hisG Gene Research Articles

Effect of Biochar on the Production of L-Histidine From Glucose Through Escherichia coli Metabolism.

The organic compounds from biochar play a role of hormone analogs, stimulating the expression of metabolites by controlling related gene and protein. In this experiment, we reported the L-histidine biosysthesis was promoted by biochar treatment in E. coli unlike genetic engineering of the traditional method. The related results indicated the most optimal concentration was found to be 3%, and 7% is the lethal dose. E. coli was inhibited in the high-concentration treatment. On the other hand, docking technology was usually used as drug screening, basing on Lock-and-key model of protein in order to better understand mechanisms. So the organic compounds of biochar from GC-MS analysis that acted as ligands were connected to HisG protein controlling L-histidine biosysthesis in E. coli. The result showed that the three organic molecules interacted with HisG protein by hydrogen bond. So we considered that these three compounds play regulatory roles in L-histidine biosysthesis, and the hisG gene expression fully supports this conclusion.

Salmonella Proteomic Profiling during Infection Distinguishes the Intracellular Environment of Host Cells.

Essential to bacterial pathogenesis, Salmonella enterica serovar Typhimurium (S. Typhimurium) has evolved the capacity to quickly sense and adapt to specific intracellular environment within distinct host cells. Here we examined S. Typhimurium proteomic remodeling within macrophages, allowing direct comparison with our previous studies in epithelial cells. In addition to many shared features, our data revealed proteomic signatures highly specific to one type of host cells. Notably, intracellular S. Typhimurium differentially regulates the two type III secretion systems (T3SSs) far more quickly in macrophages than in epithelial cells; bacterial flagellar and chemotaxis systems degenerate more quickly in macrophages than in HeLa cells as well. Importantly, our comparative analysis uncovered high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells. Targeted metabolomic measurements revealed markedly lower histidine levels within macrophages. Intriguingly, further functional studies established that histidine biosynthesis that is defective (due to a hisG mutation) renders the bacterium (strain SL1344) hypersensitive to intracellular shortage of this amino acid. Indeed, another S. Typhimurium strain, namely, strain 14028s, with a fully functional biosynthetic pathway exhibited only minor induction of the his operon within infected macrophages. Our work thus provided novel insights into S. Typhimurium adaptation mechanisms within distinct host cells and also provided an elegant paradigm where proteomic profiling of intracellular pathogens is utilized to discriminate specific host environments (e.g., on the basis of nutrient availability). IMPORTANCE Salmonella Typhimurium is one of the leading causes of foodborne bacterial infection. Nevertheless, how Salmonella adapts to distinct types of host cells during infection remains poorly understood. By contrasting intracellular Salmonella proteomes from both infected macrophages and epithelial cells, we found striking proteomic signatures specific to particular types of host cells. Notably, Salmonella proteomic remodeling exhibited quicker kinetics in macrophages than in epithelial cells with respect to bacterial virulence and flagellar and chemotaxis systems. Furthermore, we unveiled high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells, which is attributable to differing intracellular levels of this amino acid. Intriguingly, we found that a defective hisG gene renders a Salmonella strain hypersensitive to histidine shortage in macrophages. Overall, our work reveals specific Salmonella adaptation mechanisms in distinct host cells, which should aid in the development of novel anti-infection strategies.

Influence of GSH S-transferase on the mutagenicity induced by dichloromethane and 1,2-dichloropropane.

It has been suggested that dichloromethane (DCM) and 1,2-dichloropropane (DCP) are responsible for occupational cholangiocarcinoma. Dihaloalkanes are metabolically activated by GSH S-transferase theta1 (GSTT1) to yield products such as episulfonium ions. However, whether the GSTT1-mediated step of these dihaloalkanes is related to occupational cholangiocarcinoma is not known. In the present study, we investigated the influence of GSTT1 activation on the mutagenicity of DCM and 1,2-DCP using GSTT1-expressing Salmonella typhimurium TA100 (TA100-GST). Since the mutagenicity of DCM was significantly increased in TA100-GST compared with mock control (TA100-pCTC), GSTT1 is thought to be involved in the mutagenicity of DCM. Mutation spectrum analysis on the hisG gene revealed that C:G to A:T transversions were the predominant form observed in DCM-treated TA100-pCTC. However, C:G to T:A transitions were dramatically increased in TA100-GST. We also analysed the DCM-DNA adduct, N2-GSH-Me-dG, and formation of N2-GSH-Me-dG was increased in TA100-GST compared with TA100-pCTC. On the other hand, 1,2-DCP did not increase the numbers of revertants in TA100-GSTT1. In mutation spectrum analysis, C:G to T:A transitions was predominant in both TA100-pCTC and TA100-GSTT1. These findings suggest that GSTT1 has little involvement in DCP mutagenicity, and other mechanisms might be more important for bioactivation and consequent genotoxicity. Clarification of the mechanisms underlying the development of DCM- and/or 1,2-DCP-related human cholangiocarcinoma may help establish risk assessment and prevention strategies against occupational cancer.

