Histidine Utilization Research Articles

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

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the HindIII site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of Tn1000 into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of Tn1000 insertions in the "plus" or gamma delta orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the Tn1000 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 hutIG 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 Tn1000 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.

Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes

The hutC gene of Klebsiella aerogenes encodes a repressor that regulates expression of the histidine utilization (hut) operons. The DNA sequence of a region known to contain hutC was determined and shown to contain two long rightward-reading open reading frames (ORFs). One of these ORFs was identified as the 3' portion of the hutG gene. The other ORF was the hutC gene. The repressor predicted from the hutC sequence contained a helix-turn-helix motif strongly similar to that seen in other DNA-binding proteins, such as lac repressor and the catabolite gene activator protein. This motif was located in the N-terminal portion of the protein, and this portion of the protein seemed to be sufficient to allow repression of the hutUH operon but insufficient to allow interaction with the inducer. The presence of a promoterlike sequence and a ribosome-binding site immediately upstream of the hutC gene explained the earlier observation that hutC can be transcribed independently of the other hut operon genes. The predicted amino acid sequence of hut repressor strongly resembled that of the corresponding protein from Pseudomonas putida (S. L. Allison and A. T. Phillips, J. Bacteriol. 172:5470-5476, 1990). An unexpected, leftward-reading ORF extending from about the middle of hutC into the preceding (hutG) gene was also detected. The deduced amino acid sequence of this leftward ORF was quite distinct from that of an unexpected ORF of similar size found immediately downstream of the P. putida hutC gene. The nonstandard codon usage of this leftward ORF and the expression of repressor activity from plasmids with deletions in this region made it unlikely that this ORF was necessary for repressor activity.

Cascading regulation of histidase activity in Streptomyces griseus

Mutants of Streptomyces griseus unable to utilize histidine as the sole nitrogen source have been isolated and characterized. Using a mutant defective in the production of histidase, we have demonstrated that urocanate functions as the inducer of the histidine utilization system. Another mutant produced histidase that was locked in an inactive form but could be activated by treatment with an extract from the wild-type strain or the histidase-negative strain. This mutant was deficient in the activity of a protein of Mr ca. 90,000 to 100,000 that is required for the activation of histidase. Histidase was synthesized constitutively but was maintained in an inactive form until after histidine or urocanate was added to the medium. At least four components were implicated in the activation of histidase: histidase, the activation protein, urocanate, and a phosphatase that is apparently inactive in cells grown without inducer. The functions of the last three factors could be supplanted in vitro by incubation of histidase with snake venom phosphodiesterase or 5' nucleotidase. The results suggest that histidine utilization by S. griseus is controlled posttranslationally by an activation cascade that involves at least two regulatory proteins.

Regulation of the galactose-inducible lac operon and the histidine utilization operons in pts mutants of Klebsiella aerogenes.

Galactose appears to be the physiological inducer of the chromosomal lac operon in Klebsiella aerogenes. Both lactose and galactose are poor inducers in strains having a functional galactose catabolism (gal) operon, but both are excellent inducers in gal mutants. Thus the slow growth of K. aerogenes on lactose reflects the rapid degradation of the inducer. Several pts mutations were characterized and shown to affect both inducer exclusion and permanent catabolite repression. The beta-galactosidase of pts mutants cannot be induced at all by lactose, and pts mutants appear to have a permanent and constitutive inducer exclusion phenotype. In addition, pts mutants show a reduced rate of glucose metabolism, leading to slower growth on glucose and a reduced degree of glucose-mediated permanent catabolite repression. The crr-type pseudorevertants of pts mutations relieve the constitutive inducer exclusion for lac but do not restore the full level of glucose-mediated permanent catabolite repression and only slightly weaken the glucose-mediated inducer exclusion. Except for weakening the glucose-mediated permanent catabolite repression, pts and crr mutations have no effect on expression of the histidine utilization (hut) operons.

RNA polymerase as a repressor of transcription in the hut(P) region of mutant Klebsiella aerogenes histidine utilization operons.

