Bacterial cell division: regulating Z-ring formation

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

Cell Reproduction and Morphological Changes in Mycoplasma capricolum

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.Mycoplasmas are parasitic bacteria that have extremely low G+C contents and small genomes (9). Their morphology is irregular because of the lack of a peptidoglycan layer.In Escherichia coli, initiation of chromosomal DNA replication occurs once during the cell’s replicative cycle, and the nucleoids partition before cell division (13). The chromosomal replication of E. coli initiates in a small region and proceeds in both directions. It is mainly controlled by the timing and frequency of initiation, while the velocity of replication is constant.In mycoplasmas, chromosome replication also starts at a fixed site, followed by bidirectional progression (19–21, 25, 40). As in many eubacteria (36), the dnaA gene is expressed and plays important roles in the initiation of replication (35). These observations suggest that the outline of chromosome replication of mycoplasmas is similar to that of E. coli. However, the process of mycoplasma cell reproduction has not been clarified. Moreover, the cell division cycle of E. coli cannot be simply applied to mycoplasmas because of their irregular cell morphology (4). A model has been suggested for the cell cycle of Mycoplasma mycoides (6, 30, 31), which is closely related to Mycoplasma capricolum (39). According to this model, an elementary rounded body grows into a filamentous form and then new elementary rounded bodies are developed within this filament and released, but this model has not been adequately substantiated.In this study, we analyzed the process of DNA replication, cell morphology, and viability under various conditions of M. capricolum and proposed a model of cellular reproduction for this bacterium.     

New evidence of an old problem: The coupling of genome replication to cell growth in bacteria

The problem of coordinating genome replication with cell growth in bacteria was posed over four decades ago. Unlike for eukaryotes, this problem has not been completely solved even for Escherichia coli, which has been comprehensively studied by molecular biologists, to say nothing of other bacteria. Current models of the bacterial life cycle solve the coupling problem by introducing a phenomenological hypothesis that considers the dynamic coordination of growth and replication but does not unveil the underlying molecular mechanisms. Here we review the mechanisms regulating genome replication initiation with regards to their coupling to growth processes in the three best investigated bacterial species: E. coli, Bacillus subtilis, and Caulobacter crescentus. A putative correlation between the type of cell growth laws and the actual mechanisms regulating the replication of DNA formed during the process of evolution in various classes of bacteria, is discussed, including those intracellular parasites in which degenerative evolution has discarded most of their genomes. We contemplate the concept of a universal growth law for bacterial cells and some features in the formation of a primitive negative replication regulating mechanism in the context of the coupling problem.     

A Replication-Inhibited Unsegregated Nucleoid at Mid-Cell Blocks Z-Ring Formation and Cell Division Independently of SOS and the SlmA Nucleoid Occlusion Protein in Escherichia coli

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition.     

Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.     

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

DNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ΔrecD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ΔrecD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5′-3′ helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.     

Differential Damage in Bacterial Cells by Microwave Radiation on the Basis of Cell Wall Structure

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.     

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.     

In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400.

A DNA segment which encodes resistance to tetracycline, and cannot replicate autonomously, was excised by HindIII endonuclease from plasmid pT127 and joined to the cryptic Bacillus subtilis plasmid pHV400. The analysis of resulting chimerae has allowed us to identify a 1.8 × 10⁶ segment of pHV400 which carried the replication functions of the cryptic plasmid. Another DNA segment, designated pHV32, which can replicate in Escherichia coli but not in B. subtilis has also been used for genetic labeling of the replication region of pHV400. pHV32 is convenient for use in isolating cryptic replicons active in B. subtilis because (1) it can be prepared in large quantities, free from any interferring B. subtilis replicons, from an appropriate E. coli strain; (2) it carries unique sites for various restriction endonucleases; (3) the chloramphenicol resistance gene which it specifies can transform B. subtilis at a high efficiency (10⁶–10⁷ transformants/μg of DNA).     

Bacillus subtilis hlpB Encodes a Conserved Stand-Alone HNH Nuclease-Like Protein That Is Essential for Viability Unless the hlpB Deletion Is Accompanied by the Deletion of Genes Encoding the AddAB DNA Repair Complex

