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Jolanta Zakrzewskaczerwinska - One of the best experts on this subject based on the ideXlab platform.
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dynamics of Chromosome Replication and its relationship to predatory attack lifestyles in bdellovibrio bacteriovorus
Applied and Environmental Microbiology, 2019Co-Authors: łukasz Makowski, Damian Trojanowski, Carey Lambert, Elizabeth R Sockett, Rob Till, Rebecca Lowry, Jolanta ZakrzewskaczerwinskaAbstract: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.
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dynamics of Chromosome Replication and its relationship to predatory attack lifestyles in bdellovibrio bacteriovorus
bioRxiv, 2019Co-Authors: Lukasz Makowski, Damian Trojanowski, Carey Lambert, Elizabeth R Sockett, Rob Till, Rebecca Lowry, Jolanta ZakrzewskaczerwinskaAbstract:Abstract Bdellovibrio bacteriovorus is a small Gram-negative, an obligate predatory bacterium that is largely found in wet, aerobic environments (i.e. 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 non-replicative attack phase wherein 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 Chromosome Replication in B. bacteriovorus. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus. Our results confirm that in B. bacteriovorus Chromosome Replication fires only during the reproductive phase, and show for the first time that this predatory bacterium exhibits a novel spatiotemporal arrangement of Chromosome Replication. 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. This 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 lifecycle fails in some cells of B. bacteriovorus, they can instead use two prey cells sequentially 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 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 for the first time a real-time investigation into subcellular localization of Chromosome Replication in a single cells 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.
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amsacrine derivatives selectively inhibit mycobacterial topoisomerase i topa impair m smegmatis growth and disturb Chromosome Replication
Frontiers in Microbiology, 2018Co-Authors: Marcin Jan Szafran, Marta Kolodziej, Patrycja Skut, Agnieszka Domagala, Damian Trojanowski, Brahmam Medapi, Jolanta Zakrzewskaczerwinska, Dharmarajan Sriram, Dagmara JakimowiczAbstract:Amsacrine, which inhibits eukaryotic type II topoisomerase via DNA intercalation and stabilization of the cleavable topoisomerase-DNA complex, promotes DNA damage and eventually cell death. Amsacrine has also been shown to inhibit structurally distinct bacterial type I topoisomerases (TopAs), including mycobacterial TopA, the only and essential topoisomerase I in Mycobacterium tuberculosis. Here, we describe the modifications of an amsacrine sulfonamide moiety that presumably interacts with mycobacterial TopA, which notably increased the enzyme inhibition and drug selectivity in vivo. To analyse the effects of amsacrine and its derivatives treatment on cell cycle, we used time-lapse fluorescence microscopy (TLMM) and fusion of the β-subunit of DNA polymerase III with enhanced green fluorescence protein (DnaN-EGFP). We determined that treatment with amsacrine and its derivatives increased the number of DnaN-EGFP complexes and/or prolonged the time of Chromosome Replication and cell cycle notably. The analysis of TopA depletion strain confirmed that lowering TopA level results in similar disturbances of Chromosome Replication. In summary, since TopA is crucial for mycobacterial cell viability, the compounds targeting the enzyme disturbed the cell cycle and thus may constitute a new class of anti-tuberculosis drugs.
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multifork Chromosome Replication in slow growing bacteria
Scientific Reports, 2017Co-Authors: Damian Trojanowski, Dagmara Jakimowicz, Jolanta Zakrzewskaczerwinska, Joanna Holowka, Katarzyna GindaAbstract:The growth rates of bacteria must be coordinated with major cell cycle events, including Chromosome Replication. When the doubling time (Td) is shorter than the duration of Chromosome Replication (C period), a new round of Replication begins before the previous round terminates. Thus, newborn cells inherit partially duplicated Chromosomes. This phenomenon, which is termed multifork Replication, occurs among fast-growing bacteria such as Escherichia coli and Bacillus subtilis. In contrast, it was historically believed that slow-growing bacteria (including mycobacteria) do not reinitiate Chromosome Replication until the previous round has been completed. Here, we use single-cell time-lapse analyses to reveal that mycobacterial cell populations exhibit heterogeneity in their DNA Replication dynamics. In addition to cells with non-overlapping Replication rounds, we observed cells in which the next Replication round was initiated before completion of the previous Replication round. We speculate that this heterogeneity may reflect a relaxation of cell cycle checkpoints, possibly increasing the ability of slow-growing mycobacteria to adapt to environmental conditions.
