A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA

Boylan, S A; Eades, Linda J.; Janssen, K A; Lomax, Margaret I.; Bender, R.

doi:10.1007/bf00327420

Cited by 17 publications

(15 citation statements)

References 21 publications

(16 reference statements)

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“…operons of S. typhimurium, K. aerogenes, and Pseudomonas putida have also been cloned in E. coli (1,3,7,33), but their nucleotide sequences have not been determined. Genetic and biochemical studies of S. typhimurium (1, 32), K. aerogenes (3,13), and P. putida (7) revealed that the hut genes are organized into two or three transcriptional units with separated promoters. In contrast, the hut genes in B. subtilis seemed to be organized as a single transcriptional unit (6,18).…”

Section: Resultsmentioning

confidence: 99%

Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon

1988

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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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Section: Resultsmentioning

confidence: 99%

Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon

1988

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“…The DNA sequence shown in Fig. 2 was determined by using plasmid pCB101 as the source of DNA (6). The hut sequences on this plasmid had been maintained outside K. aerogenes for a considerable length of time, first as a A dhut transducing phage (28) and then as a plasmid (6).…”

Section: Resultsmentioning

confidence: 99%

“…Isolation and manipulation of DNA. Large-scale preparations of plasmid DNA were purified as described previously (6). In addition, plasmid DNA to be used for end labeling was passed over a Biogel A 1.5-m agarose (Bio-Rad) column to remove low-molecular-weight nucleic acids and nucleotides (18).…”

Section: Methodsmentioning

confidence: 99%

Bidirectional promoter in the hut(P) region of the histidine utilization (hut) operons from Klebsiella aerogenes

et al. 1988

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The hut(P) region (i.e., the region responsible for regulation of hutUH expression) of the Klebsiella aerogenes histidine utilization (hut) operons contains a bidirectional promoter. One transcript from this promoter encodes the hutUH operon; the role of the oppositely directed transcript is unknown, although it appears to be involved in regulating hutUH expression (A. J. Nieuwkoop, S. A. Boylan, and R. A. Bender, J. Bacteriol. 159:934-939, 1984 Klebsiella aerogenes can degrade the amino acid histidine enzymatically to ammonia, glutamate, and formamide (17). The histidine utilization (hut) genes are arranged in two tandem operons in the order hut(M)IGC(P)UH, where I, G, U, and H encode the four enzymes of the pathway and hutC encodes a repressor which regulates both operons (5, 11). The hut(M) and hut(P) regions represent the sequences controlling expression of the left and right operons, respectively. Therefore, the hut(P) region would be expected to include the binding site for RNA polymerase (hutUp), the binding site for repressor (hutUO), and the binding site(s) for positive effectors.Histidine can serve as the sole carbon or sole nitrogen source for K. aerogenes, and high-level expression of the hut operons requires not only the inactivation of the repressor but also the presence of a positive activator. This activator can be either the catabolite gene activator protein (CAP) in the presence of cyclic AMP (cAMP), signaling carbon or energy limitation, or an unknown factor(s) signaling nitrogen limitation (4, 23).We have previously suggested that the positive regulation of hutUH expression by CAP-cAMP might actually be a double-negative control (22) In this report we present the complete nucleotide sequence of the wild-type hut(P) region and identify the two RNA-polymerase-binding sites, Pc and PUH, within this sequence. In addition we identify consensus sequences suggesting binding sites for carbon and nitrogen effectors of hutUH expression (2,8). Thus, the data presented here support our previous double-negative control model of a bidirectional, mutually antagonistic promoter within hut(P). MATERIALS AND METHODSGrowth conditions. Plasmid-containing strains were grown in L broth or on L agar (3) with ampicillin at 100 jig/ml (LBamp). Minimal medium consisted of W salts (3) supplemented with 0.4% histidine as the sole carbon and nitrogen source. For S1 nuclease mapping, minimal medium was 2W5 salts (double-strength W salts adjusted to pH 7.5) supplemented with 0.4% glucose and 0.2% histidine for nitrogenlimited growth and 0.4% histidine alone for carbon-limited growth.Chemicals and enzymes. Restriction enzymes were purchased from

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“…We were able to resolve a remaining ambiguity in the published restriction map of the hut region (5 a Cells were grown in glucose-ammonia minimal medium with thiamine and thymidine as described previously (2) sufficient repressor (hutC product) is produced to allow normal repression of histidase formation, suggesting that hutC may be transcribed independently of hutI and hutG.…”

Section: Resultsmentioning

confidence: 99%

Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000

et al. 1990

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The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the Hindlll site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of TnlOOO into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of TnlOOO insertions in the "plus" or y8 orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the TnlOOO insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter.The hutlG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of TnlOOO insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.The histidine utilization (hut) gene cluster of Klebsiella aerogenes encodes the structural and regulatory proteins responsible for the inducible degradation of the amino acid L-histidine to glutamate, ammonia, and formamide (13). The hut genes are located between gal and bio on the K. aerogenes genetic map and are arranged in the order hut(M) IGC(P)UH (4, 7), where hut(M) and hut(P) are defined as the regions required for regulated transcription of hutI and hutU, respectively; hutl, hutG, hutU, and hutH encode the four enzymes of the pathway; and hutC encodes the hutspecific repressor which negatively regulates the entire pathway. In the closely related enteric bacterium Salmonella typhimurium, the hut cluster is arranged as two independent transcription units, one originating at hutIp [within the hut (M) region] and extending through hutIGC, and the other originating at hutUp [within the hut(P) region] and extending through hutUH (26,27). Several lines of evidence led us to expect that the arrangement of transcription units in K. aerogenes would be the same as that in S. typhimurium: (i) the gene order is identical; (ii) hutU (but not hutI) expression is coordinate with hutH (7, 14); (iii) hutU and hutH do not require the hutIGC region for expression (4); and (iv) the hut DNA from K. aerogenes and S. typhimurium are so similar that under appropriate conditions, a continuous heteroduplex molecule can be formed across the entire length of the cluster (3).To identify the transcription units within the hut cluster and to provide a mobile priming site for later DNA sequence analysis of the hut genes, we isolated and characterized a set of transposon Tn1OOO (-y)

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A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA

Cited by 17 publications

References 21 publications

Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon

Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon

Bidirectional promoter in the hut(P) region of the histidine utilization (hut) operons from Klebsiella aerogenes

Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000

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