RESUMO
Transcription termination factor Rho is known for its ubiquitous role in suppression of pervasive, mostly antisense, transcription. In the model Gram-positive bacterium Bacillus subtilis, de-repression of pervasive transcription by inactivation of rho revealed the role of Rho in the regulation of post-exponential differentiation programs. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain (Rho+) that expresses rho at a relatively stable high level in order to compensate for its decrease in the wild-type cells entering stationary phase. The RNAseq analysis of Rho+, WT and Δrho strains (expression profiles can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/) shows that Rho over-production enhances the termination efficiency of Rho-sensitive terminators, thus reducing transcriptional read-through and antisense transcription genome-wide. Moreover, the Rho+ strain exhibits global alterations of sense transcription with the most significant changes observed for the AbrB, CodY, and stringent response regulons, forming the pathways governing the transition to stationary phase. Subsequent physiological analyses demonstrated that maintaining rho expression at a stable elevated level modifies stationary phase-specific physiology of B. subtilis cells, weakens stringent response, and thereby negatively affects the cellular adaptation to nutrient limitations and other stresses, and blocks the development of genetic competence and sporulation. These results highlight the Rho-specific termination of transcription as a novel element controlling stationary phase. The release of this control by decreasing Rho levels during the transition to stationary phase appears crucial for the functionality of complex gene networks ensuring B. subtilis survival in stationary phase.
Assuntos
Bacillus subtilis , Proteínas de Bactérias , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ciclo Celular , Regulação Bacteriana da Expressão Gênica/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Fator Rho/genética , Fatores de Transcrição/genética , Terminação da Transcrição Genética , Transcrição Gênica , Bacillus subtilis/genética , Biofilmes/crescimento & desenvolvimento , Movimento Celular/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes/genética , Regiões Promotoras Genéticas , Esporos Bacterianos/genética , Transcriptoma/genéticaRESUMO
Populations of genetically identical microorganisms exhibit high degree of cell-to-cell phenotypic diversity even when grown in uniform environmental conditions. Heterogeneity is a genetically determined trait, which ensures bacterial adaptation and survival in the ever changing environmental conditions. Fluctuations in gene expression (noise) at the level of transcription initiation largely contribute to cell-to-cell variability within population. Not surprisingly, the analyses of the mechanisms driving phenotypic heterogeneity are mainly focused on the activity of promoters and transcriptional factors. Less attention is currently given to a role of intrinsic and factor-dependent transcription terminators. Here, we discuss recent advances in understanding the regulatory role of the multi-functional transcription termination factor Rho, the major inhibitor of pervasive transcription in bacteria and the emerging global regulator of gene expression. We propose that termination activity of Rho might be among the mechanisms by which cells manage the intensity of transcriptional noise, thus affecting population heterogeneity.
Assuntos
Heterogeneidade Genética , Fator Rho/genética , Transcrição Gênica , Bactérias/genética , Regiões Promotoras Genéticas , RNA Bacteriano/genéticaRESUMO
Bacillus subtilis Mrp family protein SalA has been shown to indirectly promote the production of the exoprotease AprE by inhibiting the expression of scoC, which codes for a repressor of aprE. The exact mechanism by which SalA influences scoC expression has not been clarified previously. We demonstrate that SalA possesses a DNA-binding domain (residues 1-60), which binds to the promoter region of scoC. The binding of SalA to its target DNA depends on the presence of ATP and is stimulated by phosphorylation of SalA at tyrosine 327. The B. subtilis protein-tyrosine kinase PtkA interacts specifically with the C-terminal domain of SalAâ in vivo and in vitro and is responsible for activating its DNA binding via phosphorylation of tyrosine 327. In vivo, a mutant mimicking phosphorylation of SalA (SalA Y327E) exhibited a strong repression of scoC and consequently overproduction of AprE. By contrast, the non-phosphorylatable SalA Y327F and the ΔptkA exhibited the opposite effect, stronger expression of scoC and lower production of the exoprotease. Interestingly, both SalA and PtkA contain the same ATP-binding Walker domain and have thus presumably arisen from the common ancestral protein. Their regulatory interplay seems to be conserved in other bacteria.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Exopeptidases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/metabolismo , Tirosina/metabolismoRESUMO
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.
Assuntos
Bactérias/genética , Fenômenos Fisiológicos Bacterianos , Regulação Bacteriana da Expressão Gênica , Fator Rho/metabolismo , Transcrição GênicaRESUMO
AbrB is a global gene regulator involved in transition phase phenomena in Bacillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B. subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrBâ S86D and S86A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosforilação , Fatores de Transcrição/genéticaRESUMO
Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.
