RESUMEN
Escherichia coli cyclophilin PpiB is a peptidyl-prolyl cis/trans isomerase (PPIase, EC: 5.2.1.8), involved in the negative modulation of various bacterial processes, such as swimming and swarming motility and biofilm formation ability. In this study, we show that PpiB possesses also a chaperone function as it can prevent the thermal denaturation of citrate synthase even with essentially eliminated PPIase activity. We demonstrate, using active site mutations, that the PPIase activity of PpiB is required in all processes, except for the negative effect on swimming, indicating a possible isomerase-independent function. Additionally, we show that the reduced PPIase activity of PpiB does not prevent the association with all prey proteins tested and that the PPIase active site is not involved necessarily in each association. We also used a random mutagenesis approach, to identify amino acid residues apart from the catalytic site, which are necessary for PpiB function. The combination of enzymatic studies concerning the PPIase and chaperone activities of each mutant protein, with structural analyses based on 3D models, provided further insights into the effects of the mutations on the function of PpiB and showed the importance of structural features in addition to the catalytic site, for its in vivo role.
Asunto(s)
Ciclofilinas/química , Proteínas Mutantes/química , Relación Estructura-Actividad , Secuencia de Aminoácidos/genética , Dominio Catalítico , Ciclofilinas/metabolismo , Escherichia coli/genética , Chaperonas Moleculares/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Pliegue de ProteínaRESUMEN
PpiB belongs to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), which catalyze the rate-limiting protein folding step at peptidyl-prolyl bonds and control several biological processes. In this study, we show that PpiB acts as a negative effector of motility and biofilm formation ability of Escherichia coli. We identify multicopy suppressors of each ΔppiB phenotype among putative PpiB prey proteins which upon deletion are often characterized by analogous phenotypes. Many putative preys show similar gene expression in wild-type and ΔppiB genetic backgrounds implying possible post-translational modifications by PpiB. We further conducted in vivo and in vitro interaction screens to determine which of them represent true preys. For DnaK, acetyl-CoA carboxylase, biotin carboxylase subunit (AccC) and phosphate acetyltransferase (Pta) we also showed a direct role of PpiB in the functional control of these proteins because it increased the measured enzyme activity of each protein and further interfered with DnaK localization and the correct folding of AccC. Taken together, these results indicate that PpiB is involved in diverse regulatory mechanisms to negatively modulate motility and biofilm formation via its functional association with certain protein substrates.
Asunto(s)
Acetil-CoA Carboxilasa/química , Biopelículas/crecimiento & desarrollo , Ciclofilinas/genética , Proteínas de Escherichia coli/genética , Proteínas HSP70 de Choque Térmico/genética , Acetil-CoA Carboxilasa/genética , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Ciclofilinas/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas HSP70 de Choque Térmico/química , Fosfato Acetiltransferasa/genética , Pliegue de ProteínaRESUMEN
The presence of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions are still not clearly defined. Here, we demonstrate that the ΔppiA deletion strain from Escherichia coli exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the ΔppiA strain during biofilm growth.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Ciclofilinas/química , Escherichia coli/metabolismo , Isomerasa de Peptidilprolil/química , Proteínas Recombinantes/química , Ciclofilinas/genética , Ciclofilinas/metabolismo , Cartilla de ADN , Escherichia coli/genética , Perfilación de la Expresión Génica , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Isomerasa de Peptidilprolil/genética , Conformación Proteica , Proteínas Recombinantes/genéticaRESUMEN
The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.
Asunto(s)
Islas Genómicas/genética , Fijación del Nitrógeno/genética , Pseudomonas stutzeri/genética , Pseudomonas/genética , Proteínas Bacterianas/genética , Secuencia de Bases , China , ADN Bacteriano/química , ADN Bacteriano/genética , Evolución Molecular , Variación Genética , Geografía , Alemania , Grecia , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Pseudomonas/clasificación , ARN Ribosómico 16S/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.