RESUMO
ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.
Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Citidina Trifosfato/metabolismo , DNA Bacteriano/metabolismo , Myxococcus xanthus/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Hidrólise , Mutação , Myxococcus xanthus/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de TempoRESUMO
Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.
Assuntos
Bactérias , Flagelina , Flagelina/genética , Flagelina/metabolismo , Estereoisomerismo , Bactérias/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Archaea/metabolismo , Flagelos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The acquisition of multidrug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the polarized α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (ß-lactam) antibiotics. Combining 2-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR uses a common operator of the divergent tipR and acrAB-nodT promoter for adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified diverse compounds that interfere with DNA binding by TipR or induce its dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT deforms the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, boosting pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Transporte Biológico , Cefalosporinas , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Resistência beta-Lactâmica , Proteínas de Escherichia coli/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
How DNA-dependent RNA polymerase (RNAP) acts on bacterial cell cycle progression during transcription elongation is poorly investigated. A forward genetic selection for Caulobacter crescentus cell cycle mutants unearthed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR promotes the accumulation of the essential cell cycle transcriptional activator CtrA in late S-phase but also affects transcription at a global level to protect cells from the quinolone antibiotic nalidixic acid that induces a multidrug efflux pump and from the RNAP inhibitor rifampicin that blocks transcription elongation. We show that TrcR associates with promoters and coding sequences in vivo in a rifampicin-dependent manner and that it interacts physically and genetically with RNAP. We show that TrcR function and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella spp Thus, TrcR represents a hitherto unknown antibiotic target and the founding member of the DUF1013 family, an uncharacterized class of transcriptional regulators that track with RNAP during the elongation phase to promote transcription during the cell cycle.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/crescimento & desenvolvimento , Ciclo Celular/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Bactérias/genética , Caulobacter crescentus/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , Regiões Promotoras GenéticasRESUMO
Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50â years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M.â tuberculosis, C.â difficile, and the Gram-negative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho-methyl Fdx also exhibited activity against the Gram-negative C.â crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics.
Assuntos
Clostridioides difficile , Mycobacterium tuberculosis , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fidaxomicina , Aminoglicosídeos/farmacologia , RNA Polimerases Dirigidas por DNARESUMO
The Alphaproteobacteria show a remarkable diversity of cell cycle-dependent developmental patterns, which are governed by the conserved CtrA pathway. Its central component CtrA is a DNA-binding response regulator that is controlled by a complex two-component signaling network, mediating distinct transcriptional programs in the two offspring. The CtrA pathway has been studied intensively and was shown to consist of an upstream part that reads out the developmental state of the cell and a downstream part that integrates the upstream signals and mediates CtrA phosphorylation. However, the role of this circuitry in bacterial diversification remains incompletely understood. We have therefore investigated CtrA regulation in the morphologically complex stalked budding alphaproteobacterium Hyphomonas neptunium. Compared to relatives dividing by binary fission, H. neptunium shows distinct changes in the role and regulation of various pathway components. Most notably, the response regulator DivK, which normally links the upstream and downstream parts of the CtrA pathway, is dispensable, while downstream components such as the pseudokinase DivL, the histidine kinase CckA, the phosphotransferase ChpT and CtrA are essential. Moreover, CckA is compartmentalized to the nascent bud without forming distinct polar complexes and CtrA is not regulated at the level of protein abundance. We show that the downstream pathway controls critical functions such as replication initiation, cell division and motility. Quantification of the signal flow through different nodes of the regulatory cascade revealed that the CtrA pathway is a leaky pipeline and must involve thus-far unidentified factors. Collectively, the quantitative system-level analysis of CtrA regulation in H. neptunium points to a considerable evolutionary plasticity of cell cycle regulation in alphaproteobacteria and leads to hypotheses that may also hold in well-established model organisms such as Caulobacter crescentus.