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-β-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.

Comparative proteomic analysis of Salmonella typhimurium LT2 and its hisG gene inactivated mutant

A hisG gene, encoding ATP phosphoribosyl phosphothansferase, insertion mutation was found to effected the production of four different proteins in Salmonella Typhimurium LT2 as a results of comparative analysis of two-dimensional analysis of hisG mutant (Δhis87) and the conrol strain (LT2). MALDI-TOF analysis of these protein spots which were expresed in S. Typhimurium LT2 but not its mutant Δhis87, showed that these proteins were; GMP synthase, arginine-tRNA ligase (Arginyl-tRNA synthetase), SopE and SifA. All of these proteins are virulence-associated proteins in Salmonella. Thus we have concluded that the proper function of histidine operon is very crucial for virulence of S. Typhimurium.

The 4‐cysteine zinc‐finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress

We show that thiols in the 4-cysteine zinc-finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide- or nitric oxide (NO)-mediated modifications of thiols in the DksA 4-cysteine zinc-finger motif release the metal cofactor and drive reversible changes in the α-helicity of the protein. Wild-type and relA spoT mutant Salmonella, but not isogenic dksA-deficient bacteria, experience the downregulation of r-protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA-dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc-bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine-tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys(114) , and neighbouring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress.

Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.

Cloning of the histidine biosynthetic genes fromCorynebacterium glutamicum: Organization and analysis of thehisGandhisEgenes

The physically linked hisG and hisE genes, encoding for ATP-phosphoribosyltransferase and phosphoribosyl-ATP-pyrophosphohydrolase were isolated from the Corynebacterium glutamicum gene library by complementation of Escherichia coli histidine auxotrophs. They are two of the nine genes that participate in the histidine biosynthetic pathway. Molecular genetics and sequencing analysis of the cloned 9-kb insert DNA showed that it carries the hisG and hisE genes. In combining this result with our previous report, we propose that all histidine biosynthetic genes are separated on the genome by three unlinked loci. The coding regions of the hisG and hisE genes are 279 and 87 amino acids in length with a predicted size of about 30 and 10 kDa, respectively. Computer analysis revealed that the amino acid sequences of the hisG and hisE gene products were similar to those of other bacteria.

Relief of transcriptional polarity by a mutation that creates a promoter in the hisG gene of Salmonella typhimurium LT2.

Six mutant strains were independently isolated in Salmonella typhimurium as non-polar revertants of a polar Tn10 insertion in hisG. DNA sequence analysis showed that all six mutants result from the same nucleotide alteration: a G/C to A/T transition 4 bp from the end of the hisG coding sequence. We present data suggesting that the mutation (hisG10225) acts by creating a new transcriptional initiation signal. Furthermore, the hisG10225 mutation renders the hisG gene product cold sensitive.

Isogenic strain construction and gene mapping in Candida albicans.

Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.

Cloning of mycobacterial histidine synthesis genes by complementation of a Mycobacterium smegmatis auxotroph.

Histidine-requiring auxotrophs of Mycobacterium smegmatis were isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. One of these mutants, his5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial-specific biosynthetic pathways involved in the virulence of pathogenic species to be studied.

Comparison of Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains

In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)

Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium.

Previous genetic analysis showed that the polar effects of mutations in the hisG cistron of Salmonella typhimurium are dependent on the presence of a single putative transcription termination element within the hisG gene. In fact, all proximal mutations causing translation termination are strongly polar, whereas distal ones are not. The element was mapped by isolating mutations able to relieve the polar phenotype, and they were found to be small deletions in the region downstream of the translational stop codon (M. S. Ciampi and J. R. Roth, Genetics 118:193-202, 1988). In this study, we analyzed the his-specific RNAs synthesized in vivo in different strains harboring the polar frameshift hisG2148 mutation. The nature of the polarity effects is clearly transcriptional, since shorter RNA molecules were produced. When the hisG2148 mutation was transferred in a rho background or in strains harboring the small distal deletions, an increase in readthrough transcription was observed. The transcriptional termination element was characterized in more detail by performing high-resolution S1 nuclease mapping experiments. This analysis showed that (i) termination or exonucleolytic degradation following termination produced transcripts with heterogeneous 3' ends; (ii) this process is dependent on the transcription termination factor Rho, since relief of termination occurs in a rho background; and (iii) the element appears to function as a transcription terminator, at least to some extent, even in the course of active translation of the hisG cistron.

Transcriptional occlusion of transposon targets

In Salmonella typhimurium, insertion of transposons Tn5, Tn10 and bacteriophage Mu is inhibited by transcription of some target sequences. The transcription effects on Tn5 are large when the lac operon is a target but are limited to a slight effect on the hisG gene of the his operon. The Tn10 element shows target occlusion in both operons. Phage Mu has been shown previously to be inhibited for insertion into the lac operon. In the his operon Mu is only inhibited for insertion into the hisG gene. The variability of the inhibition effect from one sequence to another suggests site or regional specificity for transcription effects. Reducing the probability of insertion into transcribed sequences may be of selective importance to transposons since it reduces the risk of killing the host while maintaining the ability to transpose.

Activation of silent genes by transposons Tn5 and Tn10.

The presence of transposons Tn10 or Tn5 in the genome increases the frequency with which a silent (promoter-less) gene (hisD) is mutationally activated. The activation frequency is increased 5-25-fold by Tn10 and 30-90-fold by Tn5. Activation of the hisD gene is achieved by transposition of the entire transposon or one of its flanking insertion sequences to a region just upstream of the silent gene, between a Rho-dependent termination site in the adjacent hisG gene and the hisD gene. For both Tn5 and Tn10 the component insertion sequences were found to transpose much more frequently than the entire composite element. Transposons Tn5 and Tn10 have previously been shown to carry promoters which direct transcripts into sequences adjacent to their insertion sites.

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline) → CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 hisG gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.

Polarity effects in the hisG gene of salmonella require a site within the coding sequence.

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.

Heterologous expression in Bacillus subtilis II. In vitro removal of the attenuator sequence of the Escherichia coli his operon allows expression of the cloned hisG gene in B. subtilis

The promoter-proximal region of the Escherichia coli histidine (his) operon, including the promoter, the attenuator and the hisG gene, as well as the first of the nine structural genes of the his operon, have been cloned in Bacillus subtilis. In this host, the hisG gene could not be expressed because its transcription appeared to be irreversibly terminated at the attenuator (Ferretti et al., 1984). When the attenuator plus various lengths of the two bordering regions were removed, one of the attenuatorless sequences cloned in B. subtilis allowed the progression of transcription and complementation of the corresponding hisA mutation in this Gram-positive host. The deletion removed a 349-bp segment which contained the his attenuator and promoter. In B. subtilis, the productive transcription of the hisG gene started at a site in pAT153 and terminated in pC194. Sequence analysis of the deletion indicates that the E. coli ribosome-binding site of the his operon was used for the translation of the E. coli hisG gene mRNA in B. subtilis cells, which can thus grow in the absence of histidine.

Mutation in Salmonella typhimurium recovered from livers and spleens of mice

Balb/c mice were inoculated intraperitoneally with TA2662, a smooth derivative of the Salmonella typhimurium Ames tester strain (TA102) which carries the mutable hisG locus on a multicopy plasmid, or TA103, which carries the same hisG gene on the chromosome. The bacteria were recovered at various times from the liver and spleens of the infected mice. Total numbers of bacteria were determined and the mutant frequency was estimated. The frequency of occurence of histidine prototrops in experiments using TA2662 was substantially above tha average found with this strain grown in vitro. The mutant frequencies in experiments using TA103 recovered from mice were also highly significantly increased above background. We did not identify factors which might suggest selection in vivo for histidine prototrophs. There is sufficient histidine in body fluids of the host for the growth of His − bacteria. The His − and His + derivatives were found to grow equally well in vitro in the presence of amounts of histidine approximating concentrations known to exist in vivo. It is probable that mutations in TA2662 are greatly underestimated, since the hisG-containing plasmid is lost at relatively high frequency during incubation in a variety of conditions.

The mutagenicity of sodium bisulfite on base‐substitution strains of Salmonella typhimurium

The mutagenic effects of sodium bisulfite on Salmonella typhimurium LT2 strains carrying the hisG46 allele were investigated. Treating the cells with 1 M sodium bisulfite in 0.2 M acetate buffer (pH 5.2) for various time intervals at 37 degrees C resulted in an increase in the yield of mutants above the spontaneous level. The fold enhancement of the mutant yield and the fold enhancement of the mutant frequency were greatest for the parent strain hisG46 with wild type DNA repair; the derived strains with reduced repair capacity responded to a lesser degree. While sodium bisulfite was mutagenic when tested in liquid suspension assays, strain hisG46 did not respond when the standard Ames plate incorporation assay was used. The greater mutagenic response in repair-proficient strains suggests that normal repair capacities may favor the mutagenic activity of sodium bisulfite. Transduction studies demonstrated that mutations of his+ were closely linked to the hisD gene, and probably were in the structural hisG gene itself.