MksB is a novel mycobacterial condensin that orchestrates spatiotemporal positioning of replication machinery.

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.

Chromosome structure and DNA replication dynamics during the life cycle of the predatory bacterium Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, an obligate predatory Gram-negative bacterium that proliferates inside and kills other Gram-negative bacteria, was discovered more than 60 years ago. However, we have only recently begun to understand the detailed cell biology of this proficient bacterial killer. Bdellovibrio bacteriovorus exhibits a peculiar life cycle and bimodal proliferation, and thus represents an attractive model for studying novel aspects of bacterial cell biology. The life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase and an intracellular reproductive phase. During the reproductive phase, B. bacteriovorus grows as an elongated cell and undergoes binary or nonbinary fission, depending on the prey size. In this review, we discuss: (1) how the chromosome structure of B. bacteriovorus is remodeled during its life cycle; (2) how its chromosome replication dynamics depends on the proliferation mode; (3) how the initiation of chromosome replication is controlled during the life cycle, and (4) how chromosome replication is spatiotemporally coordinated with the proliferation program.

Binary or Nonbinary Fission? Reproductive Mode of a Predatory Bacterium Depends on Prey Size.

Most bacteria, including model organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, reproduce by binary fission. However, some bacteria belonging to various lineages, including antibiotic-producing Streptomyces and predatory Bdellovibrio, proliferate by nonbinary fission, wherein three or more chromosome copies are synthesized and the resulting multinucleoid filamentous cell subdivides into progeny cells. Here, we demonstrate for the first time that the predatory bacterium Bdellovibrio bacteriovorus reproduces through both binary and nonbinary fission inside different prey bacteria. Switching between the two modes correlates with the prey size. In relatively small prey cells, B. bacteriovorus undergoes binary fission; the FtsZ ring assembles in the midcell, and the mother cell splits into two daughter cells. In larger prey cells, B. bacteriovorus switches to nonbinary fission and creates multiple asynchronously assembled FtsZ rings to produce three or more daughter cells. Completion of bacterial cell cycle critically depends on precise spatiotemporal coordination of chromosome replication with other cell cycle events, including cell division. We show that B. bacteriovorus always initiates chromosome replication at the invasive pole of the cell, but the spatiotemporal choreography of subsequent steps depends on the fission mode and/or the number of progeny cells. In nonbinary dividing filaments producing five or more progeny cells, the last round(s) of replication may also be initiated at the noninvasive pole. Altogether, we find that B. bacteriovorus reproduces through bimodal fission and that extracellular factors, such as the prey size, can shape replication choreography, providing new insights about bacterial life cycles. IMPORTANCE Most eukaryotic and bacterial cells divide by binary fission, where one mother cell produces two progeny cells, or, rarely, by nonbinary fission. All bacteria studied to date use only one of these two reproduction modes. We demonstrate for the first time that a predatory bacterium, Bdellovibrio bacteriovorus, exhibits bimodal fission and the mode of division depends on the size of the prey bacterium inside which B. bacteriovorus grows. This work provides key insights into the mode and dynamics of B. bacteriovorus proliferation in different pathogens that pose a major threat to human health due to their emerging antibiotic resistance (Proteus mirabilis, Salmonella enterica, and Shigella flexneri). The use of predatory bacteria such as B. bacteriovorus is currently regarded as a promising strategy to kill antibiotic-resistant pathogens. We find that B. bacteriovorus employs different chromosome replication choreographies and division modes when preying on those pathogens. Our findings may facilitate the design of efficient pathogen elimination strategies.

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin.

Nucleoid-associated proteins (NAPs) crucially contribute to organizing bacterial chromatin and regulating gene expression. Among the most highly expressed NAPs are the HU and integration host factor (IHF) proteins, whose functional homologues, HupB and mycobacterial integration host factor (mIHF), are found in mycobacteria. Despite their importance for the pathogenicity and/or survival of tubercle bacilli, the role of these proteins in mycobacterial chromosome organization remains unknown. Here, we used various approaches, including super-resolution microscopy, to perform a comprehensive analysis of the roles of HupB and mIHF in chromosome organization. We report that HupB is a structural agent that maintains chromosome integrity on a local scale, and that the lack of this protein alters chromosome morphology. In contrast, mIHF is a highly dynamic protein that binds DNA only transiently, exhibits susceptibility to the chromosomal DNA topology changes and whose depletion leads to the growth arrest of tubercle bacilli. Additionally, we have shown that depletion of Mycobacterium smegmatis integration host factor (msIHF) leads to chromosome shrinkage and replication inhibition.