The HNH domain is found in many different proteins in all phylogenetic kingdoms and in many cases confers nuclease activity. We have found that the Bacillus subtilis hlpB (yisB) gene encodes a stand-alone HNH domain, homologs of which are present in several bacterial genomes. We show that the protein we term HlpB is essential for viability. The depletion of HlpB leads to growth arrest and to the generation of cells containing a single, decondensed nucleoid. This apparent condensation-segregation defect was cured by additional hlpB copies in trans. Purified HlpB showed cooperative binding to a variety of double-stranded and single-stranded DNA sequences, depending on the presence of zinc, nickel, or cobalt ions. Binding of HlpB was also influenced by pH and different metals, reminiscent of HNH domains. Lethality of the hlpB deletion was relieved in the absence of addA and of addAB, two genes encoding proteins forming a RecBCD-like end resection complex, but not of recJ, which is responsible for a second end-resectioning avenue. Like AddA-green fluorescent protein (AddA-GFP), functional HlpB-YFP or HlpB-FlAsH fusions were present throughout the cytosol in growing B. subtilis cells. Upon induction of DNA damage, HlpB-FlAsH formed a single focus on the nucleoid in a subset of cells, many of which colocalized with the replication machinery. Our data suggest that HlpB plays a role in DNA repair by rescuing AddAB-mediated recombination intermediates in B. subtilis and possibly also in many other bacteria.     

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.     

Replication Terminator Protein-Based Replication Fork-Arrest Systems in Various Bacillus Species

The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a Bacillus terminator consensus and identification of two more terminators in the B. subtilis 168 genome sequence to bring the total to nine.     

A Moonlighting Enzyme Links Escherichia coli Cell Size with Central Metabolism

Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.     

Effect of Microwaves on Escherichia coli and Bacillus subtilis

Suspensions of Escherichia coli and Bacillus subtilis spores were exposed to conventional thermal and microwave energy at 2,450 MHz. The degrees of inactivation by the different energy sources were compared quantitatively. During the transient heating period by microwave energy, approximately a 6 log cycle reduction in viability was encountered for E. coli. This reduction was nearly identical to what is expected for the same time-temperature exposure to conventional heating. Heating of B. subtilis spores by conventional and microwave energy was also carried out at 100 C, in ice and for transient heating. The degree of inactivation by microwave energy was again identical to that by conventional heating. In conclusion, inactivation of E. coli and B. subtilis by exposure to microwaves is solely due to the thermal energy, and there is no per se effect of microwaves.     

Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in ΔdatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.Initiation of DNA replication is highly regulated to coordinate with cell proliferation. It begins with a series of events in which the replication machinery is assembled at the replication origin of the chromosomal DNA (15, 26, 28, 38). Central to this process are the initiator proteins that bind to the origin of replication and eventually lead to the unwinding of the origin and to helicase loading on the unwound region. Previous biochemical studies and recent structural studies of the bacterial initiator protein DnaA have proposed the molecular mechanism of the action of ATP-DnaA in forming a large oligomeric complex to remodel the unique origin, oriC, and trigger duplex melting (12, 26). However, it is still not clear how the timing of initiation is controlled so that it takes place at a fixed time in the cell cycle. It has been reported that a basal level of DnaA molecules is bound by high-affinity DnaA binding sites (DnaA boxes R1, R2, and R4) at oriC throughout the cell cycle (9, 37). It is also suggested that noncanonical ATP-DnaA binding sites within oriC are occupied at elevated levels of the initiator molecules prior to the initiation event (18, 25). Thus, regulation of the activity and availability of DnaA is an important factor for the initiation control.At least three schemes are known to prevent untimely initiations in Escherichia coli. First, oriC is subject to sequestration, a process that prevents reinitiation, possibly by blocking ATP-DnaA from binding to newly replicated oriC (8, 24). E. coli oriC contains 11 GATC sites that are normally methylated on both strands by Dam methyltransferase. Immediately after passage of the replication fork, GATC sites are in a hemimethylated state, with the newly synthesized strands remaining unmethylated. SeqA binds specifically to such sites and, at oriC, protects these regions from reinitiation for about one-third of the cell cycle (6, 39). Second, in a process termed regulatory inactivation of DNA (RIDA), ATP-DnaA molecules are converted to an inactive ADP-bound form after initiation by the combined action of a β subunit of DNA polymerase III holoenzyme and Hda (16, 17). Newly synthesized DnaA molecules are able to bind ATP for the next initiation event, since its cellular concentration is much higher than that of ADP. ATP-DnaA is also regenerated from the inactive ADP-DnaA later in the cell cycle (21). Finally, the chromosomal segment datA serves to reduce the level of free DnaA protein by titrating a large number of DnaA molecules after replication of the site close to oriC (20).Cytological studies would be very useful for developing our understanding of the regulation mechanisms associated with the initiation step. In the present study, we tagged E. coli DnaA with a fluorescent protein in order to monitor its behavior in live cells. Microscopic observation revealed that DnaA is distributed all over the nucleoid. Remarkably, the majority of cells bore distinct foci that emitted brighter fluorescence against a weak fluorescent background on the nucleoid. We analyzed the behavior of these foci during the cell cycle with respect to oriC and datA.