Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium.

Ohnishi, Kouhei; Hasegawa, Akira; Matsubara, Keiko; Date, Taro; Okada, Toshihiko; Kiritani, Kazuyoshi

doi:10.1266/jjg.63.343

Cited by 32 publications

(18 citation statements)

References 25 publications

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“…Since cells of MGbcaP are viable in media containing free amino acids as the only amino acid sources, an additional transport system that retrieves BCAAs from the growth medium must be active under these conditions. A homolog of the well-conserved BCAA permease BrnQ (45,59,60) is specified by the chromosome of L. lactis MG1363, and our data suggest that the product of this gene is responsible for part of the BCAA uptake in MGbcaP. A region of BcaP is homologous to a stretch of amino acids of BrnQ, which might be an indication that this region contains a substrate (BCAA) recognition domain.…”

Section: ϫ5mentioning

confidence: 58%

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

2006

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Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

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Section: ϫ5mentioning

confidence: 58%

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

2006

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“…5). Our results show that the target of their action is not the AzlE (BrnQ) product, though the facts that the three proteins are coexpressed and that proteins similar to AzlE are involved in branched-chain amino acid transport (25,37,43) suggest that AzlC or AzlD and AzlE may somehow interact functionally.…”

Section: Discussionmentioning

confidence: 74%

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport

et al. 1997

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The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.Leucine-responsive protein (Lrp) of Escherichia coli is a global regulator affecting transcription of a diverse array of genes, many of which are involved in transport, biosynthesis, and degradation of amino acids; expression of most Lrp targets is modulated by leucine availability (9, 35). Lrp also negatively regulates its own expression (45). Lrp and its homologs, including E. coli AsnC (29), form an AsnC-Lrp family of transcription regulators that at present comprises more than 20 proteins from different groups of the domains Eubacteria and Archaea.One of the E. coli operons positively regulated by Lrp is the glutamate synthase operon, gltBDF (8,16). In Bacillus subtilis, transcription of the gltA and gltB genes, encoding the subunits of glutamate synthase, is subject to positive regulation by the product of the gltC gene (6, 7) but may be under the control of other proteins as well. The GltC protein belongs to the LysR family of bacterial regulators of transcription (41). Recently, a fragment of the B. subtilis chromosome containing one more gene of this family, gltR, that may be involved in gltAB regulation was cloned (5).While determining the sequence of the gltR (233°) region (5) of the B. subtilis chromosome, we came upon an open reading frame that could encode a homolog of Lrp. Given the involvement of Lrp in regulation of glutamate synthase synthesis in E. coli, we were motivated to undertake a detailed analysis of its B. subtilis homolog. The B. subtilis lrp-like gene proved to be identical to azlB, described as a locus conferring resistance to 4-azaleucine (46), and to be the first gene of an operon apparently involved in transport of branched-chain amino acids. Two operons needed for branched-chain amino acid transport in E. coli are regulated by Lrp (22). The azlB gene was shown not to be involved in glutamate synthase regulation. MATERIALS AND METHODSBacterial strains and culture media. Bacterial strains used in this study are listed in Table 1. B. subtilis strains were grown at 37°C in TSS minimal medium (20) with 0.5% glucose and a 0.2% nitrogen source, in Spizizen minimal medium with 0.5% glucose (42), or in DS nutrient broth medium (20). The same media with addition of agar or tryptose blood agar base medium (Difco) was used for growth of bacteria on plates. Strains carrying azl-lacZ transcriptional fusions integrated at the amyE locus were isolated after transformation of strain SMY with derivatives of pJPM82 (Table 1; also, see below), with selectio...

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“…Downstream of gltR we found a convergent open reading frame (coordinates 97 to 1416) similar (38 to 42% identity at amino acid level) to several genes (brnQ of Lactobacillus delbrueckii and Salmonella typhimurium, braB and braZ of Pseudomonas aeruginosa) encoding low-affinity transport proteins for branched-chain amino acids (21,34,42). The product of this open reading frame, which we called brnQ (440 amino acids; molecular mass, 47.0 kDa; pI ϭ 10.3), is predicted to have the 12 membrane-spanning domains common to the other members of this group (data not shown).…”

Section: Isolation Of Trans-acting Gltmentioning

confidence: 92%

Altered transcription activation specificity of a mutant form of Bacillus subtilis GltR, a LysR family member

Belitsky

Sonenshein

1997

J Bacteriol

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A mutation (gltR24) that allows Bacillus subtilis glutamate synthase (gltAB) gene expression in the absence of its positive regulator, GltC, was identified. Cloning and sequencing of the gltR gene revealed that the putative gltR product belongs to the LysR family of transcriptional regulators and is thus related to GltC. A null mutation in gltR had no effect on gltAB expression under any environmental condition tested, suggesting that gltR24 is a gain-of-function mutation. GltR24-dependent transcription of gltAB, initiated at the same base pair as GltC-dependent transcription, was responsive to the nitrogen source in the medium and required the integrity of sequences upstream of the gltAB promoter that are also necessary for GltC-dependent expression. Expression of the gltC gene, transcribed divergently from gltA from an overlapping promoter, was not affected by GltR. Both wild-type GltR and GltR24 negatively regulated their own expression. The gltR gene was mapped to 233؇ on the B. subtilis chromosome, very close to the azlB locus.Glutamate synthase, encoded by the gltAB genes, synthesizes glutamate from 2-ketoglutarate and glutamine and is an essential component of the major pathway for ammonia assimilation in Bacillus subtilis (39). Glutamate synthase gene expression is positively regulated in a nitrogen source-dependent manner by the product of the gltC gene. In the absence of GltC protein, gltAB transcription is drastically reduced under normally activating growth conditions, e.g., when ammonium ions are available as nitrogen source (7,8). GltC may not be the only factor controlling gltAB expression, however, since the low, residual expression of gltA in a gltC mutant decreases further under nonactivating growth conditions, e.g., when glutamate or proline serves as sole nitrogen source (5). We sought to identify a second factor involved in gltAB regulation by looking for mutants with elevated expression of gltA despite the absence of GltC.GltC belongs to the LysR family of bacterial transcriptional regulators (20, 38), a family which now comprises about 100 members. The members of this family share some common properties (38): (i) they have near their N termini a conserved helix-turn-helix region that is thought to be responsible for DNA binding; (ii) their target genes are frequently linked to and transcribed divergently from the genes for the LysR-type regulators; (iii) binding sites for many LysR-type proteins have a consensus sequence T-N 11 -A, with elements of dyad symmetry around the two conserved nucleotides, often separated by an (AϩT)-rich core; centers of these sequences are located near position Ϫ65 with respect to the transcription start sites of the target genes; (iv) for some LysR proteins the binding site extends beyond the consensus site or they have an additional binding site between the consensus sequence and the transcription start site of the target gene; and (v) the genes encoding LysR-type proteins are frequently negatively autoregulated. All of these properties apply to GltC (7,8).Th...

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Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium.

Cited by 32 publications

References 25 publications

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport

Altered transcription activation specificity of a mutant form of Bacillus subtilis GltR, a LysR family member

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