Chromosome Organization and Replisome Dynamics in Mycobacterium smegmatis

Santi, Isabella; McKinney, John D.

doi:10.1128/mbio.01999-14

Cited by 61 publications

(92 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The observations thus obtained show that, unlike what has been claimed in several recent investigations (34)(35)(36), multifork replication occurs in M. smegmatis. Multifork replication is observed in bacteria in such cases where the mass doubling time is less than the sum of their chromosomal replication and cell division times, the C and D periods, respectively (37,38).…”

Section: Resultscontrasting

confidence: 70%

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

et al. 2016

View full text Add to dashboard Cite

Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc 2 155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ⌬DRKIN, a strain derived from mc 2 155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ⌬DRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2. IMPORTANCEMycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state. Members of the Y family DNA polymerases are capable of translesion synthesis (TLS) that allows them to catalyze the insertion of deoxyribonucleotide triphosphates (dNTPs) opposite potentially lethal replication-blocking lesions (1). The ability of these polymerases to bypass such lesions helps the cell to survive under DNA-damaging conditions. However, survival comes at a cost. Mutations are introduced more frequently than under normal conditions, as these polymerases function in an error-prone manner (2). In Escherichia coli, DinP/DinB (DNA polymerase IV [Pol IV]) and UmuC (DNA Pol V) are the two Y family polymerases that mediate TLS (2). Mutations in the genes that encode UmuC and a related protein, UmuD, result in a UV-nonmutable (umu) phenotype. In contrast, mutation of dinB, the gene that codes for the Pol IV enzyme DinB, does not lead to an apparent phenotype, and therefore, the function of this protein remains enigmatic. The expression of the E. col...

show abstract

Section: Resultscontrasting

confidence: 70%

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Using time-lapse microscopy, we observed one to two SSB-GFP foci during DNA replication (“C”) but none during pre- (“B”) and post- (“D”) replication periods (Figure 1H). We and others have observed M. smegmatis subpopulations that begin new rounds of replication after completing the first round but before division [10, 12, 17, 19]. We identified this additional replication period (“E”) in 54% of cells by observing SSB-foci near poles pre-division (Figures 1H, 1I&S1P).…”

Section: Resultsmentioning

confidence: 80%

“…Chromosome subcellular positioning is highly organized yet variable among bacterial species [2, 9, 10]. In E. coli, the origin is located midcell with left and right chromosome arms symmetrically positioned on either side [2, 11].…”

Section: Resultsmentioning

confidence: 99%

“…The ter translocation event from the new pole to midcell is rapid, typically completing in 15–30 minutes (Figures 1F&S1L; Movie S2). The distal attB chromosome site (245° from ori) was also reported to translocate rapidly to the replisome, indicating that the replisome “reels in” chromosomal loci before they are replicated [10]. Ter positioning is approximately proportional to cell length at birth and division (Figures 1G, S1G–J).…”

Section: Resultsmentioning

confidence: 99%

“…The ori and ter are positioned closer to the new pole before division so asymmetric division gives rise to identical chromosome locations in unequally sized daughter cells (Figure 1J). M. smegmatis chromosome subcellular organization has been previously explored, however orientation and symmetry conclusions were inconsistent between studies [10, 13, 14]. Our finding of ori-ter-ter-ori orientation is similar to the longitudinal pattern of chromosome organization of C. crescentus and M. xanthus [9, 16] and reported for M. smegmatis [14], but unlike the L-ori-R chromosome organization and midcell localization of E. coli [2] also suggested for M. smegmatis [10, 13].…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

A Parallel Adder Coordinates Mycobacterial Cell-Cycle Progression and Cell-Size Homeostasis in the Context of Asymmetric Growth and Organization

et al. 2017

View full text Add to dashboard Cite

Summary In model bacteria, such as E. coli and B. subtilis, regulation of cell cycle progression and cellular organization achieves consistency in cell size, replication dynamics, and chromosome positioning [1–3]. Mycobacteria elongate and divide asymmetrically, giving rise to significant variation in cell size and elongation rate among closely related cells [4, 5]. Given the physical asymmetry of mycobacteria, the models that describe coordination of cellular organization and cell cycle progression in model bacteria are not directly translatable [1, 2, 6–8]. Here we used time-lapse microscopy and fluorescent reporters of DNA replication and chromosome positioning to examine the coordination of growth, division, and chromosome dynamics at a single-cell level in Mycobacterium smegmatis (M. smegmatis) and Mycobacterium bovis Bacillus Calmette–Guérin (BCG). By analyzing chromosome and replisome localization, we demonstrated that chromosome positioning is asymmetric and proportional to cell size. Furthermore, we found that cellular asymmetry is maintained throughout the cell cycle and is not established at division. Using measurements and stochastic modeling of mycobacterial cell size and cycle timing in both slow and fast growth conditions, we found that well-studied cell size control models are insufficient to explain the mycobacterial cell cycle. Instead, we showed that mycobacterial cell cycle progression is regulated by an unprecedented mechanism involving parallel adders (i.e. constant growth increments) that start at replication initiation. Together, these adders enable mycobacterial populations to regulate cell size, growth, and heterogeneity in the face of varying environments.

show abstract

Lessons from a Minimal Genome: What Are the Essential Organizing Principles of a Cell Built from Scratch?

et al. 2019

View full text Add to dashboard Cite

One of the primary challenges facing synthetic biology is reconstituting a living system from its component parts. A particularly difficult landmark is reconstituting a self‐organizing system that can undergo autonomous chromosome compaction, segregation, and cell division. Here, we discuss how the syn3.0 minimal genome can inform us of the core self‐organizing principles of a living cell and how these self‐organizing processes can be built from the bottom up. The review underscores the importance of fundamental biology in rebuilding life from its molecular constituents.

show abstract

Chromosome Organization and Replisome Dynamics in Mycobacterium smegmatis

Cited by 61 publications

References 50 publications

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

A Parallel Adder Coordinates Mycobacterial Cell-Cycle Progression and Cell-Size Homeostasis in the Context of Asymmetric Growth and Organization

Lessons from a Minimal Genome: What Are the Essential Organizing Principles of a Cell Built from Scratch?

Contact Info

Product

Resources

About