Chromosome Organization and Replisome Dynamics in Mycobacterium smegmatis

Subcellular organization of the bacterial nucleoid and spatiotemporal dynamics of DNA replication and segregation have been studied intensively, but the functional link between these processes remains poorly understood. Here we use quantitative time-lapse fluorescence microscopy for single-cell analysis of chromosome organization and DNA replisome dynamics in Mycobacterium smegmatis. We report that DNA replication takes place near midcell, where, following assembly of the replisome on the replication origin, the left and right replication forks colocalize throughout the replication cycle. From its initial position near the cell pole, a fluorescently tagged chromosomal locus (attB, 245 degrees from the origin) moves rapidly to the replisome complex just before it is replicated. The newly duplicated attB loci then segregate to mirror-symmetric positions relative to midcell. Genetic ablation of ParB, a component of the ParABS chromosome segregation system, causes marked defects in chromosome organization, condensation, and segregation. ParB deficiency also results in mislocalization of the DNA replication machinery and SMC (structural maintenance of chromosome) protein. These observations suggest that ParB and SMC play important and overlapping roles in chromosome organization and replisome dynamics in mycobacteria. IMPORTANCE We studied the spatiotemporal organization of the chromosome and DNA replication machinery in Mycobacterium smegmatis, a fast-growing relative of the human pathogen Mycobacterium tuberculosis. We show that genetic ablation of the DNA-binding proteins ParB and SMC disturbs the organization of the chromosome and causes a severe defect in subcellular localization and movement of the DNA replication complexes. These observations suggest that ParB and SMC provide a functional link between chromosome organization and DNA replication dynamics. This work also reveals important differences in the biological roles of the ParABS and SMC systems in mycobacteria versus better-characterized model organisms, such as Escherichia coli and Bacillus subtilis.

Single-cell studies of Mycobacterium smegmatis cell cycle using time-lapse fluorescence microscopy

Joëlle Xiao Yuan Ven

Cell division is one of the most primordial cellular processes and is essential for life propagation. It is composed of crucial events, namely genome duplication, chromosomes segregation and membrane separation. Failure of these processes will have dramatic impacts on the development of any organisms, including cell death, polyploidy, or cell cycle arrest. In the case of bacterial pathogens, blocking cell division efficiently using targeted drugs is of interest to cure bacterial infections. However, despite its ancestral origin, the mechanisms of cell division have diverged amongst organisms. Given its crucial role, as well as the potential therapeutic applications, cell division is an intensive field of research and many aspects are yet to be fully understood. Amongst bacteria, the phyla Proteobacteria and Firmicutes have been intensively studied using their respective model organisms Escherichia coli and Bacillus subtilis. However, there is a paucity of research on cell division in Actinobacteria, the phylum that contains the worldâs leading pathogen, Mycobacterium tuberculosis. In particular, the molecular players involved in the process of chromosome segregation and the timing of cell division have not been fully investigated. In the well-studied bacteria E. coli and B. subtilis the Min system determines the division site localization, while the nucleoid occlusion system (Noc or SlmA, respectively) determines the timing of division. In mycobacteria, however, no homologs for the Min or Noc systems have been identified thus far. However, the partitioning (par) genes are currently thought to be responsible for symmetric chromosome segregation. In addition, FtsK has been identified as a homolog of the B. subtilis protein SpoIIIE, which is responsible for rescuing DNA in the forming septum and pulling it in the forespore during starvation-induced sporulation. Overall, the role and dynamics of DNA, where FtsK might play a role, during mycobacterial cell division, still remain to be uncovered. Current methods to study DNA localization using snapshot microscopy typically utilise DNA dyes, such as DAPI, Hoechst, or SYTO. However, these compounds impair cell growth, preventing the dynamic study of mycobacterial division. To circumvent this shortcoming, I constructed a Mycobacterium smegmatis reporter strain expressing a Dendra2 fusion protein in-frame to the histone-like protein Hlp, and compared it to conventional DNA dyes. Additionally, I characterized FtsK and, for the first time, demonstrated its essentiality in M. smegmatis. As FtsK is essential I adapted a dual repression system to knock down FtsK, which proved to be a synthetic rescue with the par genes deletion. Using a custom-made microfluidic device and long-term time-lapse microscopy, the distribution and dynamics of both Hlp and FtsK fluorescent reporter proteins were followed at the single-cell level. This approach allowed the spatial and temporal characterization of these proteins with respect to other septal genes in wild-type cells as well as in Îpar mutants. Finally, I propose a new model where the par genes are important for chromosome orientation and FtsK is important for chromosome segregation. Altogether, this thesis represents a step forward towards a better understanding of the mechanisms underlying cell division in mycobacteria and in the long-term, a potential source of novel treatments to control the division of M. tuberculosis, for instance by targeting FtsK.

