The degradation of l-histidine, imidazolyl-l-lactate and imidazolylpropionate by Pseudomonas testosteroni

1. Imidazol-5-ylpropionate and imidazol-5-yl-lactate are degraded by Pseudomonas testosteroni via inducible pathways. 2. Growth on either compound as the sole source of carbon results in the induction of the enzymes for histidine catabolism. 3. The pathway of histidine degradation in this organism, a non-fluorescent Pseudomonad, is shown to be the same as that operating in Pseudomonas fluorescens and Pseudomonas putida. It consists of the successive formation of urocanate, imidazol-4-on-5-ylpropionate, N-formimino-l-glutamate, N-formyl-l-glutamate and glutamate. 4. Whole cells of P. testosteroni accumulate urocanate in the reaction mixture when incubated with imidazolylpropionate, but only after an adaptive lag period which is removed by previous growth on imidazolylpropionate as the source of carbon. 5. Imidazolyl-lactate is oxidized to imidazolylpyruvate, which then gives rise to histidine by specific transamination with l-glutamate. 6. Cells grown on histidine, urocanate or imidazolylpropionate are also able to degrade imidazolyllactate. 7. Mutants lacking urocanase are unable to grow on imidazolylpropionate, imidazolyl-lactate, histidine or urocanate. One with impaired histidase activity cannot utilize histidine or imidazolyl-lactate, but grows normally on imidazolylpropionate or urocanate. A mutant unable to grow on imidazolylpropionate is indistinguishable from the wild-type with respect to growth on histidine, imidazolyl-lactate or urocanate. 8. Thus it is established that imidazolyl-lactate is metabolized via histidine whereas imidazolylpropionate enters the histidine degradation pathway after conversion into urocanate.     

The control of the enzymes degrading histidine and related imidazolyl derivatives in Pseudomonas testosteroni

1. The induction of the enzymes for the degradation of l-histidine, imidazolylpropionate and imidazolyl-l-lactate in Pseudomonas testosteroni was investigated. 2. The activities of histidine ammonia-lyase, histidine-2-oxoglutarate aminotransferase and urocanase are consistent with these enzymes being subject to co-ordinate control under most growth conditions. However, a further regulatory mechanism may be superimposed for histidase alone under conditions where degradation of histidine must take place for growth to occur. 3. Experiments with a urocanase(-) mutant show that urocanate is an inducer for the enzymes given above and also for N-formiminoglutamate hydrolyase and N-formylglutamate hydrolase. 4. N-Formiminoglutamate hydrolase and N-formylglutamate hydrolase are also induced by their substrates, and it is suggested that these two enzymes may be different gene products from those expressed in the presence of urocanate. 5. Induction of the enzyme system for the oxidation of imidazolylpropionate is dependent on exposure of cells to this compound.     

Genome-wide screen reveals novel mechanisms for regulating cobalt uptake and detoxification in fission yeast

Cobalt is an essential micronutrient but is toxic when present in excess. To study cobalt homeostasis we performed a genome-wide screen for deletion strains that show sensitivity or resistance to CoCl(2). Among 54 cobalt-sensitive strains, 18 are supersensitive strains, which are involved in histidine biosynthetic process, ubiquitination, mitochondria function, membrane trafficking, transporter and a variety of other known functions or still unknown functions. Furthermore, we identified 56 cobalt-resistant deletion strains, which are mainly involved in mitochondria function, signal transduction, ubiquitination, and gene expression and chromatin remodeling. Notably, deletion of the zhf1 (+) gene, encoding a zinc ion transporter, confers supersensitivity to cobalt and overexpression of the zhf1 (+) gene confers marked tolerance to cobalt, indicating that Zhf1 play key roles in cobalt detoxification. Interestingly, all the histidine-auxotrophic mutants displayed cobalt sensitivity and deletion of cationic amino acid transporter Cat1, which was shown to be involved in histidine uptake, suppressed the CoCl(2)-sensitive growth defect of the his2 mutants, suggesting that CoCl(2) may be transported into the cell together with histidine via histidine transporters including Cat1. In addition, we obtained results suggesting that the E2 ubiquitin conjugating enzyme Rhp6 and Sty1 stress MAP kinase pathway are involved in the regulation of cobalt homeostasis. Altogether, our genome-wide study demonstrates for the first time the mechanisms of cobalt homeostasis, particularly its uptake and detoxification in fission yeast.     

Regulation of histidine catabolism by succinate in Pseudomonas putida

The regulation of the histidine-degrading pathway is known to involve induction and repression. Our studies have shown that succinate may control the histidine-degrading pathway by sequential negative feedback inhibition. Succinate inhibited urocanase, and urocanate in turn inhibited histidase. Crude preparations of the two enzymes were made from Pseudomonas putida grown on l-histidine. Succinate was a competitive inhibitor of urocanase (K(i), 1.8 mm). Lactate, pyruvate, alpha-ketoglutarate, and glutamate did not inhibit urocanase. Urocanate inhibited histidase competitively (K(i), 0.13 mm). A multienzyme system (histidine to glutamate), when incubated with histidine and succinate, exhibited the combined effect. Succinate caused the level of accumulated urocanate to increase and indirectly blocked histidine disappearance. Growth of cells on urocanate as a nitrogen source was inhibited by 1% succinate. Succinate may play a physiological role in the biological regulation of histidine metabolism.     