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recent advances in helicobacter pylori Replication possible implications in adaptation to a pathogenic lifestyle and perspectives for drug design
Current Topics in Microbiology and Immunology, 2017Co-Authors: Anna Zawilakpawlik, Jolanta ZakrzewskaczerwinskaAbstract:DNA Replication is an important step in the life cycle of every cell that ensures the continuous flow of genetic information from one generation to the next. In all organisms, Chromosome Replication must be coordinated with overall cell growth. Helicobacter pylori growth strongly depends on its interaction with the host, particularly with the gastric epithelium. Moreover, H. pylori actively searches for an optimal microniche within a stomach, and it has been shown that not every microniche equally supports growth of this bacterium. We postulate that besides nutrients, H. pylori senses different, unknown signals, which presumably also affect Chromosome Replication to maintain H. pylori propagation at optimal ratio allowing H. pylori to establish a chronic, lifelong infection. Thus, H. pylori Chromosome Replication and particularly the regulation of this process might be considered important for bacterial pathogenesis. Here, we summarize our current knowledge of Chromosome and plasmid Replication in H. pylori and discuss the mechanisms responsible for regulating this key cellular process. The results of extensive studies conducted thus far allow us to propose common and unique traits in H. pylori Chromosome Replication. Interestingly, the repertoire of proteins involved in Replication in H. pylori is significantly different to that in E. coli, strongly suggesting that novel factors are engaged in H. pylori Chromosome Replication and could represent attractive drug targets.
Leise Riber - One of the best experts on this subject based on the ideXlab platform.
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bacterial Chromosome Replication and dna repair during the stringent response
Frontiers in Microbiology, 2020Co-Authors: Anurag Kumar Sinha, Anders Lobnerolesen, Leise RiberAbstract: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.
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inhibition of escherichia coli Chromosome Replication by rifampicin treatment or during the stringent response is overcome by de novo dnaa protein synthesis
Molecular Microbiology, 2020Co-Authors: Leise Riber, Anders LobnerolesenAbstract:Initiation of Escherichia coli Chromosome Replication is controlled by the DnaA initiator protein. Both rifampicin-mediated inhibition of transcription and ppGpp-induced changes in global transcription stops Replication at the level of initiation. Here, we show that continued DnaA protein synthesis allows for Replication initiation both during the rifampicin treatment and during the stringent response when the ppGpp level is high. A reduction in or cessation of de novo DnaA synthesis, therefore, causes the initiation arrest in both cases. In accordance with this, inhibition of translation with chloramphenicol also stops initiations. The initiation arrest caused by rifampicin was faster than that caused by chloramphenicol, despite of the latter inhibiting DnaA accumulation immediately. During chloramphenicol treatment transcription is still ongoing and we suggest that transcriptional events in or near the origin, that is, transcriptional activation, can allow for a few extra initiations when DnaA becomes limiting. We suggest, for both rifampicin treated cells and for cells accumulating ppGpp, that a turn-off of initiation from oriC requires a stop in de novo DnaA synthesis and that an additional lack of transcriptional activation enhances this process, that is, leads to a faster initiation stop.
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multiple dna binding proteins contribute to timing of Chromosome Replication in e coli
Frontiers in Molecular Biosciences, 2016Co-Authors: Leise Riber, Jakob Frimodtmoller, Godefroid Charbon, Anders LobnerolesenAbstract:Chromosome Replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. DnaA associated with either ATP or ADP binds to a set of strong DnaA binding sites in oriC, whereas only DnaAATP is capable of binding additional and weaker sites to promote initiation. Additional DNA binding proteins act to ensure that initiation occurs timely by affecting either the cellular mass at which DNA Replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: i) binding directly to oriC to affect DnaA binding, ii) altering the DNA topology in or around oriC, iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of Replication, other DNA-binding proteins act not only on oriC for modulation of its activity but also at additional regulatory sites to control the nucleotide bound status of DnaA. Here we review the contribution of key DNA binding proteins to the tight regulation of Chromosome Replication in E. coli cells.
Anders Lobnerolesen - One of the best experts on this subject based on the ideXlab platform.
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bacterial Chromosome Replication and dna repair during the stringent response
Frontiers in Microbiology, 2020Co-Authors: Anurag Kumar Sinha, Anders Lobnerolesen, Leise RiberAbstract: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.