Mutants of Klebsiella aerogenes able to express the hutUH operon in the absence of positive effectors were isolated and characterized. These mutations improve the hutUH promoter (PUH) by changing the -10 region to match the consensus sequence more closely. These mutations also affect another, oppositely oriented promoter in this region, PC. Although the mutations lie far outside PC, they cause PC to be inactive, apparently because binding of RNA polymerase to the PUH promoter blocks the overlapping PC site. Thus, in the mutants, RNA polymerase bound at the strong (mutant) PUH site effectively repress the PC promoter.

Organization and multiple regulation of histidine utilization genes in Pseudomonas putida.

The arrangement of the histidine utilization (hut) genes in Pseudomonas putida was established by examining the structure of a DNA segment that had been cloned into Escherichia coli via a cosmid vector. Southern blot analysis revealed that the restriction patterns of the hut genes cloned into E. coli and present in the P. putida genome were identical, indicating that no detectable DNA rearrangement took place during the cloning. Expression of the hut genes from a series of overlapping clones indicated the gene order to be hutG-hutI-hutH-hutU-hutC-hutF. The transcription directions of the different hut genes were determined by cloning the genes under control of the lambda pL promoter. This showed that hutF, encoding formiminoglutamate hydrolase, was transcribed in a direction opposite to that of the other genes. Inactivation of the cloned hut genes by Tn1000 insertion revealed that the hut genes were divided into three major transcriptional units (hutF, hutC [the repressor gene], and hut UHIG), but hutG may also be independently transcribed. When cloned individually with hutC on the same vector, hutF and hutU (which encodes urocanase) expression was induced by urocanate, indicating that these two genes each possess an operator-promoter element. Tn1000 insertions (in the cloned genes) or Tn5 insertions (in the P. putida genome) affecting the hutI or hutH gene only partially eliminated hutG expression. Furthermore, hutG, which specifies N-formylglutamate amidohydrolase, was regulated by the hutC product when the two genes were cloned on the same vector and expressed in E. coli. Therefore, hutG can be expressed independently from its own promoter, in keeping with earlier observations that N-formylglutamate amidohydrolase synthesis is not coordinated with that of urocanase and histidase and can be induced by N-formylglutamate or urocanate.

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

An 8-kilobase HindIII 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 phi 105. Histidine utilization was restored in a hutH1 mutant by the specialized transducing phage (phi 105hutH11). The histidase gene in phi 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 hutP1 mutation was determined as a substitution of an amino acid in ORF1. By using a specialized transducing phage containing the wild-type ORF1 gene, it was demonstrated that the presence of ORF1 protein in trans was absolutely required for the induction of the hut operon in a hutP1 mutant. These data strongly suggested that ORF1 encodes a positive regulator of the hut operon.

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

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). A 247-base-pair (bp) fragment containing hut(P) carries two RNA-polymerase-binding sites agree with the start sites of the two transcripts produced from hut(P) DNA in vitro and in vivo. The binding sites share a 4-bp region, suggesting that occupancy of the regulatory site precludes occupancy of the hutUH promoter, and vice versa. In the absence of positive effectors, the binding to the site responsible for hutUH transcription is weaker than the binding to the site responsible for regulation. The nucleotide sequence of the 250-bp fragment containing hut(P) contains two possible matches to the consensus sequence for Escherichia coli promoters, a better and worse match, corresponding in position to the stronger and weaker RNA-polymerase-binding sites, respectively. The sequence also contains a region similar to the consensus sequence for binding of the catabolite gene activator protein of E. coli. A sequence similar to the consensus for Ntr-dependent promoters was also found, overlapping both RNA-polymerase-binding sites, but it is not a functional promoter. Finally, an initiation codon preceded by a Shine-Dalgarno consensus sequence and followed by an open reading frame identifies a probable start of the hutU gene coding sequences.

Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida.

Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida. By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate. Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did. This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate. A 9.6-kilobase-pair HindIII DNA fragment containing the P. putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli. Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter. FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps. Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II). FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C. The enzyme was maximally active in the range of pH 7 to 8. FGase was found to be a monomer of molecular weight 50,000. N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively. The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.

Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida.

A library of the Pseudomonas putida chromosome, prepared through the use of the cosmid pJB8 ligated to a partial Sau3A digest of bacterial DNA, followed by in vitro packaging into bacteriophage lambda particles, was used to construct a strain of Escherichia coli which contained the genes for histidine utilization. This isolate produced a repressor product and all five enzymes required in Pseudomonas spp. for histidine dissimilation, whereas none of these could be detected in the nontransduced parent E. coli strain. When this transductant was grown on various media containing histidine or urocanate as the inducer, it was observed that production of the cloned histidine degradative enzymes was influenced somewhat by the choice of nitrogen source used but not by the carbon source. The recombinant cosmid was isolated and found to consist of 21.1 kilobase pairs of DNA, with approximately 16 kilobase pairs derived from Pseudomonas DNA and the remainder being from the pJB8 vector. Digestion of this insert DNA with EcoRI provided a 6.1-kilobase-pair fragment which, upon ligation in pUC8 and transformation into an E. coli host, was found to encode histidine ammonia-lyase and urocanase. The inducible nature of this production indicated that the hut repressor gene also was present on this fragment. Insertional inactivation of the histidine ammonia-lyase and urocanase genes by the gamma-delta transposon has permitted location of these structural genes and has provided evidence that transcription proceeds from urocanase through histidine ammonia-lyase. Mapping of the 16-kilobase-pair Pseudomonas DNA segment with restriction enzymes and subcloning of additional portions, one of which contained the gene for formiminoglutamate hydrolase and another that could constitutively express activities for both imidazolone propionate hydrolase and formylglutamate hydrolase, has provided evidence for the organization of all hut genes.

In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli

Histidine ammonia-lyase catalyzes the first step in histidine catabolism, the deamination of histidine to urocanate and ammonia. In vitro experiments have shown that histidine ammonia-lyase also can catalyze the reverse (amination) reaction, histidine synthesis, relatively efficiently under extreme reaction conditions (4 M NH4OH, pH 10). An Escherichia coli hisB deletion strain was transformed with a pBR322 derivative plasmid (pCB101) containing the entire Klebsiella aerogenes histidine utilization (hut) operon to determine whether the catabolic histidine ammonia-lyase could function biosynthetically in vivo to satisfy the histidine auxotrophy. Although the initial construct did not grow on media containing urocanate and ammonia as a source of histidine, spontaneous mutants possessing this ability were isolated. Four mutants characterized grew at doubling times of 4 h compared with 1 h when histidine was present, suggesting that histidine synthesis, although unequivocally present, remained growth limiting. Each mutant contained a plasmid-encoded mutation which eliminated urocanase activity, the second enzyme in the Hut catabolic pathway. This genetic block led to the accumulation of high intracellular levels of urocanate, which was subsequently converted to histidine via histidine ammonia-lyase, thus satisfying the histidine auxotrophic requirement.

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

The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.

Genetic and physical maps of Klebsiella aerogenes genes for histidine utilization (hut).

Deletion derivatives of the hut-containing plasmid pCB101 were tested against point mutants defective in individual genes of the histidine utilization (hut) operons using a complementation/recombination assay. Location of the genes of the right operon, hutU and hutH, was confirmed by direct assay of the gene products, urocanase and histidase; location of the repressor gene was identified by measuring the ability of the plasmid-carried genes to repress the formation of histidase from a chromosomal location. The analysis of eight deletion plasmids unambiguously confirms the map order of the hut genes as hutI-G-C-U-H, and demonstrates that, in Klebsiella aerogenes, the hutU and hutH genes are transcribed from their own promoter. In addition, the genetic map of hut can be aligned with the restriction map of the hut DNA in plasmid pCB101. One of the deletion plasmids studied apparently encodes a defective histidase subunit that is trans-dominant to active histidase. Another deletion, which completely removes the left operon, hutIG, allows high level expression of the hutUH operon and thus overproduction of a toxic intermediate.

