Replication Of Bacterial Chromosome Research Articles

Pathological R-loops in bacteria from engineered expression of endogenous antisense RNAs whose synthesis is ordinarily terminated by Rho.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.

Recollections of a Helmstetter Disciple.

Nearly fifty years ago, it became possible to construct E. coli minichromosomes using recombinant DNA technology. These very small replicons, comprising the unique replication origin of the chromosome oriC coupled to a drug resistance marker, provided new opportunities to study the regulation of bacterial chromosome replication, were key to obtaining the nucleotide sequence information encoded into oriC and were essential for the development of a ground-breaking in vitro replication system. However, true authenticity of the minichromosome model system required that they replicate during the cell cycle with chromosome-like timing specificity. I was fortunate enough to have the opportunity to construct E. coli minichromosomes in the laboratory of Charles Helmstetter and, for the first time, measure minichromosome cell cycle regulation. In this review, I discuss the evolution of this project along with some additional studies from that time related to the DNA topology and segregation properties of minichromosomes. Despite the significant passage of time, it is clear that large gaps in our understanding of oriC regulation still remain. I discuss some specific topics that continue to be worthy of further study.

★Track: Inferred counting and tracking of replicating DNA loci

Fluorescent microscopy is the primary method to study DNA organization within cells. However, the variability and low signal/noise commonly associated with live-cell time-lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present ★Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged, and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track’s abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, ★Track can help to uncover the mechanistic principles of chromosome organization and dynamics across a range of systems.

Bacteriophage Twort protein Gp168 is a β-clamp inhibitor by occupying the DNA sliding channel.

Bacterial chromosome replication is mainly catalyzed by DNA polymerase III, whose beta subunits enable rapid processive DNA replication. Enabled by the clamp-loading complex, the two beta subunits form a ring-like clamp around DNA and keep the polymerase sliding along. Given the essential role of β-clamp, its inhibitors have been explored for antibacterial purposes. Similarly, β-clamp is an ideal target for bacteriophages to shut off host DNA synthesis during host takeover. The Gp168 protein of phage Twort is such an example, which binds to the β-clamp of Staphylococcus aureus and prevents it from loading onto DNA causing replication arrest. Here, we report a cryo-EM structure of the clamp–Gp168 complex at 3.2-Å resolution. In the structure of the complex, the Gp168 dimer occupies the DNA sliding channel of β-clamp and blocks its loading onto DNA, which represents a new inhibitory mechanism against β-clamp function. Interestingly, the key residues responsible for this interaction on the β-clamp are well conserved among bacteria. We therefore demonstrate that Gp168 is potentially a cross-species β-clamp inhibitor, as it forms complex with the Bacillus subtilis β-clamp. Our findings reveal an alternative mechanism for bacteriophages to inhibit β-clamp and provide a new strategy to combat bacterial drug resistance.

Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Escherichia coli Cell Cycle by Transcriptional Modulators That Interact With Origin DNA.

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC’s lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

Bacterial Chromosome Replication and DNA Repair During the Stringent Response.

The stringent response regulates bacterial growth rate and is important for cell survival under changing environmental conditions. The effect of the stringent response is pleiotropic, affecting almost all biological processes in the cell including transcriptional downregulation of genes involved in stable RNA synthesis, DNA replication, and metabolic pathways, as well as the upregulation of stress-related genes. In this Review, we discuss how the stringent response affects chromosome replication and DNA repair activities in bacteria. Importantly, we address how accumulation of (p)ppGpp during the stringent response shuts down chromosome replication using highly different strategies in the evolutionary distant Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. Interestingly, (p)ppGpp-mediated replication inhibition occurs downstream of the origin in B. subtilis, whereas replication inhibition in E. coli takes place at the initiation level, suggesting that stringent cell cycle arrest acts at different phases of the replication cycle between E. coli and B. subtilis. Furthermore, we address the role of (p)ppGpp in facilitating DNA repair activities and cell survival during exposure to UV and other DNA damaging agents. In particular, (p)ppGpp seems to stimulate the efficiency of nucleotide excision repair (NER)-dependent repair of DNA lesions. Finally, we discuss whether (p)ppGpp-mediated cell survival during DNA damage is related to the ability of (p)ppGpp accumulation to inhibit chromosome replication.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Evolutionary Changes in DnaA-Dependent Chromosomal Replication in Cyanobacteria.