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inhibition of escherichia coli Chromosome Replication by rifampicin treatment or during the stringent response is overcome by de novo dnaa protein synthesis
Molecular Microbiology, 2020Co-Authors: Leise Riber, Anders LobnerolesenAbstract:Initiation of Escherichia coli Chromosome Replication is controlled by the DnaA initiator protein. Both rifampicin-mediated inhibition of transcription and ppGpp-induced changes in global transcription stops Replication at the level of initiation. Here, we show that continued DnaA protein synthesis allows for Replication initiation both during the rifampicin treatment and during the stringent response when the ppGpp level is high. A reduction in or cessation of de novo DnaA synthesis, therefore, causes the initiation arrest in both cases. In accordance with this, inhibition of translation with chloramphenicol also stops initiations. The initiation arrest caused by rifampicin was faster than that caused by chloramphenicol, despite of the latter inhibiting DnaA accumulation immediately. During chloramphenicol treatment transcription is still ongoing and we suggest that transcriptional events in or near the origin, that is, transcriptional activation, can allow for a few extra initiations when DnaA becomes limiting. We suggest, for both rifampicin treated cells and for cells accumulating ppGpp, that a turn-off of initiation from oriC requires a stop in de novo DnaA synthesis and that an additional lack of transcriptional activation enhances this process, that is, leads to a faster initiation stop.
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comparative activity of ceftriaxone ciprofloxacin and gentamicin as a function of bacterial growth rate probed by escherichia coli Chromosome Replication in the mouse peritonitis model
Antimicrobial Agents and Chemotherapy, 2018Co-Authors: Maria Schei Haugan, Anders Lobnerolesen, Niels FrimodtmollerAbstract:Commonly used antibiotics exert their effects predominantly on rapidly growing bacterial cells; yet, the growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, a means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated Chromosome Replication as a readout of in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (quantitative PCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average level and on a single-cell level, from one single biological specimen. Here, we asked whether the in situ growth rate predicts antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as a proof of concept. Our data demonstrate that the activities of the commonly used antibiotics ceftriaxone and gentamicin correlated with pretreatment bacterial growth rate; both drugs performed better during rapid growth than during slow growth. Conversely, ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic’s dependency on active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.
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comparative activity of ceftriaxone ciprofloxacin and gentamicin as a function of bacterial growth rate probed by escherichia coli Chromosome Replication in the mouse peritonitis model
bioRxiv, 2018Co-Authors: Maria Schei Haugan, Anders Lobnerolesen, Niels FrimodtmollerAbstract:Commonly used antibiotics exert their effect predominantly on rapidly growing bacterial cells, yet growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated Chromosome Replication as readout for in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (qPCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average and on a single-cell level; from one single biological specimen. Here, we asked whether the in situ growth rate could predict antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as proof-of-concept. Our data demonstrate that the activity of commonly used antibiotics Ceftriaxone and Gentamicin correlated with pre-treatment bacterial growth rate; both drugs performing better during rapid growth than during slow growth. Conversely, Ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic's dependency upon active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.
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Chromosome Replication as a measure of bacterial growth rate during escherichia coli infection in the mouse peritonitis model
Scientific Reports, 2018Co-Authors: Maria Schei Haugan, Godefroid Charbon, Niels Frimodtmoller, Anders LobnerolesenAbstract:The efficacy of most antibiotics is dependent on active bacterial growth, yet little is known about the growth dynamics during infection. Therefore, means to measure in-host bacterial growth rate is of importance. Here, we use Chromosome Replication as readout for in situ bacterial growth rate during infection; obtained from a single biological specimen. We have applied two independent methods: quantitative PCR (qPCR) and fluorescence microscopy, to quantify the level of Chromosome Replication present during Escherichia coli propagation in the mouse peritonitis model. We find that the methods complement each other and allow for quantification of growth rate, both on a population average and on a single-cell level. We demonstrate the presence of heterogeneous growth rates within bacterial populations propagating during infection. Also, no growth cessation was observed during the apparent stationary phase in vivo, and, by comparison of growth dynamics at different anatomical sites, we demonstrate that E. coli is unlikely to grow independently intravascularly. These findings provide novel insight into bacterial growth during host infection, and underscore the importance of pinpointing the primary site of infection in septicaemia of unknown origin and ensuring antibiotic availability at this site.
Dagmara Jakimowicz - One of the best experts on this subject based on the ideXlab platform.
