RESUMEN
Osmoregulated periplasmic glucans (OPGs) are produced by many proteobacteria and are important for bacterial-host interactions. The opgG and opgH genes involved in the synthesis of OPGs are the most widely distributed genes in proteobacterial genomes. Two other non-homologous genes, both named ndvB, are also involved in OPG biosynthesis in several species. The Pseudomonas aeruginosa genome possesses two ORFs, PA5077 and PA5078, that show similarity to opgH and opgG of Pseudomonas syringae, respectively, and one ORF, PA1163, similar to ndvB of Sinorhizobium meliloti. Here, we report that the opgGH locus of P. aeruginosa PA14 is involved in the synthesis of linear polymers with beta-1,2-linked glucosyl residues branched with a few beta-1,6 glucosyl residues. Succinyl residues also substitute this glucose backbone. Transcription of opgGH is repressed by high osmolarity. Low osmolarity promotes the formation of highly structured biofilms, but biofilm development is slower and the area of biomass is reduced under high osmolarity. Biofilm development of an opgGH mutant grown under low osmolarity presents a similar phenotype to the wild-type biofilm grown under high osmolarity. These results suggest that OPGs are important for biofilm formation under conditions of low osmolarity. A previous study suggested that the P. aeruginosa ndvB gene is involved in the resistance of biofilms to antibiotics. We have shown that ndvB is not involved in the biosynthesis of the OPG described here, and opgGH do not appear to be involved in the resistance of P. aeruginosa PA14 biofilms to antibiotics.
Asunto(s)
Genes Bacterianos , Glucanos/biosíntesis , Polisacáridos Bacterianos/biosíntesis , Pseudomonas aeruginosa/genética , Equilibrio Hidroelectrolítico/genética , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Glucanos/química , Glucanos/genética , Glucosa/análisis , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genética , Pseudomonas aeruginosa/fisiología , Pseudomonas syringae/genética , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/genética , Ácido Succínico/análisis , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
Osmoregulated periplasmic glucans (OPGs) G protein (OpgG) is required for OPGs biosynthesis. OPGs from Escherichia coli are branched glucans, with a backbone of beta-1,2 glucose units and with branches attached by beta-1,6 linkages. In Proteobacteria, OPGs are involved in osmoprotection, biofilm formation, virulence and resistance to antibiotics. Despite their important biological implications, enzymes synthesizing OPGs are poorly characterized. Here, we report the 2.5 A crystal structure of OpgG from E.coli. The structure was solved using a selenemethionine derivative of OpgG and the multiple anomalous diffraction method (MAD). The protein is composed of two beta-sandwich domains connected by one turn of 3(10) helix. The N-terminal domain (residues 22-388) displays a 25-stranded beta-sandwich fold found in several carbohydrate-related proteins. It exhibits a large cleft comprising many aromatic and acidic residues. This putative binding site shares some similarities with enzymes such as galactose mutarotase and glucodextranase, suggesting a potential catalytic role for this domain in OPG synthesis. On the other hand, the C-terminal domain (residues 401-512) has a seven-stranded immunoglobulin-like beta-sandwich fold, found in many proteins where it is mainly implicated in interactions with other molecules. The structural data suggest that OpgG is an OPG branching enzyme in which the catalytic activity is located in the large N-terminal domain and controlled via the smaller C-terminal domain.