Induction of Histidine-Degrading Enzymes in Pseudomonas aeruginosa

Urocanate but not histidine was able to induce formation of histidine-degrading enzymes in a histidine ammonia-lyase-deficient mutant of Pseudomonas aeruginosa. The results, in conjunction with others reported previously, indicate that urocanate, the first intermediate, is the physiological inducer of the pathway.     

Formation and operation of the histidine-degrading pathway in Pseudomonas aeruginosa

Histidine ammonia lyase (histidase), urocanase, and the capacity to degrade formiminoglutamate, which are respectively involved in steps I, II, and IV in the catabolism of histidine, were induced during growth of Pseudomonas aeruginosa on histidine or urocanate, and were formed gratuitously in the presence of dihydro-urocanate. Urocanase-deficient bacteria formed enzymes I and IV constitutively; presumably they accumulate enough urocanate from the breakdown of endogenous histidine to induce formation of the pathway. Urocanate did not satisfy the histidine requirement of a histidine auxotroph, indicating that it probably acted as an inducer without being converted to histidine. The results imply that urocanate is the physiological inducer of the histidine-degrading enzymes in P. aeruginosa. Enzymes of the pathway were extremely sensitive to catabolite repression; enzymes I and II, but not IV, were coordinately repressed. Our results suggest a specific involvement of nitrogenous metabolites in the repression. Mutant bacteria with altered sensitivity to repression were obtained. The molecular weight of partially purified histidase was estimated at 210,000 by sucrose gradient centrifugation. Its K(m) for histidine was 2 x 10(-3)m in tris(hydroxymethyl)aminomethane chloride buffer. Sigmoid saturation curves were obtained in pyrophosphate buffer, indicating that the enzyme might have multiple binding sites for histidine. Under certain conditions, histidase appeared to be partially inactive in vivo. These findings suggest that some sort of allosteric interaction involving histidase may play a role in governing the operation of the pathway of histidine catabolism.     

Photoactivation of Urocanase in Pseudomonas putida: Possible Role in Photoregulation of Histidine Metabolism

Irradiation by near-ultraviolet light of cells or extracts of Pseudomonas putida increased the urocanase activity. Irradiated cells exhibited enhanced catabolic activity on histidine and urocanate.     

The origin and ecological significance of multiple branches for histidine utilization in Pseudomonas aeruginosa PAO1

Pseudomonas proliferate in a wide spectrum of harsh and variable environments. In many of these environments, amino acids, such as histidine, are a valuable source of carbon, nitrogen and energy. Here, we demonstrate that the histidine uptake and utilization (hut) pathway of Pseudomonas aeruginosa PAO1 contains two branches from the intermediate formiminoglutamate to the product glutamate. Genetic analysis revealed that the four-step route is dispensable as long as the five-step route is present (and vice versa). Mutants with deletions of either the four-step (HutE) or five-step (HutFG) branches were competed against each other and the wild-type strain to test the hypothesis of ecological redundancy; that is, that the presence of two pathways confers no benefit beyond that delivered by the individual pathways. Fitness assays performed under several environmental conditions led us to reject this hypothesis; the four-step pathway can provide an advantage when histidine is the sole carbon source. An IclR-type regulator (HutR) was identified that regulates the four-step pathway. Comparison of sequenced genomes revealed that P.aeruginosa strains and P.fluorescens Pf-5 have branched hut pathways. Phylogenetic analyses suggests that the gene encoding formiminoglutamase (hutE) was acquired by horizontal gene transfer from a Ralstonia-like ancestor. Potential barriers to inter-species transfer of the hutRE module were explored by transferring it from P.aeruginosa PAO1 to P.fluorescens SBW25. Transfer of the operon conferred the ability to utilize histidine via the four-step pathway in a single step, but the fitness cost of acquiring this new operon was found to be environment dependent.     

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.     

