INTRODUCTION

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In Bacillus subtilis, the sigL gene encodes a sigma factor homologous to members of the RpoN family of sigma factors (5). Promoters recognized by an RNA polymerase associated with RpoN have common features: (i) they are devoid of typical 10 and 35 sequences but contain a strongly conserved TGGCAC N5 TTGCA sequence centered at positions 12 and 24, and (ii) they require a positive regulatory protein with a central domain (the catalytic domain) which includes a conserved nucleotide-binding pocket. This positive regulatory protein interacts with upstream activating sequences (UAS) to stimulate the isomerization of closed complexes between RNA polymerase and the promoter DNA sequences to open complexes (21). RpoN differs from other alternative sigma factors in that it is needed for the transcription of genes whose products have diverse physiological roles.

In B. subtilis, sigL mutants have a pleiotropic phenotype: transcription of the levanase operon is strongly reduced, and catabolism of several amino acids (arginine, ornithine, isoleucine, and valine) is abolished (5). Most studies of the catabolism of branched-chain amino acids have been done with Pseudomonas, Streptomyces, and Streptococcus species. In these bacteria, the catabolism of branched-chain amino acids requires the cooperation of two sequential series of reactions. The enzymes of the first series constitute a common pathway and catalyze the conversion by deamination or dehydrogenation of Leu, Val, and Ile to their respective 2-keto acids. Branched-chain 2-keto acid dehydrogenase, which catalyzes the second step in this initial process, is a multienzyme complex (BCDH complex) involved in the oxidation of the 2-keto acid derivatives of all three branched-chain amino acids. The acyl coenzyme A metabolites formed at the end of the common pathway are catabolized by a series of enzymes, one specific for each initial amino acid (19).

The BCDH complex from several sources has been characterized; these include Pseudomonas aeruginosa (20), Pseudomonas putida (32), B. subtilis (12), rabbit liver (25), and rat and bovine kidneys (24, 26). The purified complexes from B. subtilis, P. putida, P. aeruginosa, and several mammals are all composed of four polypeptides: a dehydrogenase (E1), decarboxylase (E1), a dihydrolipoamide acyltransferase (E2), and a dihydrolipoamide dehydrogenase (E3). In B. subtilis, the BCDH complex is involved in the biosynthesis of branched-chain fatty acids, which are the major acyl constituents of the cell membrane (37). A bfmB mutant of B. subtilis requiring short branched-chain carboxylic acids for growth has been described. It is defective in branched-chain 2-keto acid dehydrogenase. Three genes, bfmBAA, bfmBAB, and bfmBB, encode the E1, E1, and E2 components, respectively, of the BCDH complex involved in the biosynthesis of branched-chain fatty acids (35). Little is known concerning the regulation of the expression of the genes involved in isoleucine and valine catabolism in B. subtilis. A sigL mutant cannot use isoleucine or valine as a source of nitrogen, suggesting that the expression of one or several enzymes of the isoleucine and valine degradation pathway is controlled by a transcriptional regulator with a central domain (5).

In this paper, we characterize an operon containing seven genes involved in the isoleucine and valine degradation pathway. Transcription of this operon is induced by the presence of isoleucine or valine in the growth medium and strongly depends on SigL. We also describe a new transcriptional regulator, BkdR, which has a central domain and which is an activator of the transcription of this operon.

	MATERIALS AND METHODS

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Bacterial strains and culture media. The B. subtilis strains used in this work are listed in Table 1. Escherichia coli TGI [K-12 (lac-pro) supE thi hsd5/F' traD36 proA⁺B⁺ lacI^q lacZM15] was used for cloning experiments. E. coli was grown in LB broth (27), and B. subtilis was grown in SP medium (8 g of nutrient broth/liter, 1 mM MgSO₄, 10 mM KCl, 0.5 mM CaCl₂, 10 µM MnCl₂, 2 µM FeSO₄) or MM minimal medium [60 mM K₂HPO₄, 44 mM KH₂PO₄, 15 mM (NH₄)₂SO₄, 3 mM trisodium citrate, 2 mM MgSO₄, 2.2 mg of ferric ammonium citrate per liter] supplemented with carbon sources (0.1%) and auxotrophic requirements (at 100 mg/liter). TPK minimal medium contains 50 mM glucose, 100 mM potassium phosphate (pH 7.0), 0.5 mM MgSO₄, 0.01 mM MnSO₄, 0.02 mM FeCl, 50 µg of tryptophan per ml, and 20 mM nitrogen source.

