live-cell-imaging-life-cycle-bacterial-predator-bdellovibrio

Live-Cell Imaging of the Life Cycle of Bacterial Predator <em>Bdellovibrio bacteriovorus</em> using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...

University of Wroclaw

Sublimons are substoichiometric DNA molecules which are generated by recombinations across short repeats, located in main mitochondrial genome of plants. Since short repeats are believed to recombine irreversibly and to be usually inactive, it is unknown how substoichiometric sequences are maintained. Occasionally, sublimons are amplified during substoichiometric shifting (SSS) and take the role of the main genome. Using the Phaseolus vulgaris system, we have addressed the questions concerning accumulation of sublimons, the role of recombination in their maintenance and selective amplification during SSS. We found that sublimons accompanied by parental recombination sequences were maintained by constant recombination across a short 314-bp repeat. The abundance of these sublimons was three orders of magnitude higher than accumulation of those which could not be maintained by recombination because their parental forms were absent from the main genome. As expected for active recombination, two recombination-derived sublimons were equimolar and so were their parental forms. One parental and one substoichiometric form shared the A/C polymorphism indicating their frequent inter-conversion. Only the C variant of the sublimon was amplified during substoichiometric shift implying strong selection of DNA molecules operating during SSS.

Counting mtDNA molecules in Phaseolus vulgaris: sublimons are constantly produced by recombination via short repeats and undergo rigorous selection during substoichiometric shifting.

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.

Choreography of the Mycobacterium replication machinery during the cell cycle.

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.

Multifork chromosome replication in slow-growing bacteria.

In bacteria, chromosomal DNA must be efficiently compacted to fit inside the small cell compartment while remaining available for the proteins involved in replication, segregation, and transcription. Among the nucleoid-associated proteins (NAPs) responsible for maintaining this highly organized and yet dynamic chromosome structure, the HU protein is one of the most conserved and highly abundant. HupB, a homologue of HU, was recently identified in mycobacteria. This intriguing mycobacterial NAP is composed of two domains: an N-terminal domain that resembles bacterial HU, and a long and distinctive C-terminal domain that contains several PAKK/KAAK motifs, which are characteristic of the H1/H5 family of eukaryotic histones. In this study, we analyzed the  binding of HupB on the chromosome scale. By using PALM (photoactivated localization microscopy) and ChIP-Seq (chromatin immunoprecipitation followed by deep sequencing), we observed that the C-terminal domain is indispensable for the association of HupB with the nucleoid. Strikingly, the  binding of HupB displayed a bias from the origin () to the terminus () of the mycobacterial chromosome (numbers of binding sites decreased toward ). We hypothesized that this binding mode reflects a role for HupB in organizing newly replicated  regions. Thus, HupB may be involved in coordinating replication with chromosome segregation. We currently know little about the organization of the mycobacterial chromosome and its dynamics during the cell cycle. Among the mycobacterial nucleoid-associated proteins (NAPs) responsible for chromosome organization and dynamics, HupB is one of the most intriguing. It contains a long and distinctive C-terminal domain that harbors several PAKK/KAAK motifs, which are characteristic of the eukaryotic histone H1/H5 proteins. The HupB protein is also known to be crucial for the survival of tubercle bacilli during infection. Here, we provide  experimental evidence showing that the C-terminal domain of HupB is crucial for its DNA binding. Our results suggest that HupB may be involved in organizing newly replicated regions and could help coordinate chromosome replication with segregation. Given that tuberculosis (TB) remains a serious worldwide health problem (10.4 million new TB cases were diagnosed in 2015, according to WHO) and new multidrug-resistant  strains are continually emerging, further studies of the biological function of HupB are needed to determine if this protein could be a prospect for novel antimicrobial drug development.

HupB Is a Bacterial Nucleoid-Associated Protein with an Indispensable Eukaryotic-Like Tail.

The bacterial chromosome undergoes dynamic changes in response to ongoing cellular processes and adaptation to environmental conditions. Among the many proteins involved in maintaining this dynamism, the most abundant is the nucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, possesses an additional C-terminal domain that resembles that of eukaryotic histones H1/H5. Recently, we demonstrated that the highly abundant HupB protein occupies the entirety of the  chromosome and that the HupB-binding sites exhibit a bias from the origin () to the terminus (). In this study, we used HupB fused with enhanced green fluorescent protein (EGFP) to perform the first analysis of chromosome dynamics and to track the  and replication machinery directly on the chromosome during the mycobacterial cell cycle. We show that the chromosome is located in an off-center position that reflects the unequal division and growth of mycobacterial cells. Moreover, unlike the situation in , the sister  regions of  move asymmetrically along the mycobacterial nucleoid. Interestingly, in this slow-growing organism, the initiation of the next round of replication precedes the physical separation of sister chromosomes. Finally, we show that HupB is involved in the precise timing of replication initiation. Although our view of mycobacterial nucleoid organization has evolved considerably over time, we still know little about the dynamics of the mycobacterial nucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously suggested as a potential target for antibiotic therapy against tuberculosis. Here, we fused HupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the mycobacterial chromosome in real time and to monitor the replication process directly on the chromosome. Our results reveal that, unlike the situation in , the nucleoid of an apically growing mycobacterium is positioned asymmetrically within the cell throughout the cell cycle. We show that HupB is involved in controlling the timing of replication initiation. Since tuberculosis remains a serious health problem, studies concerning mycobacterial cell biology are of great importance.

The Origin of Chromosomal Replication Is Asymmetrically Positioned on the Mycobacterial Nucleoid, and the Timing of Its Firing Depends on HupB.

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 . 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 . 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.

Amsacrine Derivatives Selectively Inhibit Mycobacterial Topoisomerase I (TopA), Impair  Growth and Disturb Chromosome Replication.

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.

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

Bacterial chromosomes have a single, unique replication origin (named ), from which DNA synthesis starts. This study describes methods of visualizing  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.

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

 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  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  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  Our results show that in , 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  life cycle fails in some cells of , they can instead use second prey cells to complete their life cycle. New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium , which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The  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  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  This process initiates at the invasion pole of  and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of  require two prey cells sequentially to complete their life cycle.

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

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  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.).

Damian Trojanowski

Department of Molecular Microbiology,
Faculty of Biotechnology,
Department of Molecular Microbiology, Faculty of Biotechnology

University of Wroclaw

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

Damian Trojanowski

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Microbiology,Faculty of Biotechnology,Department of Molecular Microbiology, Faculty of Biotechnology

University of Wroclaw

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

Department of Molecular Microbiology,
Faculty of Biotechnology,
Department of Molecular Microbiology, Faculty of Biotechnology