Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria: Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern

ABSTRACT Among actinomycetes, chromosome organization and segregation studies have been limited to Streptomyces coelicolor, Corynebacterium glutamicum, and Mycobacterium spp. There are differences with respect to ploidy and chromosome organization pattern in these bacteria. Here, we report on chromosome replication, organization, and segregation in Rhodococcus erythropolis PR4, which has a circular genome of 6.5 Mbp. The origin of replication of R. erythropolis PR4 was identified, and the DNA content in the cell under different growth conditions was determined. Our results suggest that the number of origins increases as the growth medium becomes rich, suggesting an overlapping replication cell cycle in this bacterium. Subcellular localization of the origin region revealed polar positioning in minimal and rich media. The terminus, which is the last region to be replicated and segregated, was found to be localized at the cell center in large cells. The middle markers corresponding to the 1.5-Mb and 4.7-Mb loci did not overlap, suggesting discontinuity in the segregation of the two arms of the chromosome. Chromosome segregation was not affected by inhibiting cell division. Deletion of parA or parB affected chromosome segregation. Unlike in C. glutamicum and Streptomyces spp., diploidy or polyploidy was not observed in R. erythropolis PR4. Our results suggest that R. erythropolis is different from other members of Actinobacteria; it is monoploid and has a unique chromosome segregation pattern. This is the first report on chromosome organization, replication, and segregation in R. erythropolis PR4. IMPORTANCE Rhodococci are highly versatile Gram-positive bacteria with high bioremediation potential. Some rhodococci are pathogenic and have been suggested as emerging threats. No studies on the replication, segregation, and cell cycle of these bacteria have been reported. Here, we demonstrate that the genus Rhodococcus is different from other actinomycetes, such as members of the genera Corynebacterium, Mycobacterium, and Streptomyces, with respect to ploidy and chromosome organization and segregation. Such studies will be useful not only in designing better therapeutics pathogenic strains in the future but also for studying genome maintenance in strains used for bioremediation.

Download Full-text

Chromosome segregation in B. subtilis is highly heterogeneous

BMC Research Notes ◽

10.1186/s13104-020-05322-9 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Nina El Najjar ◽

Peter L. Graumann

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Chromosome Segregation ◽

Growth Rates ◽

Slow Growth ◽

Growth Conditions ◽

Single Chromosome ◽

Copy Numbers ◽

Initiation Of Replication ◽

Bacterial Cell Cycle

Abstract Objective The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level. Results B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

Download Full-text

Chromosome Dynamics in Bacteria: Triggering Replication at the Opposite Location and Segregation in the Opposite Direction

mBio ◽

10.1128/mbio.01002-19 ◽

2019 ◽

Vol 10 (4) ◽

Author(s):

Ady B. Meléndez ◽

Inoka P. Menikpurage ◽

Paola E. Mera

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Opposite Direction ◽

Caulobacter Crescentus ◽

Chromosome Replication ◽

Opposite Orientation ◽

Circular Chromosome ◽

Content Type ◽

Cell Pole ◽

Protein Gradients

ABSTRACT Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell. IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

Download Full-text

Novel chromosome organization pattern in actinomycetales–overlapping replication cycles combined with diploidy

10.1101/094169 ◽

2016 ◽

Author(s):

Kati Böhm ◽

Fabian Meyer ◽

Agata Rhomberg ◽

Jörn Kalinowski ◽

Catriona Donovan ◽

...

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Chromosome Organization ◽

Model Species ◽

Chromosomal Organization ◽

Dna Segregation ◽

Rate Dependent ◽

Dependent Cell ◽

Underlying Mechanisms ◽

Cell Cycle Models

AbstractBacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacteriumCorynebacterium glutamicumorganizes chromosome segregation and DNA replication. Unexpectedly, we find thatC. glutamicumcells are at least diploid under all conditions tested and that these organisms have overlapping C-periods during replication with both origins initiating replication simultaneously. Based on experimentally obtained data we propose growth rate dependent cell cycle models forC. glutamicum.