Replication of the circular bacterial chromosome is initiated at a unique origin (oriC) in a DnaA-dependent manner in which replication proceeds bidirectionally from oriC to ter. The nucleotide compositions of most bacteria differ between the leading and lagging DNA strands. Thus, the chromosomal DNA sequence typically exhibits an asymmetric GC skew profile. Further, free-living bacteria without genomes encoding dnaA were unknown. Thus, a DnaA-oriC-dependent replication initiation mechanism may be essential for most bacteria. However, most cyanobacterial genomes exhibit irregular GC skew profiles. We previously found that the Synechococcus elongatus chromosome, which exhibits a regular GC skew profile, is replicated in a DnaA-oriC-dependent manner, whereas chromosomes of Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, which exhibit an irregular GC skew profile, are replicated from multiple origins in a DnaA-independent manner. Here we investigate the variation in the mechanisms of cyanobacterial chromosome replication. We found that the genomes of certain free-living species do not encode dnaA and such species, including Cyanobacterium aponinum PCC 10605 and Geminocystis sp. NIES-3708, replicate their chromosomes from multiple origins. Synechococcus sp. PCC 7002, which is phylogenetically closely related to dnaA-lacking free-living species as well as to dnaA-encoding but DnaA-oriC-independent Synechocystis sp. PCC 6803, possesses dnaA. In Synechococcus sp. PCC 7002, dnaA was not essential and its chromosomes were replicated from a unique origin in a DnaA-oriC independent manner. Our results also suggest that loss of DnaA-oriC-dependency independently occurred multiple times during cyanobacterial evolution and raises a possibility that the loss of dnaA or loss of DnaA-oriC dependency correlated with an increase in ploidy level.

Inference of Bacterial Pathogen Instantaneous Population Growth Dynamics.

Although bacterial host colonization is a dynamic process that requires population growth, studies often focus on comparing bacterial populations at a given time point. However, this may not reflect the dynamics of the colonization process. Time-course assays provide important insights into the dynamics of host colonization but are laborious and may still lack resolution for immediate processes affecting populations. An alternative way to address this issue, using widely accessible tools (such as quantitative PCR [qPCR]), is to take advantage of the relationship between bacterial chromosomal replication and cell division to determine population growth status at the sampling time. Conceptually, the ratio between the number of copies at the origin of replication and that at the terminus of replication should be correlated with the measured bacterial growth rate. This peak-to-trough ratio (PTR) to estimate instantaneous population growth status was tested with the slow-growing plant-pathogenic bacterium Xylella fastidiosa. We found no correlation between PTR and the measured growth rate when using genome-level data but overall sequencing depth of coverage trends matched theoretical expectations. On the other hand, the population growth status of X. fastidiosa was predicted by PTR when using qPCR data, which was improved by the pretreatment of cells with a photoreactive DNA-binding dye. Our results suggest that PTR could be used to determine X. fastidiosa growth status both in planta and in insect vectors. We expect PTR will perform better with fast-growing bacterial pathogens, potentially becoming a powerful tool for easily and quickly assessing population growth status.

Application of UV-Visible Spectrophotometric Method for the Estimation of Ciprofloxacin HCl and Levofloxacin Hemihydrate (Antibiotics) in Marketed Drugs

Ciprofloxacin HCl, 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin1-yl)-1,4-dihydroquinoline-3-carboxylic acid, is a synthetic broad antibacterial compound belonging to the group of fluoroquinolones which is antimicrobial drug act through the inhibition of DNA-gyrase, an enzyme that is critical to bacterial chromosome replication. It is used in a wide range of infections of the urinary, respiratory and gastrointestinal tracts, as well as in skin structure and ocular infections. Levofloxacin Hemihydrate is a fluoroquinolone, has a broad-spectrum antibiotic activity against gram-positive and gram-negative bacteria and used in respiratory and urinary tract infections. Various Pakistani branded antibiotic drug samples containing Ciprofloxacin HCl and Levofloxacin Hemihydrate were collected from local market and their working standards from the material suppliers of Karachi city. Samples were analyzed by using UV-Vis spectrophotometer. Then the method was validated by linearity, accuracy, precision (reproducibility, intermediate precision) and range which suggested that method is appropriate and proper to determine the potency of these various brands of Ciprofloxacin HCl and Levofloxacin Hemihydrate. The analytical data suggest that the drug samples analyzed were within the limit of the Pharmacopeia requirement and criteria.

Application of UV-Visible Spectrophotometric Method for the Estimation of Ciprofloxacin HCl and Levofloxacin Hemihydrate (Antibiotics) in Marketed Drugs

Ciprofloxacin HCl, 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin1-yl)-1,4-dihydroquinoline-3-carboxylic acid, is a synthetic broad antibacterial compound belonging to the group of fluoroquinolones which is antimicrobial drug act through the inhibition of DNA-gyrase, an enzyme that is critical to bacterial chromosome replication. It is used in a wide range of infections of the urinary, respiratory and gastrointestinal tracts, as well as in skin structure and ocular infections. Levofloxacin Hemihydrate is a fluoroquinolone, has a broad-spectrum antibiotic activity against gram-positive and gram-negative bacteria and used in respiratory and urinary tract infections. Various Pakistani branded antibiotic drug samples containing Ciprofloxacin HCl and Levofloxacin Hemihydrate were collected from local market and their working standards from the material suppliers of Karachi city. Samples were analyzed by using UV-Vis spectrophotometer. Then the method was validated by linearity, accuracy, precision (reproducibility, intermediate precision) and range which suggested that method is appropriate and proper to determine the potency of these various brands of Ciprofloxacin HCl and Levofloxacin Hemihydrate. The analytical data suggest that the drug samples analyzed were within the limit of the Pharmacopeia requirement and criteria.