Assuntos
Proteínas de Bactérias/química , Proteínas Repressoras/química , Tirosina/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sequências Hélice-Volta-Hélice , Dados de Sequência Molecular , Óperon , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismoRESUMO
The nonessential regions in bacterial chromosomes are ill-defined due to incomplete functional information. Here, we establish a comprehensive repertoire of the genome regions that are dispensable for growth of Bacillus subtilis in a variety of media conditions. In complex medium, we attempted deletion of 157 individual regions ranging in size from 2 to 159 kb. A total of 146 deletions were successful in complex medium, whereas the remaining regions were subdivided to identify new essential genes (4) and coessential gene sets (7). Overall, our repertoire covers ~76% of the genome. We screened for viability of mutant strains in rich defined medium and glucose minimal media. Experimental observations were compared with predictions by the iBsu1103 model, revealing discrepancies that led to numerous model changes, including the large-scale application of model reconciliation techniques. We ultimately produced the iBsu1103V2 model and generated predictions of metabolites that could restore the growth of unviable strains. These predictions were experimentally tested and demonstrated to be correct for 27 strains, validating the refinements made to the model. The iBsu1103V2 model has improved considerably at predicting loss of viability, and many insights gained from the model revisions have been integrated into the Model SEED to improve reconstruction of other microbial models.
Assuntos
Bacillus subtilis/genética , Cromossomos Bacterianos , Modelos Biológicos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Deleção Cromossômica , Mapeamento Cromossômico , Redes e Vias Metabólicas/genética , FenótipoRESUMO
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 vivoâ 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.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Recombinases Rec A/metabolismo , Esporos/enzimologia , Esporos/crescimento & desenvolvimento , Bacillus subtilis/genética , Deleção de Genes , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Esporos/genéticaRESUMO
Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.
RESUMO
Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C14 isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using 1 H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C14 FA chain.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Ácidos Graxos/biossíntese , Lipopeptídeos/metabolismo , Redes e Vias Metabólicas , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Deleção de Genes , Engenharia Genética , Lipopeptídeos/genética , Óperon , Isoformas de Proteínas/metabolismo , Espectrometria de Massas em TandemRESUMO
Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis.
RESUMO
Blocking replication forks in the Escherichia coli chromosome by ectopic Ter sites renders the RecBCD pathway of homologous recombination and SOS induction essential for viability. In this work, we show that the E. coli helicase II (UvrD) is also essential for the growth of cells where replication forks are arrested at ectopic Ter sites. We propose that UvrD is required for Tus removal from Ter sites. The viability of a SOS non-inducible Ter-blocked strain is fully restored by the expression of the two SOS-induced proteins UvrD and RecA at high level, indicating that these are the only two SOS-induced proteins required for replication across Ter/Tus complexes. Several observations suggest that UvrD acts in concert with homologous recombination and we propose that UvrD is associated with recombination-initiated replication forks and that it removes Tus when a PriA-dependent, restarted replication fork goes across the Ter/Tus complex. Finally, expression of the UvrD homologue from Bacilus subtilis PcrA restores the growth of uvrD-deficient Ter-blocked cells, indicating that the capacity to dislodge Tus is conserved in this distant bacterial species.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Modelos Genéticos , Mutação/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Resposta SOS em Genética/genética , Regiões Terminadoras Genéticas/genéticaRESUMO
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.
Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , DNA Topoisomerase IV/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/crescimento & desenvolvimento , DNA Girase/fisiologia , Replicação do DNA , Escherichia coli/enzimologia , Mutação , Recombinases Rec A/metabolismoRESUMO
Replication forks arrested by inactivation of the main Escherichia coli DNA polymerase (polymerase III) are reversed by the annealing of newly synthesized leading- and lagging-strand ends. Reversed forks are reset by the action of RecBC on the DNA double-strand end, and in the absence of RecBC chromosomes are linearized by the Holliday junction resolvase RuvABC. We report here that the UvrD helicase is essential for RuvABC-dependent chromosome linearization in E. coli polymerase III mutants, whereas its partners in DNA repair (UvrA/B and MutL/S) are not. We conclude that UvrD participates in replication fork reversal in E. coli.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Replicação do DNA/genética , Escherichia coli/genética , Dano ao DNA/fisiologia , DNA Polimerase III/metabolismo , Replicação do DNA/fisiologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , MutaçãoRESUMO
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.
Assuntos
Replicação do DNA , Escherichia coli/genética , Regiões Terminadoras Genéticas , Sequência de Bases , DNA Bacteriano/genéticaRESUMO
Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability. Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery. Several strategies for the processing of inactivated replication forks before replisome reassembly have been described. Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest. The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage. These various restart pathways may reflect different structures at stalled forks. We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes.