DNA and origin region segregation are not affected by the transition from rod to sphere after inhibition of Escherichia coli MreB by A22

The bacterial actin homologue MreB forms a helix underneath the cytoplasmic membrane and was shown to be essential in the morphogenesis of the rod-shaped cells. Additionally, MreB was implicated to be involved in DNA segregation. However, in our hands the mreBCD deletion strain (PA340-678) grew without apparent DNA segregation defect, suggesting that the reported chromosome segregation inhibition could be caused by a temporarily effect of MreB inhibition or depletion. To assess the involvement of MreB in DNA segregation during the transition from rod to sphere, we compared the effect of A22 and the PBP2 inhibitor mecillinam on the percentage of cells with segregated nucleoids and the number of oriC foci in wild-type Escherichia coli cells. Cells became spherical in the same time window during both treatments and we could not detect any difference in the chromosome or oriC segregation between these two treatments. Additionally, flow cytometric analyses showed that A22 and mecillinam treatment gave essentially the same chromosome segregation pattern. We conclude that MreB is not directly involved in DNA segregation of E. coli.     

Distinct segregation dynamics of the two Vibrio cholerae chromosomes

The study of prokaryotic chromosome segregation has focused primarily on bacteria with single circular chromosomes. Little is known about segregation in bacteria with multipartite genomes. The human diarrhoeal pathogen Vibrio cholerae has two circular chromosomes of unequal sizes. Using static and time-lapse fluorescence microscopy, we visualized the localization and segregation of the origins of replication of the V. cholerae chromosomes. In all stages of the cell cycle, the two origins localized to distinct subcellular locations. In newborn cells, the origin of chromosome I (oriCIvc) was located near the cell pole while the origin of chromosome II (oriCIIvc) was at the cell centre. Segregation of oriCIvc occurred asymmetrically from a polar position, with one duplicated origin traversing the length of the cell towards the opposite pole and the other remaining relatively fixed. In contrast, oriCIIvc segregated later in the cell cycle than oriCIvc and the two duplicated oriCIIvc regions repositioned to the new cell centres. DAPI staining of the nucleoid demonstrated that both origin regions were localized to the edge of the visible nucleoid and that oriCIvc foci were often associated with specific nucleoid substructures. The differences in localization and timing of segregation of oriCIvc and oriCIIvc suggest that distinct mechanisms govern the segregation of the two V. cholerae chromosomes.     

Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins

Actin-like proteins MreB and Mbl are required for proper cell shape and for viability in B. subtilis and form dynamic helical filaments underneath the cell membrane. We have found that depletion of MreB and Mbl proteins leads to a rapid defect in chromosome segregation before a defect in cell shape becomes detectable. Under these conditions, the SMC chromosome segregation complex that is essential for proper chromosome arrangement and segregation loses its specific subcellular localization, and replication origins fail to localize in a regular bipolar manner as in wild type cells. Time-lapse microscopy showed that during depletion of MreB, origin regions can move towards the same cell pole, showing that bipolar orientation of origin separation is lost. Contrarily, depletion of three other cell shape determinants, MreC, MreD, or MreBH (the third B. subtilis actin homolog) had no effect on chromosome segregation but varying effects on cell morphology. Depletion of MreC and MreD resulted in formation of round cells, while depletion of MreBH led to formation of vibrio-shaped cells. The data show that actin proteins Mbl and MreB are required for proper chromosome segregation and that Mre proteins affect different aspects in cell shape.     

A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole

Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.     

Dynamic localization and interaction with other Bacillus subtilis actin-like proteins are important for the function of MreB

Bacterial actin-like proteins play a key role in cell morphology and in chromosome segregation. Many bacteria, like Bacillus subtilis, contain three genes encoding actin-like proteins, called mreB, mbl and mreBH in B. subtilis. We show that MreB and Mbl colocalize extensively within live cells, and that all three B. subtilis actin paralogues interact with each other underneath the cell membrane. A mutation in the phosphate 2 motif of MreB had a dominant negative effect on cell morphology and on chromosome segregation. Expression of this mutant allele of MreB interfered with the dynamic localization of Mbl. These experiments show that the interaction between MreB and Mbl has physiological significance. An mreB deletion strain can grow under special media conditions, however, depletion of Mbl in this mutant background abolished growth, indicating that actin paralogues can partially complement each other. The membrane protein MreC was found to interact with Mbl, but not with MreB, revealing a clear distinction between the function of the two paralogues. The phosphate 2 mutant MreB protein allowed for filament formation of mutant or wild-type MreB, but abolished the dynamic reorganization of the filaments. The latter mutation led to a strong reduction, but not complete loss, of function of MreB, both in terms of chromosome segregation and of cell morphology. Our work shows that that the dynamic localization of MreB is essential for the proper activity of the actin-like protein and that the interactions between MreB paralogues have important physiological significance.     

Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.     

MreB actin-mediated segregation of a specific region of a bacterial chromosome

Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.     

Dysfunctional MreB inhibits chromosome segregation in Escherichia coli

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape-determining factor in rod-shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild-type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB filament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreB-depleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB filaments participate in directional chromosome movement and segregation.     

Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.     

The chromosome cycle of prokaryotes

In both eukaryotes and prokaryotes, chromosomal DNA undergoes replication, condensation–decondensation and segregation, sequentially, in some fixed order. Other conditions, like sister‐chromatid cohesion (SCC), may span several chromosomal events. One set of these chromosomal transactions within a single cell cycle constitutes the ‘chromosome cycle’. For many years it was generally assumed that the prokaryotic chromosome cycle follows major phases of the eukaryotic one: –replication–condensation–segregation–(cell division)–decondensation–, with SCC of unspecified length. Eventually it became evident that, in contrast to the strictly consecutive chromosome cycle of eukaryotes, all stages of the prokaryotic chromosome cycle run concurrently. Thus, prokaryotes practice ‘progressive’ chromosome segregation separated from replication by a brief SCC, and all three transactions move along the chromosome at the same fast rate. In other words, in addition to replication forks, there are ‘segregation forks’ in prokaryotic chromosomes. Moreover, the bulk of prokaryotic DNA outside the replication–segregation transition stays compacted. I consider possible origins of this concurrent replication–segregation and outline the ‘nucleoid administration’ system that organizes the dynamic part of the prokaryotic chromosome cycle.     

Bacterial chromosome segregation: evidence for DNA gyrase involvement in decatenation

Nucleoids isolated from a temperature-sensitive gyrB mutant of E. coli, incubated at restrictive temperatures, exhibit increased sedimentation rates and an abnormal doublet or dumbbell-shaped morphology. Shifting cells from restrictive to permissive temperature prior to nucleoid isolation leads to decreases in the percentage of doublet nucleoids and in nucleoid sedimentation rates. When nucleoids isolated from mutant cells exposed to restrictive temperature are incubated with purified gyrase, the percentage of doublet nucleoids decreases as the total number of nucleoids increases. These results, together with the demonstrated ability of gyrase to decatenate small circular DNA molecules in vitro, suggest that gyrase participates in bacterial chromosome segregation through its decatenating activity.     

Delayed nucleoid segregation in Escherichia coli

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.     

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.     

A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis

MreB shares a common prokaryotic ancestor with actin and is present in almost all rod-shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co-transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in-frame deletion of mreB and its complementation by mreB(+) only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP-MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.     

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes

The mechanisms driving bacterial chromosome segregation remain poorly characterized. While a number of factors influencing chromosome segregation have been described in recent years, none of them appeared to play an essential role in the process comparable to the eukaryotic centromere/spindle complex. The research community involved in bacterial chromosome was becoming familiar with the fact that bacteria have selected multiple redundant systems to ensure correct chromosome segregation. Over the past few years a new perspective came out that entropic forces generated by the confinement of the chromosome in the crowded nucleoid shell could be sufficient to segregate the chromosome. The segregating factors would only be required to create adequate conditions for entropy to do its job. In the article by Yazdi et al. ( 2012 ) in this issue of Molecular Microbiology, this model was challenged experimentally in live Escherichia coli cells. A Fis–GFP fusion was used to follow nucleoid choreography and analyse it from a polymer physics perspective. Their results suggest strongly that E. coli nucleoids behave as self‐adherent polymers. Such a structuring and the specific segregation patterns observed do not support an entropic like segregation model. Are we back to the pre‐entropic era?     

Independent segregation of the two arms of the Escherichia coli ori region requires neither RNA synthesis nor MreB dynamics

The mechanism of Escherichia coli chromosome segregation remains elusive. We present results on the simultaneous tracking of segregation of multiple loci in the ori region of the chromosome in cells growing under conditions in which a single round of replication is initiated and completed in the same generation. Loci segregated as expected for progressive replication-segregation from oriC, with markers placed symmetrically on either side of oriC segregating to opposite cell halves at the same time, showing that sister locus cohesion in the origin region is local rather than extensive. We were unable to observe any influence on segregation of the proposed centromeric site, migS, or indeed any other potential cis-acting element on either replication arm (replichore) in the AB1157 genetic background. Site-specific inhibition of replication close to oriC on one replichore did not prevent segregation of loci on the other replichore. Inhibition of RNA synthesis and inhibition of the dynamic polymerization of the actin homolog MreB did not affect ori and bulk chromosome segregation.     

Internal structure and dynamics of isolated Escherichia coli nucleoids assessed by fluorescence correlation spectroscopy

The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from approximately 50 kbp to approximately 3 kbp).