Blocking the Trigger: Inhibition of the Initiation of Bacterial Chromosome Replication as an Antimicrobial Strategy.

All bacterial cells must duplicate their genomes prior to dividing into two identical daughter cells. Chromosome replication is triggered when a nucleoprotein complex, termed the orisome, assembles, unwinds the duplex DNA, and recruits the proteins required to establish new replication forks. Obviously, the initiation of chromosome replication is essential to bacterial reproduction, but this process is not inhibited by any of the currently-used antimicrobial agents. Given the urgent need for new antibiotics to combat drug-resistant bacteria, it is logical to evaluate whether or not unexploited bacterial processes, such as orisome assembly, should be more closely examined for sources of novel drug targets. This review will summarize current knowledge about the proteins required for bacterial chromosome initiation, as well as how orisomes assemble and are regulated. Based upon this information, we discuss current efforts and potential strategies and challenges for inhibiting this initiation pharmacologically.

Acinetobacter and Moraxella OriCs are Functional as Chromosomal Replication Origin Transplants in E. coli

Bacterial chromosome replication is triggered by the initiator protein, DnaA, which assembles into a multimeric complex (orisome) that unwinds the replication origin, oriC, and helps to load the replicative DNA helicase. DnaA is highly conserved and the oriCs from nearly all bacteria carry clusters of oriC‐encoded DnaA recognition sites that guide step‐wise and properly timed orisome assembly. However, the arrangement of these sites is widely divergent among bacterial types and prior studies show that heterologous oriCs are only functional in extremely close relatives with nearly identical origin nucleotide sequence. To investigate the basis for DnaA recognition site diversity and possibly identify shared steps in orisome assembly, we replaced E. coli's chromosomal oriC with heterologous donor oriCs from its distant Gammaproteobacterial relatives and measured replication activity in vivo. Despite carrying little sequence similarity to the host, oriCs from Acinetobacter baylyi (ADP1) and Moraxella catarrhalis (ATCC 25240) were active in E. coli and used alternative pathways to assemble E. coli DnaA into functional orisomes. Mapping studies revealed the transplanted oriCs lacked the distinctive DNA unwinding element comprising 13mer repeat motifs as well as the arrayed low affinity DnaA recognition sites that are both hallmarks of E. coli oriC. Using flow cytometry analysis, we found that ADP1 and M. catarrhalis oriCs were also unable to initiate DNA synthesis at the proper cell cycle time in their E. coli hosts. Thus, the mechanisms for initiation timing must be distinct from those that permit replication fork assembly on these origins. We propose that E. coli and ADP1‐type orisomes share a sub‐architecture required for mechanical function (unwinding and helicase loading), but also carry type‐specific variations in the arrangement and affinity of DnaA recognition sites that are required for proper initiation timing using their cognate DnaAs. We propose that replication origin diversity reflects the many varieties of cell cycle‐coupled regulatory mechanisms used to control the availability/accessibility of DnaA at the oriCs of different bacterial types. Further studies of DnaA‐oriC interactions on heterologous origins may also reveal novel targets for broad spectrum bacterial growth inhibitors as well as those for type‐specific inhibition.Support or Funding InformationNational Institutes of Health (GM54042 to AL) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Crosstalk Regulation Between Bacterial Chromosome Replication and Chromosome Partitioning.

Despite much effort, the bacterial cell cycle has proved difficult to study and understand. Bacteria do not conform to the standard eukaryotic model of sequential cell-cycle phases. Instead, for example, bacteria overlap their phases of chromosome replication and chromosome partitioning. In “eukaryotic terms,” bacteria simultaneously perform “S-phase” and “mitosis” whose coordination is absolutely required for rapid growth and survival. In this review, we focus on the signaling “crosstalk,” meaning the signaling mechanisms that advantageously commit bacteria to start both chromosome replication and chromosome partitioning. After briefly reviewing the molecular mechanisms of replication and partitioning, we highlight the crosstalk research from Bacillus subtilis, Vibrio cholerae, and Caulobacter crescentus. As the initiator of chromosome replication, DnaA also mediates crosstalk in each of these model bacteria but not always in the same way. We next focus on the C. crescentus cell cycle and describe how it is revealing novel crosstalk mechanisms. Recent experiments show that the novel nucleoid associated protein GapR has a special role(s) in starting and separating the replicating chromosomes, so that upon asymmetric cell division, the new chromosomes acquire different fates in C. crescentus’s distinct replicating and non-replicating cell types. The C. crescentus PopZ protein forms a special cell-pole organizing matrix that anchors the chromosomes through their centromere-like DNA sequences near the origin of replication. We also describe how PopZ anchors and interacts with several key cell-cycle regulators, thereby providing an organized subcellular environment for more novel crosstalk mechanisms.

