An 8-kilobase HindlIl fragment carrying the histidase gene (hutH) and its regulatory region (hutP), from the Bacillus subtilis histidine utilization (hut) operon, was cloned in the temperate bacteriophage +105. Histidine utilization was restored in a hutHl mutant by the specialized transducing phage (+lO5hutH11). The histidase gene in +105hutH11 was inducible and was shown to be under catabolite repression. The nucleotide sequence of 3,932 base pairs including the hutH and hutP loci revealed three open reading frames (ORFs). The molecular weights of ORF1 and ORF2 proteins were calculated to be 16,576 (151 amino acid residues) and 55,675 (508 amino acid residues), respectively. Reverse transcriptase mapping experiments showed that the putative promoter for the hut operon could be recognized by RNA polymerase sigma 43. The transcript starts at an adenosine residue 32 base pairs upstream from the initiation codon of ORF1. hutH+-transforming activity was found in ORF2, indicating that ORF2 encoded the histidase. A hutPI mutation was determined as a substitution of an amino acid in ORF1. By using a specialized transducing phage containing the wild-type ORFi gene, it was demonstrated that the presence of ORF1 protein in trans was absolutely required for the induction of the hut operon in a hutPI mutant. These data strongly suggested that ORF1 encodes a positive regulator of the hut operon.The enzymes involved in histidine catabolism have been studied for many bacterial strains, such as Salmonella typhimurium, Klebsiella aerogenes, Pseudomonas species, and Bacillus subtilis. In all cases except the Pseudomonas species, histidine is catabolized to glutamic acid, ammonia, and formamide by the action of four enzymes (20); in the Pseudomonas species, five enzymes are involved (19). Genetic and biochemical studies have revealed that the genes responsible for histidine catabolism are organized as operons (hut operon) (13,18,20).hut genes in all cases are inducible and controlled by catabolite repression (19,20). The molecular basis of the regulation is well characterized for S. typhimurium and K. aerogenes. The expression of hut genes is regulated negatively by urocanic acid and positively by the catabolite activator protein with cyclic AMP (20). In addition, a third regulatory mechanism has been described for K. aerogenes and Pseudomonas aeruginosa, in which catabolite repression is relieved during nitrogen-limited growth (26,27
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