Transformation and phenotype characterization. Standard procedures were used to transform E. coli (27), and transformants were selected on LB broth plates containing ampicillin (100 µg/ml). B. subtilis was transformed with plasmid or chromosomal DNA as previously described (16), and transformants were selected on SP medium plates containing chloramphenicol (5 µg/ml), kanamycin (5 µg/ml), erythromycin (1 µg/ml), and lincomycin (10 µg/ml). Amylase activity in B. subtilis was detected after growth on tryptose blood agar base (Difco) containing 10 g of hydrolyzed starch per liter (Connaught). Starch degradation was detected by sublimating iodine onto the plates.

DNA manipulation. Standard procedures were used to extract plasmid from E. coli (27). Restriction enzymes, phage T4 DNA polymerase, phage T4 DNA ligase, and T4 polynucleotide kinase were used as recommended by the manufacturers. DNA fragments were purified from the agarose gel with a Prep-A-Gene kit (Bio-Rad Laboratories, Richmond, Calif.). The PCR technique with Thermus aquaticus DNA polymerase was used for amplification. The oligonucleotide primers used included mismatches allowing the creation of EcoRI and BamHI restriction sites or HindIII and BamHI restriction sites.

Plasmid constructions. pAC5 (17), a derivative of pAF1 (6), carries the pC194 chloramphenicol resistance gene cat and a lacZ gene between two fragments of the B. subtilis amyE gene. PCR was used to introduce EcoRI restriction sites at various positions upstream from ptb. PCR was performed with one oligonucleotide (5'-GGAGGATCCTCAGCATGAGCAAC-3') corresponding to the coding sequence of the ptb gene (codons 20 to 25) and the other one corresponding to various positions in the ptb promoter region. The EcoRI-BamHI restriction fragments generated in this way were inserted between the EcoRI and BamHI restriction sites of pAC5, creating translational fusions between codon 25 of ptb and codon 8 of lacZ. The DNA sequences of the different PCR fragments were verified by direct sequencing of the various corresponding plasmids. The resulting plasmids were linearized at the single PstI restriction site and integrated into the chromosome of strain 168 by homologous recombination at the amyE locus by use of chloramphenicol selection. Integrants carrying the translational fusions were named QB7501, QB7517, QB7518, QB7519, and QB7520 (see Fig. 5 and Table 1).

Reverse transcriptase mapping of the mRNA start point in the ptb gene. Total RNA was isolated from B. subtilis 168 grown in MM medium supplemented with glucose and tryptophan and with or without 20 mM isoleucine as the inducer. Exponentially growing cells were harvested at an optical density at 600 nm of 1, and RNA was extracted as previously described (8). Two oligonucleotides, O₁ (5'-CCTCAGCATGAGCAACCGCAATGGTC-3') and O₂ (5'-GTGTATCGACGCTTTGCCGATTAAATC-3'), complementary to the ptb coding sequence were labelled with 10 U of polynucleotide kinase and 0.37 MBq of [-³²P]ATP (15 TBq/mmol; Amersham). DNA primers were elongated, and the products were analyzed as previously described (15) and shown to have the same transcriptional start sites.

-Galactosidase assays. B. subtilis cells containing lacZ fusions were grown to an optical density at 600 nm of 1. -Galactosidase specific activities were determined as previously described and are expressed as Miller units per milligram of protein (5). The values reported represent averages from at least three independent assays.

	RESULTS

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Identification of an activator involved in isoleucine utilization in B. subtilis. Members of the family of activators of ⁵⁴-dependent transcription are composed of three distinct functional domains, an NH₂-terminal domain, a central domain, and a COOH-terminal domain, involved in signal reception, transcriptional activation, and DNA binding, respectively. The central domain and the C-terminal domain are the most highly conserved among these regulators (30). Degenerate oligonucleotide primers have been used to amplify DNA fragments encoding central domains from the genomes of diverse bacteria, including B. subtilis. This procedure led to the identification of both known genes (levR and rocR) and two novel gene fragments, called 70-Bsu and 81-Bsu (9). The 70-Bsu DNA fragment (a gift from I. Kaufman and T. Nixon) was cloned in pHT181, an integrative plasmid (11). The recombinant plasmid was used to transform B. subtilis 168. Since the 70-Bsu DNA fragment is an internal fragment of the gene, the integration led to a null mutation in the corresponding chromosomal gene. The B. subtilis mutant strain containing pHT181::70-Bsu inserted by homologous recombination in the corresponding gene was tested for growth in TPK minimal medium containing isoleucine (20 mM) as the sole nitrogen source. This strain grew much more slowly than the wild-type strain in the same medium containing isoleucine (data not shown). The mutant strain and the wild-type strain grew well with (NH₄)₂SO₄ as the sole nitrogen source. These results strongly suggest that the DNA fragment encoding a new central domain is part of a regulatory gene controlling isoleucine utilization in B. subtilis.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Proposed pathway for the degradation of branched-chain amino acids in B. subtilis.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Organization of the structural genes of the bkd operon of B. subtilis. The proposed functions of the gene products are based on similarities to the corresponding genes from Enterococcus faecalis (36). CoA, coenzyme A.

Promoter and control regions located upstream from the ptb gene. In gram-negative and gram-positive bacteria, all promoters recognized by holoenzymes containing ⁵⁴ possess at least a conserved GG doublet located at position 24 upstream from the transcriptional start site, followed by a GC doublet at position 12, with a spacing region of 10 bp. They do not have the typical 10 and 35 hexamers recognized by the major housekeeping sigma factor (21). The transcriptional start site of ptb was mapped by primer extension with reverse transcriptase and two oligonucleotides, O₁ and O₂. The same 5' ends were identified with each oligonucleotide (Fig. 3). Transcription started at two adjacent nucleotides. The promoter region contains a region consistent with the consensus sequence (12 and 24) of ⁵⁴-dependent promoters (Fig. 4).

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Reverse transcriptase mapping of the transcriptional start site of the ptb gene in B. subtilis 168 grown in the presence (lane A) or absence (lane B) of 20 mM isoleucine. The positions of the cDNA-extended fragments identified with oligonucleotide O1 were compared with those obtained by sequencing of an M13 recombinant phage containing the promoter region with the same oligonucleotide used as a primer (lanes at left, TCGA, respectively, from left to right). Transcriptional start sites are indicated by the asterisks.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Nucleotide sequences of promoter regions of the levanase operon (plev), the rocABC operon (procABC), the rocDEF operon (procDEF), and the bkd operon (pbkd). The transcriptional start sites are indicated by arrows. Boxes indicate strictly conserved DNA sequences around positions 12 and 24 with respect to the transcriptional start sites.

View larger version (48K): [in this window] [in a new window]   FIG. 5.   Nucleotide sequence of the ptb upstream region. Deletion end points are indicated by arrows and numbered with respect to the transcriptional start site (indicated by asterisks). The sequences at positions 12 and 24 are indicated. Boxed regions indicate putative UAS. The effects of upstream deletions on the expression of the ptb'-'lacZ translational fusions are expressed as -galactosidase specific activities, which were determined with extracts prepared from cells growing exponentially in MM minimal medium containing glucose and 20 mM isoleucine as the inducer. In the absence of isoleucine, the basal level was about 5 U/mg of protein for each strain. For deletion end points A (232), B (122), C (107), D (92), and E (77), -galactosidase activities were 2,350, 2,135, 205, 5, and 10 U/mg of protein, respectively.

Induction of the expression of the bcd, lpd, and bkdB genes. pMutin4 (34) was used to construct transcriptional fusions with the lacZ gene. These constructions were integrated by homologous recombination into the bcd, lpd, and bkdB genes, leading to strains QB7514, QB7507, and QB7521, respectively. The recombination event was mediated by DNA fragments generated by PCR, inserted into the vector, and corresponding to the internal part of each gene. Integration of the plasmids (i) inactivates the corresponding gene; (ii) fuses the target gene to the lacZ gene; and (iii) allows the expression of downstream genes from an IPTG-inducible promoter, pspac. The -galactosidase activities of strains QB7514, QB7507, and QB7521 were assayed after culturing in MM glucose minimal medium containing or not containing 20 mM isoleucine as the inducer (Table 4). The -galactosidase expression in strains QB7514, QB7507, and QB7521 was induced by 20 mM isoleucine in the growth medium. The uninduced level of -galactosidase expression was about 300 U/mg of protein. The addition of 1 mM IPTG to the growth medium led to a strong decrease in lacZ expression from the bcd'-lacZ fusion, suggesting that the product of one or several genes downstream from bcd represses the 12, 24 promoter (see Discussion). A bkdR::aphA3 null mutation was introduced into the bcd'-lacZ strain and into the lpd'-lacZ strain by transformation, leading to strains QB7515 and QB7513, respectively. -Galactosidase was not expressed in cultures of these strains, whether or not they contained isoleucine as the inducer. Attempts to combine a bkdB::pMutin4 null mutation with either a sigL or a bkdR null mutation were unsuccessful, suggesting that three of the four subunits of the BCDH complex are essential for cell viability.

Disruption of bkdR and phenotypes of bcd, lpd, and bkdB mutants. B. subtilis QB7512 (containing the aphA3 gene inserted into bkdR) cannot grow in the presence of the branched-chain amino acids isoleucine and valine as the sole nitrogen sources. Thus, the product of the bkdR gene is involved in the control of the catabolism of these amino acids (Table 2). A similar phenotype was observed for strain QB7514, in which the pMutin4 vector was integrated into the bcd gene (Table 2). This strain was unable to use isoleucine or valine as a sole nitrogen source. Moreover, the doubling time with ammonia as a nitrogen source was unaffected. This result indicates that the bcd gene product, which shows similarities to leucine dehydrogenases, is involved in isoleucine and valine catabolism.

Regulation of the operon in response to nitrogen or amino acid availability. Two B. subtilis proteins, TnrA and CodY, are known to be involved in regulation by the nitrogen source and by an excess of amino acids, respectively. TnrA is a transcriptional factor required for global nitrogen regulation in B. subtilis (38). During nitrogen limitation, the TnrA protein is required for activation of the transcription of gabP, nasB, and nrgAB and negatively regulates glnRA expression. We introduced a trnA::erm mutation into strain QB7501 to give strain QB7505. lacZ expression in cultures of this strain during growth with the poor nitrogen source glutamate was assayed. In the absence of an inducer, lacZ expression in MN (7) glucose minimal medium containing glutamate was 4.5-fold higher in strain QB7505 than in strain QB7501. These results suggest that the tnrA gene product may act as a repressor of the operon. The presence of 20 mM isoleucine did not affect the induced level of expression in tnrA mutants (data not shown; see Discussion).

	DISCUSSION

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The product of sigL is involved in the catabolism of isoleucine or valine present as the sole nitrogen source (5). In this work, we have identified an operon, the bkd operon, containing seven genes and involved in the deamination of isoleucine and valine and in the oxidative decarboxylation of branched-chain -keto acids. Recently, a cluster of seven genes with a similar organization and involved in the breakdown of branched-chain amino acids was found in Enterococcus faecalis. Four of these genes, ldp, bkdA1, bkdA2, and bkdB, encode the E3, E1, E1, and E2 components of a BCDH complex. The genes preceding lpd in E. faecalis, ptb and buk, are similar to phosphotransbutyrylase and butyrate kinase genes, respectively, and are probably involved in ATP generation (36).

