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A conserved two-component signal transduction system controls the response to phosphate starvation in Bifidobacterium breve UCC2003

ABSTRACT: This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by phoRP in Bifidobacterium breve UCC2003, which controls the response to phosphate (Pi) starvation. The response regulator PhoP was shown to bind to the promoter region of pstSCAB, specifying a predicted Pi transporter system, as well as that of phoU, which specifies a putative Pi-responsive regulatory protein. This interaction is assumed to cause transcriptional activation under conditions of Pi limitation. The phoRP genes appear to be subject to positive auto-regulation, and with pstSCAB and phoU, represent the complete regulon controlled by the phoRP-encoded 2CRS in B. breve UCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together with phoRP is conserved among many high GC-content Gram+ bacteria. The importance of the phoRP 2CRS in the response of B. breve to Pi starvation conditions was confirmed by generating a B. breve::phoP insertion mutant which exhibited decreased growth under phosphate-limiting conditions as compared to its parent strain UCC2003.

ORGANISM(S): Bifidobacterium breve UCC2003

PROVIDER: GSE34983 | GEO | 2012/09/06

SECONDARY ACCESSION(S): PRJNA151101

REPOSITORIES: GEO

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Project description:In this study, we describe the functional characterisation of the B. breve UCC2003 gal locus, which is dedicated to the utilisation of galactan, a plant-derived polysaccharide. Using a combination of molecular approaches we conclude that the galA gene of B. breve UCC2003 encodes a beta-1,4-endogalactanase producing galacto-oligosaccharides, which are specifically internalised by an ABC transport system, encoded by galBCDE, and which are then hydrolysed to galactose moieties by a dedicated intracellular beta-galactosidase, specified by galG. The generated galactose molecules are presumed to be fed into the fructose-6-phosphate phosphoketolase pathway via the Leloir pathway, thereby allowing B. breve UCC2003 to use galactan as its sole carbon and energy source. In addition to these findings we demonstrate that GalR is a LacI-type DNA-binding protein, which not only appears to control transcription of the galCDEGR operon, but also that of the galA gene. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

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A conserved two-component signal transduction system controls the response to phosphate starvation in Bifidobacterium breve UCC2003

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