and its implications for various models proposed for the control of timing of mitosis are discussed.     

Monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors

Continuous growth of individual bacteria has been previously studied by direct observation using optical imaging. However, optical microscopy studies are inherently diffraction limited and limited in the number of individual cells that can be continuously monitored. Here we report on the use of the asynchronous magnetic bead rotation (AMBR) sensor, which is not diffraction limited. The AMBR sensor allows for the measurement of nanoscale growth dynamics of individual bacterial cells, over multiple generations. This torque-based magnetic bead sensor monitors variations in drag caused by the attachment and growth of a single bacterial cell. In this manner, we observed the growth and division of individual Escherichia coli, with 80-nm sensitivity to the cell length. Over the life cycle of a cell, we observed up to a 300% increase in the rotational period of the biosensor due to increased cell volume. In addition, we observed single bacterial cell growth response to antibiotics. This work demonstrates the non-microscopy limited AMBR biosensor for monitoring individual cell growth dynamics, including cell elongation, generation time, lag time, and division, as well as their sensitivity to antibiotics.     

Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO₂ production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.     

A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and cell division

Homologues of CgtA, the common GTP-binding protein of Vibrio harveyi, are present in diverse organisms ranging from bacteria to humans. In bacteria, proteins homologous to CgtA form a subfamily of small GTP-binding proteins, called Obg/Gtp1. Similarity between bacterial members of this subfamily and their eukaryotic homologues is as high as about 50%. Nevertheless, specific functions of these proteins remain largely unknown. Genes coding for CgtA-like proteins are essential in almost all species of bacteria. The only known exception is V. harveyi, whose cells survive disruption of the cgtA gene. Therefore, the V. harveyi cgtA insertional mutant is a very useful tool for studies on functions of CgtA. Here we demonstrate that under normal growth conditions, cells of the cgtA mutant are slightly larger than wild-type cells, whereas indirect inhibition of DNA replication initiation by addition of rifampicin results in significantly higher differences in average cell size between these two strains as measured by flow cytometry. These differences decreased when cell division was inhibited by cephalexin. DNA synthesis per cell mass was found to be increased in the cgtA mutant relative to wild-type V. harveyi strain, whereas the mutant cells grew slower than bacteria with functional cgtA gene. Kinetics of DNA replication after inhibition of cell division was also considerably different in wild-type and cgtA mutant strains. These results suggest that the cgtA gene product plays a role in coupling of DNA replication to cell growth and cell division.     

Predicted steady-state cell size distributions for various growth models

The question of how an individual bacterial cell grows during its life cycle remains controversial. In 1962 Collins and Richmond derived a very general expression relating the size distributions of newborn, dividing and extant cells in steady-state growth and their growth rate; it represents the most powerful framework currently available for the analysis of bacterial growth kinetics. The Collins-Richmond equation is in effect a statement of the conservation of cell numbers for populations in steady-state exponential growth. It has usually been used to calculate the growth rate from a measured cell size distribution under various assumptions regarding the dividing and newborn cell distributions, but can also be applied in reverse--to compute the theoretical cell size distribution from a specified growth law. This has the advantage that it is not limited to models in which growth rate is a deterministic function of cell size, such as in simple exponential or linear growth, but permits evaluation of far more sophisticated hypotheses. Here we employed this reverse approach to obtain theoretical cell size distributions for two exponential and six linear growth models. The former differ as to whether there exists in each cell a minimal size that does not contribute to growth, the latter as to when the presumptive doubling of the growth rate takes place: in the linear age models, it is taken to occur at a particular cell age, at a fixed time prior to division, or at division itself; in the linear size models, the growth rate is considered to double with a constant probability from cell birth, with a constant probability but only after the cell has reached a minimal size, or after the minimal size has been attained but with a probability that increases linearly with cell size. Each model contains a small number of adjustable parameters but no assumptions other than that all cells obey the same growth law. In the present article, the various growth laws are described and rigorous mathematical expressions developed to predict the size distribution of extant cells in steady-state exponential growth; in the following paper, these predictions are tested against high-quality experimental data.     

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.     

Organic Solvents as Probes for the Structure and Function of the Bacterial Membrane: Effects of Ethanol on the Wild Type and an Ethanol-Resistant Mutant of Escherichia coli K-12

The effects of ethanol on the growth of a wild-type Escherichia coli K-12 are described. These effects include a reduction of the steady-state growth rate and an interference with the division process. They appear as an immediate response to the addition of ethanol and are rapidly reversed by removal of ethanol. Mutants were selected that could grow at a concentration of ethanol that stopped wild-type growth. The growth of one of the mutants we studied (strain S9L100) is stimulated by the presence of ethanol, methanol, or dimethyl sulfoxide. This strain exhibits pleiotropic growth defects including abnormal cell division and morphology. It also appears to have an altered lac permease function which is not due to a mutation in the Y gene itself. We conclude that this mutant has an altered membrane and that the membrane defect may be the cause of the abnormal growth properties. The use of compounds which serve as general membrane perturbants and mutants resistant to these perturbants form a system accessible to both genetic and physical-biochemical techniques.     

The dynamics of continuous microbial culture described by cell age distribution and concentration of one substrate

A mathematical model has been considered in which the known equation of McKendrick and Von Foerster for cell age distribution is combined with that for substrate concentration. The dependence of cell division rate on cell age has been taken as a step function. The interrelation between culture parameters describing the substrate consumption and cell division has been found. The shape of cell age distribution as well as the values of substrate and cell concentrations in steady and transient states have been investigated. Stationary regimes at the initial culture state synchronized by ages have been found to be established as damped oscillations and age waves. Under definite conditions the transition from one steady growth regime to another includes sharp single-time age synchronization of the culture.     

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.