AdpA Positively Regulates Morphological Differentiation and Chloramphenicol Biosynthesis in Streptomyces venezuelae.

In members of genus Streptomyces, AdpA is a master transcriptional regulator that controls the expression of hundreds of genes involved in morphological differentiation, secondary metabolite biosynthesis, chromosome replication, etc. However, the function of AdpASv, an AdpA ortholog of Streptomyces venezuelae, is unknown. This bacterial species is a natural producer of chloramphenicol and has recently become a model organism for studies on Streptomyces. Here, we demonstrate that AdpASv is essential for differentiation and antibiotic biosynthesis in S. venezuelae and provide evidence suggesting that AdpASv positively regulates its own gene expression. We speculate that the different modes of AdpA-dependent transcriptional autoregulation observed in S. venezuelae and other Streptomyces species reflect the arrangement of AdpA binding sites in relation to the transcription start site. Lastly, we present preliminary data suggesting that AdpA may undergo a proteolytic processing and we speculate that this may potentially constitute a novel regulatory mechanism controlling cellular abundance of AdpA in Streptomyces. IMPORTANCEStreptomyces are well-known producers of valuable secondary metabolites which include a large variety of antibiotics and important model organisms for developmental studies in multicellular bacteria. The conserved transcriptional regulator AdpA of Streptomyces exerts a pleiotropic effect on cellular processes, including the morphological differentiation and biosynthesis of secondary metabolites. Despite extensive studies, the function of AdpA in these processes remains elusive. This work provides insights into the role of a yet unstudied AdpA ortholog of Streptomyces venezuelae, now considered a novel model organism. We found that AdpA plays essential role in morphological differentiation and biosynthesis of chloramphenicol, a broad-spectrum antibiotic. We also propose that AdpA may undergo a proteolytic processing that presumably constitutes a novel mechanism regulating cellular abundance of this master regulator.

Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis.

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that can adapt to the various nutrients available during its life cycle. However, in the nutritionally stringent environment of the macrophage phagolysosome, Mtb relies mainly on cholesterol. In previous studies, we demonstrated that Mtb can accumulate and utilize cholesterol as the sole carbon source. However, a growing body of evidence suggests that a lipid-rich environment may have a much broader impact on the pathogenesis of Mtb infection than previously thought. Therefore, we applied high-resolution transcriptome profiling and the construction of various mutants to explore in detail the global effect of cholesterol on the tubercle bacillus metabolism. The results allow re-establishing the complete list of genes potentially involved in cholesterol breakdown. Moreover, we identified the modulatory effect of vitamin B12 on Mtb transcriptome and the novel function of cobalamin in cholesterol metabolite dissipation which explains the probable role of B12 in Mtb virulence. Finally, we demonstrate that a key role of cholesterol in mycobacterial metabolism is not only providing carbon and energy but involves also a transcriptome remodeling program that helps in developing tolerance to the unfavorable host cell environment far before specific stress-inducing phagosomal signals occur.

Lsr2 and Its Novel Paralogue Mediate the Adjustment of Mycobacterium smegmatis to Unfavorable Environmental Conditions.