Download Full-text

Chromosome segregation in B. subtilis is highly heterogeneous

10.21203/rs.3.rs-57280/v1 ◽

2020 ◽

Author(s):

Nina El Najjar ◽

Peter Graumann

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Chromosome Segregation ◽

Growth Rates ◽

Slow Growth ◽

Growth Conditions ◽

Single Chromosome ◽

Copy Numbers ◽

Initiation Of Replication ◽

Bacterial Cell Cycle

Download Full-text

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by Soj/ParA during growth and sporulation

10.1101/2021.12.18.473321 ◽

2021 ◽

Author(s):

David M Roberts ◽

Anna Anchimiuk ◽

Tomas G Kloosterman ◽

Heath Murray ◽

Ling Juan Wu ◽

...

Keyword(s):

Cell Cycle ◽

Bacillus Subtilis ◽

Chromosome Segregation ◽

Chromosome Structure ◽

Replication Initiation ◽

Chromosome Organization ◽

Filament Formation ◽

Axial Filament ◽

Dna Replication Initiation ◽

The Web

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving ATP-dependent dimerization and DNA binding, leading to chromosome segregation and SMC loading. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation, and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. We now show that a major redistribution of SMC complexes drives axial filament formation, in a process regulated by ParA/Soj. Unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyse ATP. These results reveal a new role for ParA/Soj proteins in the regulation of SMC dynamics in bacteria, and yet further complexity in the web of interactions involving chromosome replication, segregation, and organization, controlled by ParAB and SMC.

Download Full-text

Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

mBio ◽

10.1128/mbio.02125-14 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 39

Author(s):

Damian Trojanowski ◽

Katarzyna Ginda ◽

Monika Pióro ◽

Joanna Hołówka ◽

Partycja Skut ◽

...

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Mycobacterium Smegmatis ◽

Chromosome Replication ◽

Specific Cell ◽

Replication Machinery ◽

Content Type ◽

Chromosome Dynamics ◽

Mycobacterial Cell ◽

Slow Growing

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.

Download Full-text

Chromosome segregation in B. subtilis is highly heterogeneous

10.21203/rs.3.rs-57280/v2 ◽

2020 ◽

Author(s):

Nina El Najjar ◽

Peter Graumann

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Chromosome Segregation ◽

Growth Rates ◽

Slow Growth ◽

Growth Conditions ◽

Single Chromosome ◽

Copy Numbers ◽

Initiation Of Replication ◽

Bacterial Cell Cycle

Abstract Objective: The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level.Results: B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

Download Full-text

The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

Eukaryotic Cell ◽

10.1128/ec.05093-11 ◽

2011 ◽

Vol 10 (10) ◽

pp. 1295-1305 ◽

Cited By ~ 27

Author(s):

Jitendra Thakur ◽

Kaustuv Sanyal

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Candida Albicans ◽

Fission Yeast ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Mitotic Chromosome ◽

Content Type ◽

Spindle Elongation ◽

Dam1 Complex

ABSTRACTA fungus-specific outer kinetochore complex, the Dam1 complex, is essential inSaccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. BothCandida albicansandSchizosaccharomyces pombehave regional centromeres as opposed to the short-point centromeres ofS. cerevisiae. The interaction of one microtubule per kinetochore is established both inS. cerevisiaeandC. albicansearly during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis inS. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle inS. cerevisiaeandC. albicansbut only during mitosis inS. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation inC. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochore-microtubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.

Download Full-text

Chromosome Segregation in Bacillus subtilis Follows an Overall Pattern of Linear Movement and Is Highly Robust against Cell Cycle Perturbations

mSphere ◽

10.1128/msphere.00255-20 ◽

2020 ◽

Vol 5 (3) ◽

Cited By ~ 2

Author(s):

Nina El Najjar ◽

David Geisel ◽

Felix Schmidt ◽

Simon Dersch ◽

Benjamin Mayer ◽

...

