RESUMO
In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coliâ ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S.â enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA.
Assuntos
Proteínas de Bactérias/metabolismo , Celulose/biossíntese , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glucosiltransferases/genética , Proteínas de Bactérias/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Glucosiltransferases/metabolismo , Glicosiltransferases/metabolismo , Klebsiella pneumoniae/metabolismo , Mutagênese Sítio-Dirigida , Óperon , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Salmonella typhimurium/metabolismo , Transdução de SinaisRESUMO
Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative σ factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa.
Assuntos
Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Regulon , Fator sigma/metabolismo , Fatores de Virulência/biossíntese , Sítios de Ligação , Imunoprecipitação da Cromatina , Meios de Cultura/química , Deleção de Genes , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Pressão Osmótica , Ligação Proteica , Fator sigma/genéticaRESUMO
The ubiquitous second messenger c-di-GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c-di-GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL-like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT-29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL-like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c-di-GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4 C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S. typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Flagelos/fisiologia , Salmonella typhimurium/fisiologia , Salmonella typhimurium/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Células HT29 , Humanos , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Movimento , Fenótipo , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Infecções por Salmonella , Salmonella typhimurium/genética , VirulênciaRESUMO
The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.
Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/metabolismo , Fibrose Cística/microbiologia , Proteínas de Membrana/metabolismo , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa , Transdução de Sinais/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fibrose Cística/complicações , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Conformação Proteica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Infecções Respiratórias/genética , Infecções Respiratórias/microbiologiaRESUMO
Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3',5'-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5' untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses.