RESUMO
Ectoine and hydroxyectoine are effective microbial osmostress protectants, but can also serve as versatile nutrients for bacteria. We have studied the genetic regulation of ectoine and hydroxyectoine import and catabolism in the marine Roseobacter species Ruegeria pomeroyi and identified three transcriptional regulators involved in these processes: the GabR/MocR-type repressor EnuR, the feast and famine-type regulator AsnC and the two-component system NtrYX. The corresponding genes are widely associated with ectoine and hydroxyectoine uptake and catabolic gene clusters (enuR, asnC), and with microorganisms predicted to consume ectoines (ntrYX). EnuR contains a covalently bound pyridoxal-5'-phosphate as a co-factor and the chemistry underlying the functioning of MocR/GabR-type regulators typically requires a system-specific low molecular mass effector molecule. Through ligand binding studies with purified EnuR, we identified N-(alpha)-L-acetyl-2,4-diaminobutyric acid and L-2,4-diaminobutyric acid as inducers for EnuR that are generated through ectoine catabolism. AsnC/Lrp-type proteins can wrap DNA into nucleosome-like structures, and we found that the asnC gene was essential for use of ectoines as nutrients. Furthermore, we discovered through transposon mutagenesis that the NtrYX two-component system is required for their catabolism. Database searches suggest that our findings have important ramifications for an understanding of the molecular biology of most microbial consumers of ectoines.
Assuntos
Diamino Aminoácidos/metabolismo , Elementos Reguladores de Transcrição/genética , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Transativadores/genética , Aminobutiratos/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Sinais (Psicologia) , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/genética , Família MultigênicaRESUMO
The Cag Type IV secretion system, which contributes to inflammation and cancerogenesis during chronic infection, is one of the major virulence factors of the bacterial gastric pathogen Helicobacter pylori. We have generated and characterized a series of non-marked site-directed chromosomal mutants in H. pylori to define domains of unknown function of the essential tip protein CagL of the Cag secretion system. Characterizing the CagL mutants, we determined that their function to activate cells and transport the effector CagA was reduced to different extents. We identified three novel regions of the CagL protein, involved in its structural integrity, its possible interaction with the CagPAI T4SS pilus protein CagI, and in its binding to integrins and other host cell ligands. In particular two novel variable CagL motifs were involved in integrin binding, TSPSA, and TASLI, which is located opposite of its integrin binding motif RGD. We thereby defined functionally important subdomains within the CagL structure, which can be used to clarify CagL contributions in the context of other CagPAI proteins or for inhibition of the CagT4SS. This structure-function correlation of CagL domains can also be instructive for the functional characterization of other potential VirB5 orthologs whose structure is not yet known.
Assuntos
Proteínas de Bactérias/genética , Helicobacter pylori/genética , Sistemas de Secreção Tipo IV/genética , Antígenos de Bactérias/genética , Linhagem Celular Tumoral , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Integrinas/genética , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica/genética , Transporte Proteico/genética , Estômago/microbiologiaRESUMO
Helicobacter pylori colonizes about half of the world's population. It is a causative agent of stomach diseases, including malignant tumors. We report the genome sequence of strain N6, which is widely used in H. pylori research and appreciated for its large cell size and high transformation efficiency.
Assuntos
Genoma Bacteriano , Helicobacter pylori/genética , França/epidemiologia , Gastrite/epidemiologia , Gastrite/microbiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Humanos , Dados de Sequência MolecularRESUMO
Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.