Where and When Bacterial Chromosome Replication Starts: A Single Cell Perspective.

Bacterial chromosomes have a single, unique replication origin (named oriC), from which DNA synthesis starts. This study describes methods of visualizing oriC regions and the chromosome replication in single living bacterial cells in real-time. This review also discusses the impact of live cell imaging techniques on understanding of chromosome replication dynamics, particularly at the initiation step, in different species of bacteria.

Segregation but Not Replication of the Pseudomonas aeruginosa Chromosome Terminates at Dif

Coordination between chromosome replication and segregation is essential for equal partitioning of genetic material between daughter cells. In bacteria, this is achieved through the proximity of the origin of replication, oriC, and the chromosome partitioning site, parS We report here that in Pseudomonas aeruginosa, segregation but not replication is also controlled at the terminus region of the chromosome. Using the fluorescent repressor operator system (FROS), we investigated chromosome segregation in P. aeruginosa strain PAO1-UW, wherein the chromosome dimer resolution site, dif, is asymmetrically positioned relative to oriC In these cells, segregation proceeded sequentially along the two chromosomal arms and terminated at dif In contrast, chromosome replication terminated elsewhere, opposite from oriC We further found two large domains on the longer arm of the chromosome, wherein DNA segregated simultaneously. Notably, GC-skew, which reflects a bias in nucleotide usage between the leading and lagging strands of the chromosome, switches polarity at the dif locus but not necessarily at the terminus of replication. These data demonstrate that termination of chromosome replication and segregation can be physically separated without adverse effects on bacterial fitness. They also reveal the critical role of the dif region in defining the global layout of the chromosome and the progression of chromosome segregation and suggest that chromosome packing adapts to its subcellular layout.IMPORTANCE Segregation of genetic information is a central event in cellular life. In bacteria, chromosome segregation occurs concurrently with replication, sequentially along the two arms from oriC to dif How the two processes are coordinated is unknown. We explored here chromosome segregation in an opportunistic human pathogen, Pseudomonas aeruginosa, using its strain with markedly unequal chromosomal arms. We found that replication and segregation diverge in this strain and terminate at very different locations, whereas the longer chromosomal arm folds into large domains to align itself with the shorter arm. The significance of this research is in establishing that segregation and replication of bacterial chromosomes are largely uncoupled from each other and that the large-scale structure of the chromosome adapts to its subcellular layout.

Transcriptional Regulation Buffers Gene Dosage Effects on a Highly Expressed Operon in Salmonella.

Highly expressed genes are commonly located close to the origin of replication of bacterial chromosomes (OriC). This location skew is thought to reflect selective advantages associated with gene dosage effects during the replication cycle. The expression of constitutively expressed genes can vary up to fivefold based on chromosomal location, but it is not clear what level of variation would occur in naturally regulated operons. We tested the magnitude of the chromosome location effect using EF-Tu (tufA, tufB), an abundant protein whose cellular level correlates with, and limits, the maximum growth rate. We translocated the Salmonella tufB operon to four locations across the chromosome. The distance from OriC had only a small effect on growth rate, consistent with this operon having the natural ability to upregulate expression and compensate for reduced gene dosage. In contrast, when the total EF-Tu concentration was limiting for the growth rate (tufA deleted), we observed a strong gene dosage effect when tufB was located further from OriC. However, only a short period of experimental evolution was required before the bacteria adapted to this EF-Tu starvation situation by acquiring genetic changes that increased expression levels from the translocated tufB gene, restoring growth rates. Our findings demonstrate that, at least for the tufB operon, gene dosage is probably not the dominant force selecting for a chromosomal location close to OriC. We suggest that the colocation of highly expressed genes close to OriC might instead be selected because it enhances their coregulation during various growth states, with gene dosage being a secondary benefit.IMPORTANCE A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB, to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufB were easily and rapidly selected. We conclude, at least for tufB, that forces other than gene dosage may be significant in selecting for chromosomal location.

The Chlamydia muridarum plasmid revisited : new insights into growth kinetics

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence. Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes. There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum. We have proven that the differences described are solely due to the plasmid pNigg.

Phosphorylation of the Bacillus subtilis Replication Controller YabA Plays a Role in Regulation of Sporulation and Biofilm Formation.

Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

The Chlamydia muridarum plasmid revisited : new insights into growth kinetics.

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence. Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes. There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum. We have proven that the differences described are solely due to the plasmid pNigg.