OsHT, a Rice Gene Encoding for a Plasma-Membrane Localized Histidine Transporter

Using a degenerative probe designed according to the most conservative region of a known Lys- and His-specific amino acid transporter (LHT1) from Arabidopsis, we isolated a full-length cDNA named OsHT (histidine transporter of Oryza sativa L.) by screening the rice cDNA library. The cDNA is 1.3kb in length and the open reading frame encodes for a 441 amino acid protein with a calculated molecular mass of 49 kDa. Multiple sequence alignments showed that OsHT shares a high degree of sequence conservation at the deduced amino acid level with the Arabidopsis LHT1 and six putative lysine and histidine transporters. Computational analysis indicated that OsHT is an integral membrane protein with 11 putative transmembrane helices. This was confirmed by the transient expression assay because the OsHT-GFP fusion protein was, indeed, localized mainly in the plasma membrane of onion epidermal cells. Functional complementation experiments demonstrated that OsHT was able to work as a histidine transporter in Saccharomyces cerevisiae, suggesting that OsHT is a gene that encodes for a histidine transporter from rice.This is the first time that an LHT-type amino acid transporter gene has been cloned from higher plants other than A rabidopsis.     

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

AIMS: Four well-described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula. Methods and RESULTS: The effect of Pseudomonas strains on the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium was assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains. Strains F113, SBW25 and CHAO increased shoot weights (by 20%, 22% and 35%, respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%). Strains SBW25 and CHAO increased root lengths (19% and 69%) and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, while SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain-specific. Pythium increased the activity of C, N and P cycle enzymes, while the Pseudomonas strains reduced this effect, indicating reduced plant damage. CONCLUSION: Strains SBW25 and CHAO had the greatest beneficial characteristics, as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonization ability in the rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first critical comparison of such important strains of Ps. fluorescens showing disease biocontrol potential.     

OsHT, a Rice Gene Encoding for a Plasma-Membrane Localized Histidine Transporter

Using a degenerative probe designed according to the most conservative region of a known Lys- and His-specific amino acid transporter (LHT 1) from Arabidopsis, we isolated a full-length cDNA named OsHT (histidine transporter of Oryza sativa L.) by screening the rice cDNA library. The cDNA is 1.3 kb in length and the open reading frame encodes for a 441 amino acid protein with a calculated molecular mass of 49 kDa. Multiple sequence alignments showed that OsHT shares a high degree of sequence conservation at the deduced amino acid level with the Arabidopsis LHT1 and six putative lysine and histidine transporters. Computational analysis indicated that OsHT is an integral membrane protein with 11 putative transmembrane helices. This was confirmed by the transient expression assay because the OsHT-GFP fusion protein was, indeed, localized mainly in the plasma membrane of onion epidermal cells. Functional complementation experiments demonstrated that OsHT was able to work as a histidine transporter in Saccharomyces cerevisiae, suggesting that OsHT is a gene that encodes for a histidine transporter from rice.This is the first time that an LHT-type amino acid transporter gene has been cloned from higher plants other than Arabidopsis.     

Heterologous functional analysis of the Malus xiaojinensis MxIRT1 gene and the His-box motif by expression in yeast

Malus xiaojinensis is an important, iron-efficient rootstock germplasm. Iron uptake is an elaborately controlled process in plant roots, involving specialized transporters. MxIRT1, a Fe(II) transporter gene of M. xiaojinensis, is homologous to other iron transporters at the amino acid level. In the current study, the plasmid pYES2.0-MxIRT1, containing MxIRT1 cDNA, was constructed and transformed into yeast mutants. The results indicated that it could reverse the phenotype of yeast strain DEY1453, an iron uptake mutant. Complementation tests suggested that it might not be a specific transporter, as it was able to restore the phenotypes of other yeast mutant strains, including Mn, Cu and Zn uptake mutants. The functions of the critical histidine residues in the His-box of MxIRT1 were tested by transforming mutant yeast strain DEY1453 with different His residues altered by directed mutagenesis. The His-box of MxIRT1 was found to be necessary for iron transport, with different histidine residues (H_1–4) playing different roles in the transport.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders.

Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders. The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols. In contrast, DNA extracted from inoculated soils hybridized to this probe. Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils. This increase was related kinetically to the growth of biphenyl degraders. Pseudomonas sp. strain AW, a dominant biphenyl degrader, was selected as a possible parental strain. Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity. Two hybrid strains, P. aeruginosa JB2-3 and Pseudomonas sp. JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242). Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW. These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.     

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.     

The benefits and risks of expressing the POT and FOT family of oligopeptide transporters in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, all strains possess a gene for the evolutionarily conserved POT family peptide transporter, Ptr2; however, the genes for a novel FOT family transporter were found only in some wine brewing strains. The substrate specificity of the POT and FOT family of transporters was compared. Among the naturally occurring oligopeptides that were tested, Lys-Leu and Arg-Phe were Ptr2-specific substrates. Artificial dipeptide aspartame was imported specifically through the FOT transporter, but the structurally similar Asp-Phe was a substrate of both FOT and Ptr2 transporters. Furthermore, only the FOT transporter was important for high sensitivity to an antibiotic puromycin. These results demonstrate that the POT and FOT family of transporters have distinct substrate preferences although both transporters import overlapping dipeptide substrates. Having POT and FOT transporters is advantageous for cells to acquire nutrients, but also detrimental when these cells are exposed to the toxic molecules of their substrates.