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Identification of two-component regulatory systems in Bifidobacterium infantis by functional complementation and degenerate PCR approaches.

ABSTRACT: Two-component signal transduction systems (2CSs) are widely used by bacteria to sense and adapt to changing environmental conditions. With two separate approaches, three different 2CSs were identified on the chromosome of the probiotic bacterium Bifidobacterium infantis UCC 35624. One locus was identified by means of functional complementation of an Escherichia coli mutant. Another two were identified by PCR with degenerate primers corresponding to conserved regions of one protein component of the 2CS. The complete coding regions for each gene cluster were obtained, which showed that each 2CS-encoding locus specified a histidine protein kinase and an assumed cognate response regulator. Transcriptional analysis of the 2CSs by Northern blotting and primer extension identified a number of putative promoter sequences for this organism while revealing that the expression of each 2CS was growth phase dependent. Analysis of the genetic elements involved revealed significant homology with several distinct regulatory families from other high-G+C-content bacteria. The conservation of the genetic organization of these three 2CSs in other bacteria, including a number of recently published Bifidobacterium genomes, was investigated.

SUBMITTER: MacConaill LE

PROVIDER: S-EPMC165215 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Identification of two-component regulatory systems in Bifidobacterium infantis by functional complementation and degenerate PCR approaches.

MacConaill Laura E LE Butler Derek D O'Connell-Motherway Mary M Fitzgerald Gerald F GF van Sinderen Douwe D

Applied and environmental microbiology 20030701 7

Two-component signal transduction systems (2CSs) are widely used by bacteria to sense and adapt to changing environmental conditions. With two separate approaches, three different 2CSs were identified on the chromosome of the probiotic bacterium Bifidobacterium infantis UCC 35624. One locus was identified by means of functional complementation of an Escherichia coli mutant. Another two were identified by PCR with degenerate primers corresponding to conserved regions of one protein component of t ...[more]

PMID: 12839803

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Project description:This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by serRK, which is believed to control the expression of the ser2003 locus in Bifidobacterium breve UCC2003. The ser2003 locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region of ser2003 and the probable recognition sequence of SerR was determined by a combinatorial approach of in vitro site-directed mutagenesis, coupled to transcriptional fusion and EMSA assays. The importance of the serRK 2CRS in the response of B. breve to protease-mediated induction was confirmed by generating B. breve-s-serR and B. breve-::serU insertion mutants, which exhibited altered ser2003 transcriptional induction patterns as compared to their parent strain UCC2003. DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) 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.

Project description:Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass of 76 kDa. A rho-independent transcription terminator-like sequence was found 25 bp downstream of the termination codon. The amino acid sequences of beta-GalI and beta-GalIII are homologous to those found in the LacZ and the LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).

Dataset Information

Identification of two-component regulatory systems in Bifidobacterium infantis by functional complementation and degenerate PCR approaches.

Publications

Identification of two-component regulatory systems in Bifidobacterium infantis by functional complementation and degenerate PCR approaches.

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