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amsacrine derivatives selectively inhibit mycobacterial topoisomerase i topa impair m smegmatis growth and disturb Chromosome Replication
Frontiers in Microbiology, 2018Co-Authors: Marcin Jan Szafran, Marta Kolodziej, Patrycja Skut, Agnieszka Domagala, Damian Trojanowski, Brahmam Medapi, Jolanta Zakrzewskaczerwinska, Dharmarajan Sriram, Dagmara JakimowiczAbstract:Amsacrine, which inhibits eukaryotic type II topoisomerase via DNA intercalation and stabilization of the cleavable topoisomerase-DNA complex, promotes DNA damage and eventually cell death. Amsacrine has also been shown to inhibit structurally distinct bacterial type I topoisomerases (TopAs), including mycobacterial TopA, the only and essential topoisomerase I in Mycobacterium tuberculosis. Here, we describe the modifications of an amsacrine sulfonamide moiety that presumably interacts with mycobacterial TopA, which notably increased the enzyme inhibition and drug selectivity in vivo. To analyse the effects of amsacrine and its derivatives treatment on cell cycle, we used time-lapse fluorescence microscopy (TLMM) and fusion of the β-subunit of DNA polymerase III with enhanced green fluorescence protein (DnaN-EGFP). We determined that treatment with amsacrine and its derivatives increased the number of DnaN-EGFP complexes and/or prolonged the time of Chromosome Replication and cell cycle notably. The analysis of TopA depletion strain confirmed that lowering TopA level results in similar disturbances of Chromosome Replication. In summary, since TopA is crucial for mycobacterial cell viability, the compounds targeting the enzyme disturbed the cell cycle and thus may constitute a new class of anti-tuberculosis drugs.
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multifork Chromosome Replication in slow growing bacteria
Scientific Reports, 2017Co-Authors: Damian Trojanowski, Dagmara Jakimowicz, Jolanta Zakrzewskaczerwinska, Joanna Holowka, Katarzyna GindaAbstract:The growth rates of bacteria must be coordinated with major cell cycle events, including Chromosome Replication. When the doubling time (Td) is shorter than the duration of Chromosome Replication (C period), a new round of Replication begins before the previous round terminates. Thus, newborn cells inherit partially duplicated Chromosomes. This phenomenon, which is termed multifork Replication, occurs among fast-growing bacteria such as Escherichia coli and Bacillus subtilis. In contrast, it was historically believed that slow-growing bacteria (including mycobacteria) do not reinitiate Chromosome Replication until the previous round has been completed. Here, we use single-cell time-lapse analyses to reveal that mycobacterial cell populations exhibit heterogeneity in their DNA Replication dynamics. In addition to cells with non-overlapping Replication rounds, we observed cells in which the next Replication round was initiated before completion of the previous Replication round. We speculate that this heterogeneity may reflect a relaxation of cell cycle checkpoints, possibly increasing the ability of slow-growing mycobacteria to adapt to environmental conditions.
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choreography of the mycobacterium Replication machinery during the cell cycle
Mbio, 2015Co-Authors: Damian Trojanowski, Patrycja Skut, Dagmara Jakimowicz, Jolanta Zakrzewskaczerwinska, Joanna Holowka, Katarzyna Ginda, Monika PioroAbstract:ABSTRACT It has recently been demonstrated that bacterial Chromosomes are highly organized, with specific positioning of the Replication initiation region. Moreover, the positioning of the Replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of Chromosome Replication in relation to the cell cycle, as well as cell division and Chromosome segregation events, revealed that chromosomal origin-of-Replication (oriC) regions segregate soon after the start of Replication. Moreover, our data demonstrate that organization of the Chromosome by ParB determines the replisome choreography. IMPORTANCE Despite significant progress in elucidating the basic processes of bacterial Chromosome Replication and segregation, understanding of Chromosome dynamics during the mycobacterial cell cycle remains incomplete. Here, we provide in vivo experimental evidence that replisomes in Mycobacterium smegmatis are highly dynamic, frequently splitting into two distinct Replication forks. However, unlike in Escherichia coli, the forks do not segregate toward opposite cell poles but remain in relatively close proximity. In addition, we show that Replication cycles do not overlap. Finally, our data suggest that ParB participates in the positioning of newly born replisomes in M. smegmatis cells. The present results broaden our understanding of Chromosome segregation in slow-growing bacteria. In view of the complexity of the mycobacterial cell cycle, especially for pathogenic representatives of the genus, understanding the mechanisms and factors that affect Chromosome dynamics will facilitate the identification of novel antimicrobial factors.
Gregory T. Marczynski - One of the best experts on this subject based on the ideXlab platform.