Keyword(s):

Cell Cycle ◽

Bacillus Subtilis ◽

Chromosome Segregation ◽

Large Fraction ◽

Directed Motion ◽

Content Type ◽

Linear Movement ◽

Cell Cycle Perturbation ◽

Random Patterns

ABSTRACT Although several proteins have been identified that facilitate chromosome segregation in bacteria, no clear analogue of the mitotic machinery in eukaryotic cells has been identified. In order to investigate if recognizable patterns of segregation exist during the cell cycle, we tracked the segregation of duplicated origin regions in Bacillus subtilis for 60 min in the fastest practically achievable resolution, achieving 10-s intervals. We found that while separation occurred in random patterns, often including backwards movement, overall, segregation of loci near the origins of replication was linear for the entire cell cycle. Thus, the process of partitioning can be best described as directed motion. Simulations with entropy-driven separation of polymers synthesized by two polymerases show sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, showing that for Bacillus, segregation patterns can be modeled based on entropic forces. To test if obstacles for replication forks lead to an alteration of the partitioning pattern, we challenged cells with chemicals inducing DNA damage or blocking of topoisomerase activity. Both treatments led to a moderate slowing down of separation, but linear segregation was retained, showing that chromosome segregation is highly robust against cell cycle perturbation. IMPORTANCE We have followed the segregation of origin regions on the Bacillus subtilis chromosome in the fastest practically achievable temporal manner, for a large fraction of the cell cycle. We show that segregation occurred in highly variable patterns but overall in an almost linear manner throughout the cell cycle. Segregation was slowed down, but not arrested, by treatment of cells that led to transient blocks in DNA replication, showing that segregation is highly robust against cell cycle perturbation. Computer simulations based on entropy-driven separation of newly synthesized DNA polymers can recapitulate sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, indicating that for Bacillus, segregation patterns may include entropic forces helping to separate chromosomes during the cell cycle.

Download Full-text

CytochromebdOxidase Has an Important Role in Sustaining Growth and Development ofStreptomyces coelicolorA3(2) under Oxygen-Limiting Conditions

Journal of Bacteriology ◽

10.1128/jb.00239-18 ◽

2018 ◽

Vol 200 (16) ◽

Cited By ~ 4

Author(s):

Marco Fischer ◽

Dörte Falke ◽

Carolin Naujoks ◽

R. Gary Sawers

Keyword(s):

Streptomyces Coelicolor ◽

Reduction Rate ◽

Growth Conditions ◽

Developmental Cycle ◽

Wild Type ◽

Mycelium Growth ◽

Developmental Defects ◽

Content Type ◽

Mycelia Growth ◽

Genes Encoding

ABSTRACTStreptomyces coelicolorA3(2) is a filamentously growing, spore-forming, obligately aerobic actinobacterium that uses both a copperaa3-type cytochromecoxidase and a cytochromebdoxidase to respire oxygen. Using defined knockout mutants, we demonstrated that either of these terminal oxidases was capable of allowing the bacterium to grow and complete its developmental cycle. The genes encoding thebcccomplex and theaa3oxidase are clustered at a single locus. Using Western blot analyses, we showed that thebcc-aa3oxidase branch is more prevalent in spores than thebdoxidase. The level of the catalytic subunit, CydA, of thebdoxidase was low in spore extracts derived from the wild type, but it was upregulated in a mutant lacking thebcc-aa3supercomplex. This indicates that cytochromebdoxidase can compensate for the lack of the other respiratory branch. Components of both oxidases were abundant in growing mycelium. Growth studies in liquid medium revealed that a mutant lacking thebcc-aa3oxidase branch grew approximately half as fast as the wild type, while the oxygen reduction rate of the mutant remained close to that of the wild type, indicating that thebdoxidase was mainly functioning in controlling electron flux. Developmental defects were observed for a mutant lacking the cytochromebdoxidase during growth on buffered rich medium plates with glucose as the energy substrate. Evidence based on using the redox-cycling dye methylene blue suggested that cytochromebdoxidase is essential for the bacterium to grow and complete its developmental cycle under oxygen limitation.IMPORTANCERespiring with oxygen is an efficient means of conserving energy in biological systems. The spore-forming, filamentous actinobacteriumStreptomyces coelicolorgrows only aerobically, synthesizing two enzyme complexes for O2reduction, the cytochromebcc-aa3cytochrome oxidase supercomplex and the cytochromebdoxidase. We show in this study that the bacterium can survive with either of these respiratory pathways to oxygen. Immunological studies indicate that thebcc-aa3oxidase is the main oxidase present in spores, but thebdoxidase compensates if thebcc-aa3oxidase is inactivated. Both oxidases are active in mycelia. Growth conditions were identified, revealing that cytochromebdoxidase is essential for aerial hypha formation and sporulation, and this was linked to an important role of the enzyme under oxygen-limiting conditions.

Download Full-text