EPFL2019

The Phosphate Specific Transport (Pst) System in Fast- and Slow-Growing Mycobacteria

Cyntia De Piano

Although mycobacteria are known to accumulate polyphosphate (PolyP), a linear polymer of orthophosphate, when subjected to stresses, the exact roles of this molecule are not yet clearly understood. We found that deletion of the pstA gene encoding for a transmembrane domain of the high-affinity phosphate-specific ABC transporter system (Pst system) resulted in accumulation of high amounts of PolyP during growth in phosphate (Pi)-replete conditions in Mycobacterium tuberculosis and Mycobacterium smegmatis. These PolyP stores were gradually used during Pi starvation by the ApstA mutants in order to sustain growth and this allowed the mutant strains to reach a higher cell density than the wild-type (WT) parental strain. Neither the mRNA nor the protein levels of polyphosphate kinase 1 (PPK1) could explain the PolyP accumulation observed in the mutant, suggesting that another regulatory mechanism is involved. The ApstA mutant of M. smegmatis was hypersensitive to several in vitro stresses, including detergent, oxidative stress, and cell wall targeting antibiotics. Deletion of the main enzyme involved in PolyP synthesis, PPK1, in the ApstA mutant background of M. smegmatis completely reversed the PolyP accumulation and partially reversed the stress sensitivity as well as the derepression of PHO regulon genes observed in the ApstA mutant. The mechanism of PolyP accumulation in M. smegmatis was investigated using a genetic approach. We found that this mechanism differs from the (p)ppGpp-mediated PolyP accumulation observed in Escherichia coli, since deletion of the RelA enzyme in a ApstA mutant background did not prevent PolyP accumulation. The mechanism of PolyP degradation during Pi starvation in the M. smegmatis ApstA mutant was also analyzed. Deletion of both polyphosphatase enzymes (PPX1 and PPX2) as well as the polyphosphate kinase 2 (PPK2) enzyme did not have any impact on PolyP degradation. PPK1 is a bifunctional enzyme that can either synthesize or degrade PolyP. Our current hypothesis is that PPK1 might perform the reverse reaction (PolyP degradation) during Pi starvation. The aim of the second part of my project was to make a comparative study of the expression and regulation of the phosphate specific transporter (pst) operon in fast-growing [M. smegmatis] and slow-growing [M. tuberculosis] mycobacteria. To achieve this goal, pst reporter strains of M. smegmatis and M. tuberculosis were constructed by precise insertion of a modified dest_gfp gene (provided by Giulia Manina) encoding a destabilized green fluorescent protein (dest_GFP) into the corresponding pst locus as transcriptional ) fusions. The transcriptional reporter construct [p) was introduced in both the WT and the ApstA mutant backgrounds. The kinetics of induction of the pst-gfp reporters in WT and ApstA mutant strains in response to Pi starvation was analyzed. Population-averaged measurements were made using qRT-PCR and Western blotting. Single-cell measurements using flow cytometry and timelapse fluorescence microscopy were also performed. For the microscopy experiments, the bacteria were cultured in custom-made microfluidic devices that permit rapid switching of the culture medium, e.g., switching between Pi-replete and Pi-depleted media. Because these studies require precise environmental control and resolution of individual and temporal phenotypes, my principal experimental tool was the integrated microfluidics and timelapse fluorescence microscopy systems that have recently been developed in the laboratory. Although there have been many studies published on the population-averaged behavior of bacteria responding to external stresses such as Pi limitation, we are not aware of any studies addressing the individuality of such responses using a real-time single-cell approach. We observed that the kinetics of induction of the Pst operon upon Pi starvation scaled with the growth rate of the bacteria and peak induction occurred within about two generations for both smegmatis and tuberculosis. Interestingly, we found that the basal level of Pst expression and the steady state level reached upon induction by Pi starvation were similar in tuberculosis and M. smegmatis, although the kinetics of induction were much faster in M. smegmatis. These studies contributed to improve the understanding of the regulation and the kinetics of response of the PHO regulon in fast- and slow-growing mycobacteria. The finding that PolyP plays a role in this process could also apply to other organisms where the PHO regulon is regulated in a similar way.

EPFL2013

The studies of ParA and ParB dynamics reveal asymmetry of chromosome segregation in mycobacteria

Djenet Bousbaine, John McKinney, Isabella Santi

Active segregation of bacterial chromosomes usually involves the action of ParB proteins, which bind in proximity of chromosomal origin (oriC) regions forming nucleoprotein complexes - segrosomes. Newly duplicated segrosomes are moved either uni- or bidirectionally by the action of ATPases - ParA proteins. In Mycobacterium smegmatis the oriC region is located in an off-centred position and newly replicated segrosomes are segregated towards cell poles. The elimination of M. smegmatis ParA and/or ParB leads to chromosome segregation defects. Here, we took advantage of microfluidic time-lapse fluorescent microscopy to address the question of ParA and ParB dynamics in M. smegmatis and M. tuberculosis cells. Our results reveal that ParB complexes are segregated in an asymmetrical manner. The rapid movement of segrosomes is dependent on ParA that is transiently associated with the new pole. Remarkably in M. tuberculosis, the movement of the ParB complex is much slower than in M. smegmatis, but segregation as in M. smegmatis lasts approximately 10% of the cell cycle, which suggests a correlation between segregation dynamics and the growth rate. On the basis of our results, we propose a model for the asymmetric action of segregation machinery that reflects unequal division and growth of mycobacterial cells.