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. 2003 Jul;69(7):4219-26.
doi: 10.1128/AEM.69.7.4219-4226.2003.

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

Laura E MacConaill  1 , Derek Butler, Mary O'Connell-Motherway, Gerald F Fitzgerald, Douwe van Sinderen
Affiliations

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

Laura E MacConaill et al. Appl Environ Microbiol. 2003 Jul.

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.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of three 2CSs identified on the chromosome of B. infantis UCC 35624 and their surrounding ORFs. Arrows represent each ORF, with the gene name positioned above. The lengths of the transcripts identified by Northern analysis are indicated underneath each system by a thin arrow. Positions of promoter sequences deduced from primer extension and/or Northern blot analysis are indicated by bent arrows. The positions of putative transcriptional terminator structures are indicated by “lollipops.”
FIG. 2.
FIG. 2.
Alignment of genetic organization of system A from B. infantis UCC 35624 with corresponding loci in M. tuberculosis CDC1551 (accession no. AE007145), Mycobacterium avium subsp. paratuberculosis (AF10884), B. longum NCC2705 (AE014617), B. longum DJO10A (NZ_AABM02000022), and B. breve NCIMB8807 (unpublished data). The names of the genes are indicated within arrows for UCC 35624. The percent identities for each protein-encoding gene compared to the corresponding ORF from UCC 35624 are indicated within the arrows for each genome. The degree of amino acid identity (>90%, >80%, >70%, and <70%) is indicated by the color of the arrow (red, yellow, green, and blue, respectively).
FIG. 3.
FIG. 3.
Northern analysis of systems A and B with RNA isolated from B. infantis UCC 35624 at different optical densities at 600 nm (indicated above each lane). The estimated sizes of the transcripts are indicated on the right. (a) Transcription of system A with an internal 500-bp fragment of bikA as a probe. Similar results were obtained with probes birA and lipA. (b) Transcription of system A with probe gtpA. Similar results were obtained with probes biaA, biaB, and biaC. (c) Transcription of system B with probe bikB. Similar results were obtained with probe birB. Northern blots also revealed a 3-kb transcript for system C (not shown) with probes bikC, birC, and B. infantis orfC.
FIG. 4.
FIG. 4.
Primer extension (PE) analysis of transcriptional start site of 11-kb transcript of system A. The assumed ribosome-binding site (RBS) and start codon (ATG) of gtpA are indicated in bold. The transcriptional start site is indicated by a solid triangle, and the name of the gene is indicated in italics over the initial methionine residue. The translated amino acid residues of GtpA are shown underneath the corresponding DNA sequence. The arrow indicates the position of the extension product. Proposed −10 and −35 motifs are boxed.
FIG. 5.
FIG. 5.
Primer extension (PE) analysis of transcriptional start site of 4-kb transcript of system A. The assumed ribosome-binding site (RBS) and start codon (ATG) of birA are indicated in bold. The transcriptional start site is indicated by a solid triangle, and the name of the gene is indicated in italics over the initial methionine residue. The translated amino acid residues of BirA are shown underneath the corresponding DNA sequence. The arrow indicates the position of the extension product. Proposed −10 and −35 motifs are boxed.
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