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

Abstract

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...

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

“…It encodes a neutral 16.4-kDa protein, LrpC, that forms tetramers in solution. Based on amino acid sequence identity, it has been assigned to the Lrp/AsnC family of transcriptional regulators, which in B. subtilis includes seven Lrp/AsnC-like proteins (12)(13)(14). The N-terminal region of LrpC is predicted to form a typical helix-turn-helix DNA binding domain, characteristic of numerous transcriptional regulators (15).…”

A novel streptococcal surface protease promotes virulence, resistance to opsonophagocytosis, and cleavage of human fibrinogen

“…A gene designated sblA (for streptococcus group B lipoprotein) is located 5′ of cspA and is oriented in the opposite transcriptional direction as cspA. The predicted product shares 33% identity with AzlC from Deinococcus radiodurans (37); the precise biological function of the AzlC protein is currently unknown (38). Adjacent to cspA and oriented in the same direction are two putative genes (designated sbrA and sbsA) that encode products with homology to the response regulator and sensor proteins of two-component regulatory systems (39).…”

Induced Transcriptional Expression of <i>Bacillus subtilis</i> Amino Acid Permease <i>yvbW</i> in Response to Leucine Limitation

“…Alternatively, glutamate is utilized as an amine donor in the final step of leucine, isoleucine and valine biosynthesis; therefore glutamate is also a potential substrate for YvbW transport. Several additional transporters with putative substrate specificity for leucine and glutamate have also been identified in B. subtilis, suggesting a functional redundancy [21] [22] [26]. It is also possible that YvbW may transport valine, isoleucine or one of their precursors, which could be converted to leucine.…”

“…No difference in growth was observed between the yvbW knockout strain and the control strain. These strains were also tested for resistance to the leucine analog 4-aza-leucine (Sigma) [26]. Spizizen minimal media (leucine at 5 µg/ml) was supplemented with 2, 20 and 200 µg/ml 4-aza-leucine and growth curves were monitored.…”