RuvAB Acts at Arrested Replication Forks

AbstractReplication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage

Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria.

Factor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species

Replication fork collapse at replication terminator sequences

Replication fork arrest is a source of genome re arrangements, and the recombinogenic properties of blocked forks are likely to depend on the cause of blockage. Here we study the fate of replication forks blocked at natural replication arrest sites. For this purpose, Escherichia coli replication terminator sequences Ter were placed at ectopic positions on the bacterial chromosome. The resulting strain requires recombinational repair for viability, but replication forks blocked at Ter are not broken. Linear DNA molecules are formed upon arrival of a second round of replication forks that copy the DNA strands of the first blocked forks to the end. A model that accounts for the requirement for homologous recombination for viability in spite of the lack of chromosome breakage is proposed. This work shows that natural and accidental replication arrests sites are processed differently

PriA Is Essential for Viability of the Escherichia coli Topoisomerase IV parE10(Ts) Mutant

The parE10(Ts) mutation, which renders Escherichia coli thermosensitive for growth by inactivation of the essential E. coli topoisomerase topo IV, is lethal at all temperatures when PriA, the main replication restart protein, is absent. This lethality is suppressed by the activation of a PriA-independent replication restart pathway (dnaC809 mutation). This result suggests that topo IV acts prior to full-chromosome replication completion

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Abstract Transcription termination factor Rho controls pervasive, mainly antisense, transcription initiated at cryptic signals or resulting from read-through at weak terminators in various bacterial species. In Bacillus subtilis , Rho is intricately involved in the regulation of phenomena associated with the adaptation to stationary phase and cell differentiation including the ultimate survival program of sporulation. While knockout or overexpression of the rho gene alters global transcription and modifies cell physiology, in wild-type B. subtilis cells, the reduction of Rho levels during the transition to stationary phase is necessary for both initiation and implementation of the sporulation program. However, the mechanisms that govern Rho expression throughout the cell cycle remain largely unknown. Here, we demonstrate that, besides the previously identified vegetative SigA-dependent promoter active during exponential growth, two distinct mechanisms ensure a spatiotemporal expression of the rho gene during sporulation. In the mother cell of the sporangium, rho expression occurs through the read-through transcription initiated at the distal SigH-dependent and Spo0A∼P-regulated promoter of the spo0F gene. In the forespore, rho is transcribed from a genuine promoter recognized by the alternative sigma factor SigF. These regulatory elements compensate for the inactivation of SigA-dependent rho expression at the end of exponential growth and allow the critical “refueling” of Rho protein in both compartments of the sporangium. We show that altering rho expression in the mother cell or in the forespore affects differently the properties and the morphology of mature spores. Moreover, spores formed in the absence of Rho are impaired in their ability to revive under favorable growth conditions, exhibiting accelerated germination and slow outgrowth. Finally, we show that optimal outgrowth of the wild-type spores requires the expression of rho during spore maturation and additionally after spore germination

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis.

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks

Bacillus subtilis serine/threonine protein kinase YabT is involved in spore development via phosphorylation of a bacterial recombinase.

We characterized YabT, a serine/threonine kinase of the Hanks family, from Bacillus subtilis. YabT is a putative transmembrane kinase that lacks the canonical extracellular signal receptor domain. We demonstrate that YabT possesses a DNA-binding motif essential for its activation. In vivo YabT is expressed during sporulation and localizes to the asymmetric septum. Cells devoid of YabT sporulate more slowly and exhibit reduced resistance to DNA damage during sporulation. We established that YabT phosphorylates DNA-recombinase RecA at the residue serine 2. A non-phosphorylatable mutant of RecA exhibits the same phenotype as the ΔyabT mutant, and a phosphomimetic mutant of RecA complements ΔyabT, suggesting that YabT acts via RecA phosphorylation in vivo. During spore development, phosphorylation facilitates the formation of transient and mobile RecA foci that exhibit a scanning-like movement associated to the nucleoid in the mother cell. In some cells these foci persist at the end of spore development. We show that persistent RecA foci, which presumably coincide with irreparable lesions, are mutually exclusive with the completion of spore morphogenesis. Our results highlight similarities between the bacterial serine/threonine kinase YabT and eukaryal kinases C-Abl and Mec1, which are also activated by DNA, and phosphorylate proteins involved in DNA damage repair

Rho inactivation increases expression of KinA and KinB kinases.

<p>WT (W) and RM (r) cells containing <i>kinA</i>-SPA or <i>kinB</i>-SPA translational fusions at natural chromosomal loci were grown in LB (lanes 1–4) or sporulation-inducing DS medium (lanes 5–10) to mid-exponential (expo; OD₆₀₀ ∼ 0.5) or stationary (stat; OD₆₀₀∼ 1.5) phases and analyzed for KinA and KinB proteins using ANTI-FLAG M2 monoclonal antibodies. Equal amounts of protein were loaded onto the gel as quantified by the Bradford assay. To control equilibrium between the samples, total protein extracts from cells with <i>kinB</i>-SPA fusion were analyzed for MreB protein using anti-MreB specific antibodies.</p