Assuntos
Alphaproteobacteria/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Fatores de Transcrição/genética , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Divisão Celular , Movimento Celular , Replicação do DNA , Evolução Molecular , Fatores de Transcrição/metabolismoRESUMO
Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, ΔcdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, α-ketoglutarate, ATP, NAD+, UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that ΔcdnL cells are glutamate auxotrophs, and ΔcdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Homeostase , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Divisão Celular , Sequência Conservada , Metaboloma , Fatores de Transcrição/genéticaRESUMO
Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation phenotype may have been prevalent and subsequently lost by laboratory culturing in other Firmicutes currently considered as asporogenic. Cryptosporulation might thus represent an ancestral but unstable and adaptive developmental state in Firmicutes that is under selection under harsh environmental conditions.
Assuntos
Bacillus , Firmicutes , Esporos Bacterianos/genética , FilogeniaRESUMO
The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Caulobacter crescentus/enzimologia , Proteínas de Ligação a DNA/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Difusão , Espectrometria de Massa com Troca Hidrogênio-Deutério , Mutação , Ligação ProteicaRESUMO
A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.
Assuntos
Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Proteínas de Escherichia coli/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Antibacterianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Humanos , Óperon/genética , RNA Mensageiro/genética , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Especificidade por Substrato , Transcriptoma/genéticaRESUMO
Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA and a proximal residue can direct CtrA towards activation of the dispersal (G1-phase) program. Hence, we propose that this conserved determinant in the CtrA primary structure dictates promoter reprogramming during the growth transition in other alpha-proteobacteria that differentiate from replicative cells into dispersal cells.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/metabolismo , Ciclo Celular , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Caulobacter crescentus/citologia , DNA Bacteriano/metabolismo , Fase G1 , Guanosina Tetrafosfato/metabolismo , Movimento , Mutação/genética , Regiões Promotoras Genéticas , Ligação Proteica , Fase S , Supressão Genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger--bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)--that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 â S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Ciclo Celular/fisiologia , Flagelos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Polaridade Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Alinhamento de Sequência , Transdução de SinaisRESUMO
Peptidoglycan is the predominant stress-bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted 'peptidoglycan-intermediate' organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan-negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan-intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan-like sacculus and/or division septum.
Assuntos
Bactérias , Interações entre Hospedeiro e Microrganismos , Espaço Intracelular/microbiologia , Peptidoglicano/genética , Peptidoglicano/metabolismo , Anaplasma marginale/classificação , Anaplasma marginale/genética , Anaplasma marginale/imunologia , Anaplasma marginale/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Bactérias/metabolismo , Parede Celular/metabolismo , Chlamydia/classificação , Chlamydia/genética , Chlamydia/imunologia , Chlamydia/metabolismo , Citoplasma/metabolismo , Genoma Bacteriano/genética , Humanos , Imunidade Inata/imunologia , Orientia tsutsugamushi/classificação , Orientia tsutsugamushi/genética , Orientia tsutsugamushi/imunologia , Orientia tsutsugamushi/metabolismo , Peptidoglicano/química , Filogenia , Wolbachia/classificação , Wolbachia/genética , Wolbachia/imunologia , Wolbachia/metabolismoRESUMO
We searched for regulators of chromosome replication in the cell cycle model Caulobacter crescentus and found a novel DNA-binding protein (GapR) that selectively aids the initiation of chromosome replication and the initial steps of chromosome partitioning. The protein binds the chromosome origin of replication (Cori) and has higher-affinity binding to mutated Cori-DNA that increases Cori-plasmid replication in vivo. gapR gene expression is essential for normal rapid growth and sufficient GapR levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified dynamic DNA-binding distributions for GapR, with the strongest associations at the partitioning (parABS) locus near Cori. Using molecular-genetic and fluorescence microscopy experiments, we showed that GapR also promotes the first steps of chromosome partitioning, the initial separation of the duplicated parS loci following replication from Cori. This separation occurs before the parABS-dependent partitioning phase. Therefore, this early separation, whose mechanisms is not known, coincides with the poorly defined mechanism(s) that establishes chromosome asymmetry: C. crescentus chromosomes are partitioned to distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that GapR coordinates chromosome replication with asymmetry-establishing chromosome separation, noting that both roles are consistent with the phylogenetic restriction of GapR to asymmetrically dividing bacteria.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/metabolismo , Divisão Celular/efeitos dos fármacos , Cromossomos Bacterianos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Novobiocina/farmacologia , Plasmídeos/química , Plasmídeos/metabolismo , Origem de Replicação , Rifampina/farmacologiaRESUMO