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Crosstalk Regulation Between Bacterial Chromosome Replication and Chromosome Partitioning
Frontiers Media S.A., 2019Co-Authors: Gregory T. Marczynski, Kenny Petit, Priya PatelAbstract: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
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a novel dna binding protein coordinates asymmetric Chromosome Replication and Chromosome partitioning
bioRxiv, 2016Co-Authors: James A Taylor, Gael Panis, Patrick H Viollier, Gregory T. MarczynskiAbstract:Bacterial Chromosome Replication is regulated from a single Replication origin (ori) that receives cell cycle signals. Following Replication, bacteria often use the parABS partition system with a centromere-like parS locus to place the Chromosomes into the daughter cells. Our knowledge of cell cycle regulation is incomplete and we searched for novel regulators of Chromosome Replication. Here we show that in the cell cycle model Caulobacter crescentus a novel DNA-binding protein promotes both the initiation of Chromosome Replication and the earliest step of Chromosome partitioning. We used biochemical fractionation to identify a protein (OpaA) that preferentially binds to mutated ori DNA that also increases ori-plasmid Replication in vivo. OpaA represents a previously unknown class of DNA-binding proteins. opaA gene expression is essential and sufficient OpaA levels are required for the correct timing of Chromosome Replication. Whole genome ChIP-seq identified the genomic binding sites for OpaA, with the strongest associations at the parABS locus near ori. Using molecular-genetic and fluorescence microscopy experiments, we showed that OpaA also promotes the first step of Chromosome partitioning, the initial separation of the duplicated parS loci following ori Replication. This separation occurs before the parABS mechanism and it coincides with the regulatory step that splits the symmetry of the Chromosomes so that they are placed at distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that OpaA coordinates Replication with the poorly understood mechanism of early Chromosome separation. opaA lethal suppressor and antibiotic experiments argue that future studies be focused on the mechanistic roles for transcription and translation at this critical step of the cell cycle.
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ctra response regulator binding to the caulobacter Chromosome Replication origin is required during nutrient and antibiotic stress as well as during cell cycle progression
Molecular Microbiology, 2009Co-Authors: Patrick D Bastedo, Gregory T. MarczynskiAbstract:The Caulobacter crescentus Chromosome Replication origin (Cori) has five binding sites for CtrA, an OmpR/PhoB family 'response regulator'. CtrA is degraded in replicating 'stalked' cells but is abundant in the non-replicating 'swarmer' cells, where it was proposed to repress Replication by binding to Cori. We systematically mutated all Cori CtrA binding sites, and examined their consequences in the contexts of autonomous Cori-plasmid Replication and in the natural Chromosome locus. Remarkably, the C. crescentus Chromosome tolerates severe mutations in all five CtrA binding sites, demonstrating that CtrA is not essential for Replication. Further physiological and cell cycle experiments more rigorously supported the original hypothesis that CtrA represses Replication. However, our experiments argued against another hypothesis that residual and/or replenished CtrA protein in stalked cells might prevent extra or unscheduled Chromosome Replication before cell division. Surprisingly, we also demonstrated that Cori CtrA binding sites are very advantageous and can become essential when cells encounter nutrients and antibiotics. Therefore, the CtrA cell cycle regulator co-ordinates Replication with viable cell growth in stressful and rapidly changing environments. We argue that this new role for CtrA provided the primary selective pressure for evolving control by CtrA.
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regulated degradation of Chromosome Replication proteins dnaa and ctra in caulobacter crescentus
Molecular Microbiology, 2004Co-Authors: Boris Gorbatyuk, Gregory T. MarczynskiAbstract:DnaA protein binds bacterial Replication origins and it initiates Chromosome Replication. The Caulobacter crescentus DnaA also initiates Chromosome Replication and the C. crescentus response regulator CtrA represses Chromosome Replication. CtrA proteolysis by ClpXP helps restrict Chromosome Replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each Replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.
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a dual binding site for integration host factor and the response regulator ctra inside the caulobacter crescentus Replication origin
Journal of Bacteriology, 2003Co-Authors: Rania Siam, Ann Karen C Brassinga, Gregory T. MarczynskiAbstract:The response regulator CtrA controls Chromosome Replication by binding to five sites, a, b, c, d, and e, inside the Caulobacter crescentus Replication origin (Cori). In this study, we demonstrate that integration host factor (IHF) binds Cori over the central CtrA binding site c. Surprisingly, IHF and CtrA share DNA recognition sequences. Rather than promoting cooperative binding, IHF binding hinders CtrA binding to site c and nearby site d. Unlike other CtrA binding sites, DNA mutations in the CtrA c/IHF site uniquely impair autonomous Cori plasmid Replication. These mutations also alter transcription from distant promoters more than 100 bp away. When the CtrA c/IHF site was deleted from the Chromosome, these cells grew slowly and became selectively intolerant to a CtrA phosphor-mimic allele (D51E). Since CtrA protein concentration decreases during the cell cycle as IHF protein concentration increases, we propose a model in which IHF displaces CtrA in order to bend Cori and promote efficient Chromosome Replication.