Lsr2 is a nucleoid-associated protein (NAP) that has been found strictly in actinobacteria, including mycobacteria. It is a functional homolog of histone-like nucleoid-structuring protein (H-NS); it acts as a DNA-bridging protein that plays a role in chromosomal organization and transcriptional regulation. To date, the studies on Lsr2 have focused mainly on Mycobacterium tuberculosis In this study, we analyze the role of Lsr2 as a transcription factor in Mycobacterium smegmatis, a saprophytic bacterium whose natural habitat (soil and water) substantially differs from those of the obligatory mycobacterial pathogens. Our chromatin immunoprecipitation-sequencing (ChIP-seq) data revealed that Lsr2 binds preferentially to AT-rich regions of the M. smegmatis chromosome. We found that Lsr2 acts mainly as a repressor, controlling gene expression either directly by binding promoter regions or indirectly through DNA loop formation and DNA coating. One of the Lsr2-repressed genes encodes polyketide synthase (MSMEG_4727), which is involved in the synthesis of lipooligosaccharides (LOSs). An M. smegmatis strain deprived of Lsr2 produces more LOSs, which is mirrored by changes in the smoothness of cells and their susceptibilities to antibiotics. Unlike M. tuberculosis, M. smegmatis additionally encodes a paralogue of Lsr2, MSMEG_1060, which is a novel member of the mycobacterial NAP family. The Lsr2 and MSMEG_1060 proteins exhibit different DNA binding specificities and chromosomal localizations. Our results suggest that these proteins help M. smegmatis cells cope with stress conditions, including hypoxia and exposure to antibiotics. Thus, the present work provides novel insight into the role of Lsr2 paralogues in the ability of a saprophytic mycobacterial species to adjust to environmental changes.IMPORTANCE Nucleoid-associated proteins (NAPs) are the most abundant proteins involved in bacterial chromosome organization and global transcription regulation. The mycobacterial NAP family includes many diverse proteins; some are unique to actinobacteria, and many are crucial for survival under stress (e.g., HupB and Lsr2) and/or optimal growth conditions (e.g., mycobacterial integration host factor [mIHF]). Here, we present a comprehensive study concerning two functional homologues of mycobacterial H-NS: Lsr2 and its paralogue from M. smegmatis, MSMEG_1060. We found that Lsr2 plays a role in transcriptional regulation, mainly by repressing gene expression via DNA loop formation and/or DNA-coating mechanisms. Intriguingly, the number of Lsr2-mediated genes was found to increase under hypoxia. Compared to Lsr2, MSMEG_1060 exhibits a different DNA binding specificity and chromosomal localization. Since tuberculosis remains a serious worldwide health problem, studies on stress response-mediating agents, such as Lsr2, may contribute to the development of novel antituberculosis drugs.

Thiosemicarbazide Derivatives Decrease the ATPase Activity of Staphylococcus aureus Topoisomerase IV, Inhibit Mycobacterial Growth, and Affect Replication in Mycobacterium smegmatis.

Compounds targeting bacterial topoisomerases are of interest for the development of antibacterial agents. Our previous studies culminated in the synthesis and characterization of small-molecular weight thiosemicarbazides as the initial prototypes of a novel class of gyrase and topoisomerase IV inhibitors. To expand these findings with further details on the mode of action of the most potent compounds, enzymatic studies combined with a molecular docking approach were carried out, the results of which are presented herein. The biochemical assay for 1-(indol-2-oyl)-4-(4-nitrophenyl) thiosemicarbazide (4) and 4-benzoyl-1-(indol-2-oyl) thiosemicarbazide (7), showing strong inhibitory activity against Staphylococcus aureus topoisomerase IV, confirmed that these compounds reduce the ability of the ParE subunit to hydrolyze ATP rather than act by stabilizing the cleavage complex. Compound 7 showed better antibacterial activity than compound 4 against clinical strains of S. aureus and representatives of the Mycobacterium genus. In vivo studies using time-lapse microfluidic microscopy, which allowed for the monitoring of fluorescently labelled replisomes, revealed that compound 7 caused an extension of the replication process duration in Mycobacterium smegmatis, as well as the growth arrest of bacterial cells. Despite some similarities to the mechanism of action of novobiocin, these compounds show additional, unique properties, and can thus be considered a novel group of inhibitors of the ATPase activity of bacterial type IIA topoisomerases.

Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

Lsr2, a nucleoid-associated protein influencing mycobacterial cell cycle.