Effect of molecular hydrogen on histidine utilization by Alcaligenes eutrophus

The effect of molecular hydrogen on heterotrophic metabolism of the facultative chemolithoautotrophic bacterium Alcaligenes eutrophus strain H 16 was representatively investigated on histidine utilization. The presence of hydrogen in a histidine or urocanate-containing medium had two effects (i) growth of the cells was inhibited, and (ii) formation of histidase was repressed. Both effects were relieved by supplying the cells with exogenous carbon dioxide. Studies on mutants defective in chemolithoautotrophic metabolism revealed that growth inhibition by hydrogen was exclusively mediated by the catalytic function of the soluble hydrogenase. Mutants containing only particulate hydrogenase activity did not exhibit growth inhibition. Repression of histidase formation, however, was mediated by the catalytic activity of the soluble as well as the particulate hydrogenase. Unexpectedly, mutants defective in autotrophic carbon dioxide fixation but unaffected in hydrogen oxidation showed an inhibition of growth by hydrogen but no repression of histidase synthesis. Mutants which formed histidase constitutively were still sensitive to repression in the presence of hydrogen. The results indicate that repression of enzyme synthesis by hydrogen is dependent on the function of both, the hydrogen-oxidizing and the carbon dioxide-fixing system. It is concluded that the hydrogen effect is a transient regulatory mechanism and only relevant for unbalanced conditions of growth.

Regulation of hut enzymes and intracellular protease activities in Vibrio alginolyticus hut mutants.

The production of alkaline protease, collagenase and histidine utilization (Hut) enzymes by Vibrio alginolyticus wild-type, hutH1 and hutU1 strains was investigated. Alkaline protease synthesis was stimulated by histidine and urocanic acid in the wild-type and hutU1 strains. The hutH1 mutant alkaline protease production was stimulated by urocanic acid and not by histidine. The Hut enzymes in the wild-type strain were coordinately induced by histidine. Urocanase and formimino-hydrolase were induced by histidine in the hutH1 mutant which lacked histidase and was not able to convert histidine to urocanic acid. Collagenase production in peptone medium was inhibited in the hut mutants. It is concluded that in V. alginolyticus urocanic acid regulates alkaline protease synthesis but that the Hut enzymes are induced by histidine. The involvement of the Hut genetic system in the regulation of alkaline protease and collagenase synthesis is discussed.

Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.

Histidine dissimilation in Streptomyces coelicolor.

A growth technique that allows strains of Streptomyces coelicolor to grow dispersed in defined liquid medium has been devised and used to determine the pathway of histidine degradation by S. coelicolor. Enzymic, chromatographic and stoichiometric analyses indicated that histidine is dissimilated via N-formyl-L-glutamic acid. The enzymes for histidine utilization (hut) are induced when histidine or urocanate is included in the culture medium. Biochemical evidence suggested that urocanate, or a further metabolite, is the physiological inducer. Three hut mutants were isolated and characterized. Two of the mutants exhibit an uninducible phenotype, whereas the third mutant appears to be defective in the structural gene for formiminoglutamate iminohydrolase. Haploid recombinant analysis was employed to locate all three mutations in the left empty region of the chromosomal map.

Regulation of Extracellular Alkaline Protease Activity by Histidine in a Collagenolytic Vibrio alginolyticus Strain

Vibrio alginolyticus synthesized an inducible extracellular collagenase in a peptone medium during the stationary growth phase. These cultures also possessed extracellular alkaline serine protease activity. The alkaline protease activity did not require a specific inducer and it was produced in tryptone or minimal media. The collagenase was not produced in either the tryptone or minimal media. The alkaline protease activity was sensitive to catabolite repression by a number of carbon sources, including glucose, and by amino acids and ammonium ions. Cyclic AMP, dibutyryl cyclic AMP and cyclic GMP did not relieve catabolite repression. Histidine and urocanic acid stimulated the production of alkaline protease activity in tryptone and minimal media. Other compounds associated with the histidine utilization (hut) pathway did not increase alkaline protease activity. Histidine reversed the repression of alkaline protease activity by glucose of (NH4)2SO4 in minimal medium. Histidine and the compounds associated with the hut pathway inhibited collagenase production.

The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons.

A 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA- mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut+ and Nif+ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA- mutations and the GlnR- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA- mutations but not the GlnR- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA- strain of E. coli and a glnA- GlnR- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA- mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR- phenotype, while glnA- complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR).

Physical maps of Klebsiella aerogenes and Salmonella typhimurium hut genes.

The recognition sites for several restriction endonucleases were mapped within deoxyribonucleic acid coding for histidine utilization (hut) genes of Salmonella typhimurium and Klebsiella aerogenes. Deoxyribonucleic acid fragments containing the two hut promoters were identified by ribonucleic acid polymerase binding.