Cell fate determination in the asymmetric bacterium Caulobacter crescentus (Caulobacter) is triggered by the localization of the developmental regulator SpmX to the old (stalked) cell pole during the G1âS transition. Although SpmX is required to localize and activate the cell fate-determining kinase DivJ at the stalked pole in Caulobacter, in cousins such as Asticcacaulis, SpmX directs organelle (stalk) positioning and possibly other functions. We define the conserved σ54-dependent transcriptional activator TacA as a global regulator in Caulobacter whose activation by phosphorylation is indirectly down-regulated by SpmX. Using a combination of forward genetics and cytological screening, we uncover a previously uncharacterized and polarized component (SpmY) of the TacA phosphorylation control system, and we show that SpmY function and localization are conserved. Thus, SpmX organizes a site-specific, ancestral, and multifunctional regulatory hub integrating the in-phase oscillation of two global transcriptional regulators, CtrA (the master cell cycle transcriptional regulator A) and TacA, that perform important cell cycle functions.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Regulação Bacteriana da Expressão Gênica , Regulon/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Divisão Celular/genética , Fosforilação , Transativadores/genética , Transativadores/metabolismoRESUMO
Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of α-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5'-GANTC-3' sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different α-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.
Assuntos
Caulobacter crescentus/genética , Ciclo Celular/genética , Metilação de DNA/genética , Transcrição Gênica , Divisão Celular/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Regulação Bacteriana da Expressão Gênica , Genoma Microbiano , Metiltransferases/genética , Fosfatos/metabolismo , Regiões Promotoras Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Inanição/genética , Inanição/metabolismoRESUMO
In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Pontos de Checagem do Ciclo Celular/genética , Regulação Bacteriana da Expressão Gênica , Sinorhizobium meliloti/genética , Proteínas de Bactérias/genética , Caulobacter crescentus/citologia , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Clonagem Molecular , Replicação do DNA , Regulação para Baixo , Fabaceae/microbiologia , Deleção de Genes , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Regiões Promotoras Genéticas , Sinorhizobium meliloti/citologia , Simbiose , Transdução Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA) that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ). Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a 'nutritional override' system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.
Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Caulobacter crescentus/crescimento & desenvolvimento , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Nutrigenômica/métodos , Regiões Promotoras Genéticas , Transdução de Sinais/genéticaRESUMO
What are the minimal requirements to sustain an asymmetric cell cycle? Here we use mathematical modelling and forward genetics to reduce an asymmetric cell cycle to its simplest, primordial components. In the Alphaproteobacterium Caulobacter crescentus, cell cycle progression is believed to be controlled by a cyclical genetic circuit comprising four essential master regulators. Unexpectedly, our in silico modelling predicted that one of these regulators, GcrA, is in fact dispensable. We confirmed this experimentally, finding that ΔgcrA cells are viable, but slow-growing and elongated, with the latter mostly due to an insufficiency of a key cell division protein. Furthermore, suppressor analysis showed that another cell cycle regulator, the methyltransferase CcrM, is similarly dispensable with simultaneous gcrA/ccrM disruption ameliorating the cytokinetic and growth defect of ΔgcrA cells. Within the Alphaproteobacteria, gcrA and ccrM are consistently present or absent together, rather than either gene being present alone, suggesting that gcrA/ccrM constitutes an independent, dispensable genetic module. Together our approaches unveil the essential elements of a primordial asymmetric cell cycle that should help illuminate more complex cell cycles.
Assuntos
Caulobacter crescentus/genética , Caulobacter crescentus/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Biologia Computacional/métodos , Simulação por Computador , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Metilação , Modelos BiológicosRESUMO
Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.