Nucleoid-associated proteins (NAPs) are responsible for maintaining highly organized and yet dynamic chromosome structure in bacteria. The genus Mycobacterium possesses a unique set of NAPs, including Lsr2, which is a DNA-bridging protein. Importantly, Lsr2 is essential for the M. tuberculosis during infection exhibiting pleiotropic activities including regulation of gene expression (mainly as a repressor). Here, we report that deletion of lsr2 gene profoundly impacts the cell morphology of M. smegmatis, which is a model organism for studying the cell biology of M. tuberculosis and other mycobacterial pathogens. Cells lacking Lsr2 are shorter, wider, and more rigid than the wild-type cells. Using time-lapse fluorescent microscopy, we showed that fluorescently tagged Lsr2 forms large and dynamic nucleoprotein complexes, and that the N-terminal oligomerization domain of Lsr2 is indispensable for the formation of nucleoprotein complexes in vivo. Moreover, lsr2 deletion exerts a significant effect on the replication time and replisome dynamics. Thus, we propose that the Lsr2 nucleoprotein complexes may contribute to maintaining the proper organization of the newly synthesized DNA and therefore influencing mycobacterial cell cycle.

Functional Disassociation Between the Protein Domains of MSMEG_4305 of Mycolicibacterium smegmatis (Mycobacterium smegmatis) in vivo.

MSMEG_4305 is a two-domain protein of Mycolicibacterium smegmatis (Mycobacterium smegmatis) (Mycolicibacterium smegmatis). The N-terminal domain of MSMEG_4305 encodes an RNase H type I. The C-terminal domain is a presumed CobC, predicted to be involved in the aerobic synthesis of vitamin B12. Both domains reach their maximum at distinct pH, approximately 8.5 and 4.5, respectively. The presence of the CobC domain influenced RNase activity in vitro in homolog Rv2228c. Here, we analyzed the role of MSMEG_4305 in vitamin B12 synthesis and the functional association between both domains in vivo in M. smegmatis. We used knock-out mutant of M. smegmatis, deficient in MSMEG_4305. Whole-cell lysates of the mutants strain contained a lower concentration of vitamin B12, as it determined with immunoenzimatic assay. We observed growth deficits, related to vitamin B12 production, on media containing sulfamethazine and propionate. Removal of the CobC domain of MSMEG_4305 in ΔrnhA background hardly affected the growth rate of M. smegmatis in vivo. The strain carrying truncation showed no fitness deficit in the competitive assay and it did not show increased level of RNA/DNA hybrids in its genome. We show that homologs of MSMEG_4305 are present only in the Actinomycetales phylogenetic branch (according to the old classification system). The domains of MSMEG_4305 homologs accumulate mutations at a different rate, while the linker region is highly variable. We conclude that MSMEG_4305 is a multidomain protein that most probably was fixed in the phylogenetic tree of life due to genetic drift.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy.

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged ß-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

Nucleoid Associated Proteins: The Small Organizers That Help to Cope With Stress.

The bacterial chromosome must be efficiently compacted to fit inside the small and crowded cell while remaining accessible for the protein complexes involved in replication, transcription, and DNA repair. The dynamic organization of the nucleoid is a consequence of both intracellular factors (i.e., simultaneously occurring cell processes) and extracellular factors (e.g., environmental conditions, stress agents). Recent studies have revealed that the bacterial chromosome undergoes profound topological changes under stress. Among the many DNA-binding proteins that shape the bacterial chromosome structure in response to various signals, NAPs (nucleoid associated proteins) are the most abundant. These small, basic proteins bind DNA with low specificity and can influence chromosome organization under changing environmental conditions (i.e., by coating the chromosome in response to stress) or regulate the transcription of specific genes (e.g., those involved in virulence).

AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor.

In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilisStreptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.

Streptomycete origin of chromosomal replication with two putative unwinding elements.

DNA replication is controlled mostly at the initiation step. In bacteria, replication of the chromosome starts at a single origin of replication called oriC. The initiator protein, DnaA, binds to specific sequences (DnaA boxes) within oriC and assembles into a filament that promotes DNA double helix opening within the DNA unwinding element (DUE). This process has been thoroughly examined in model bacteria, including Escherichia coli and Bacillus subtilis, but we have a relatively limited understanding of chromosomal replication initiation in other species. Here, we reveal new details of DNA replication initiation in Streptomyces, a group of Gram-positive soil bacteria that possesses a long linear (8-10 Mbps) and GC-rich chromosome with a centrally positioned oriC. We used comprehensive in silico, in vitro and in vivo analyses to better characterize the structure of Streptomyces oriC. We identified 14 DnaA-binding motifs and determined the consensus sequence of the DnaA box. Unexpectedly, our in silico analysis using the WebSIDD algorithm revealed the presence of two putative Streptomyces DUEs (DUE1 and DUE2) located very near one another toward the 5' end of the oriC region. In vitro P1 nuclease assay revealed that DNA unwinding occurs at both of the proposed sites, but using an in vivo replication initiation point mapping, we were able to confirm only one of them (DUE2). The previously observed transcriptional activity of the Streptomyces oriC region may help explain the current results. We speculate that transcription itself could modulate oriC activity in Streptomyces by determining whether DNA unwinding occurs at DUE1 or DUE2.

Molecular mechanisms involved in bacterial chromatin organization / Molekularne podstawy organizacji chromatyny bakteryjnej.

Advances in high resolution microscopy techniques and development of high throughput DNA analyses allow to reconsider the views concerning bacterial chromosome (nucleoid). Recent reports show that nucleoid exhibits a hierarchical organization, similarly to the eukaryotic chromatin. However, bacterial chromosome undergoes constant modifications and topological rearrangements due to the ongoing DNA replication, transcription and translation processes. Organization of dynamic and highly compacted nucleoid structure depends on physical factors acting on chromosome molecule inside small cell compartment, and is a consequence of action of many different DNA-binding proteins. The main goal of this review is to present the recent reports on bacterial chromatin structure and to elucidate the physical and molecular factors influencing its intracellular organization.

Watching DNA Replication Inhibitors in Action: Exploiting Time-Lapse Microfluidic Microscopy as a Tool for Target-Drug Interaction Studies in Mycobacterium.

Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria, which are the causative agents of severe diseases and which have profound impacts on global health. Here, we used a system of microfluidics, fluorescence microscopy, and target-tagged fluorescent reporter strains of Mycobacterium smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid, and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid and that nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, while griselimycin caused replisome instability, with the subsequent overinitiation of chromosome replication and overrelaxation of the nucleoid. In addition to study target-drug interactions, our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.).

Dynamics of Chromosome Replication and Its Relationship to Predatory Attack Lifestyles in Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus degrades a host's macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus Our results show that in B. bacteriovorus, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if this B. bacteriovorus life cycle fails in some cells of Escherichia coli, they can instead use second prey cells to complete their life cycle.IMPORTANCE New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium Bdellovibrio bacteriovorus, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The B. bacteriovorus life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host's macromolecules and reuses them for its own growth. To understand the use of B. bacteriovorus as a "living antibiotic," it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell of B. bacteriovorus This process initiates at the invasion pole of B. bacteriovorus and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells sequentially to complete their life cycle.

Competition between DivIVA and the nucleoid for ParA binding promotes segrosome separation and modulates mycobacterial cell elongation.

Although mycobacteria are rod shaped and divide by simple binary fission, their cell cycle exhibits unusual features: unequal cell division producing daughter cells that elongate with different velocities, as well as asymmetric chromosome segregation and positioning throughout the cell cycle. As in other bacteria, mycobacterial chromosomes are segregated by pair of proteins, ParA and ParB. ParA is an ATPase that interacts with nucleoprotein ParB complexes - segrosomes and non-specifically binds the nucleoid. Uniquely in mycobacteria, ParA interacts with a polar protein DivIVA (Wag31), responsible for asymmetric cell elongation, however the biological role of this interaction remained unknown. We hypothesised that this interaction plays a critical role in coordinating chromosome segregation with cell elongation. Using a set of ParA mutants, we determined that disruption of ParA-DNA binding enhanced the interaction between ParA and DivIVA, indicating a competition between the nucleoid and DivIVA for ParA binding. Having identified the ParA mutation that disrupts its recruitment to DivIVA, we found that it led to inefficient segrosomes separation and increased the cell elongation rate. Our results suggest that ParA modulates DivIVA activity. Thus, we demonstrate that the ParA-DivIVA interaction facilitates chromosome segregation and modulates cell elongation.