Here, we have shown that transcription of the bkd operon in B. subtilis is induced by the presence of isoleucine or valine in the growth medium. The -keto acids -keto--methylvalerate and -ketoisovalerate are also inducers, a situation previously observed with P. putida (14). In a bcd null mutant (QB7514), the -galactosidase expression observed in the absence of isoleucine in the growth medium was decreased by the presence of IPTG, which induces the expression of the downstream genes. As branched-chain amino acids or -keto acids are probably internal inducers, it is likely that the intracellular levels of these inducers depend upon the expression of the downstream genes lpd, bkdA1, bkdA2, and bkdB. The bfmB mutant of B. subtilis requires branched short-chain carboxylic acids for growth. However, the lpd and bkdB genes could be interrupted without a loss of cell viability. Transcription of the operon is SigL dependent, and it was previously shown that mutants affected in the BCDH complex require branched-chain -keto acids for growth. sigL and bkdR null mutants are viable, and it is therefore likely that the four genes encoding the different polypeptides of the BCDH complex are constitutively transcribed at a low level to ensure the synthesis of membrane branched-chain carboxylic acids. This basal level of transcription could be due to read-through transcription from the bkdR promoter upstream from the operon. The bkdR null mutant was constructed by inserting an antibiotic resistance cassette in bkdR. This cassette might affect the basal level of transcription of the operon, which could explain the slower rate of growth of the bkdR mutant than of the sigL mutant with ammonia as the sole nitrogen source. The expression of the operon is also subject to global regulation. During exponential growth in MM glucose minimal medium containing isoleucine, the expression of the bkd operon is slightly repressed by the addition of Casamino Acids, and this repression is codY dependent. The uninduced level of the bkd operon increases 3-fold in tnrA mutants, 7-fold in the codY mutant in MM minimal medium containing glucose, and 17-fold in the presence of Casamino Acids.

CodY is a repressor which binds to promoters in AT-rich regions, known to exhibit bending and curvature. Interestingly, looped DNA structures are involved in the formation of contacts between E⁵⁴ holoenzymes and enhancer binding proteins. However, it would be interesting to test whether TnrA and CodY bind directly to the bkd promoter region. Another possibility is that the intracellular level of the inducer is higher in tnrA and codY mutants than in the wild-type strain, leading to a higher level of expression of the bkd operon in the mutants. Transcription of the operon at a high level requires the product of bkdR, a regulator with a central domain.

The different domains of members of this family of transcriptional activators have different functions and exhibit various degrees of evolutionary conservation (23). Typically, the amino-terminal domain is the signal reception domain of approximately 160 residues. There is only one known exception, the LevR protein of B. subtilis, in which the carboxy-terminal domain of the protein contains two signal reception domains (18). The amino-terminal part of BkdR is a large domain composed of 360 residues, a characteristic which is shared by two other regulators containing a central domain: AcoR from Alcaligenes eutrophus (9a) and AcoR from B. subtilis (10). This amino-terminal part contains two PAS-like domains (Fig. 6) (39). These domains, previously reported for proteins from mammals, insects, plants, fungi, and cyanobacteria, are involved in protein-protein interactions. They have been identified for a variety of sensor proteins that sense light, oxygen, and redox potential. In bacteria, the PAS domain is usually associated with the input domain of a histidine kinase or a sensor protein that regulates a histidine kinase. A PAS-like domain has also been found in the RocR regulator controlling arginine utilization in B. subtilis (39). Constitutive mutations located in the amino-terminal domain of RocR affect conserved residues of the PAS domain (3, 7), indicating that it is probably involved in the control of the activity of RocR. These PAS repeats may also be involved in the control of BkdR activity in response to the presence of the internal inducer. Site-directed mutagenesis of conserved residues in PAS domains of BkdR is under way as part of work to confirm their involvement in BkdR activity. Moreover, as nothing is known about the transcription of bkdR, further work is needed to define its regulation.

View larger version (15K): [in this window] [in a new window]   FIG. 6.   Alignment of PAS-like domains of RocR and BkdR from B. subtilis. Conserved amino acid residues are boxed. Asterisks indicated the locations of constitutive missense mutations in RocR.