The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA::kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.     

In situ analysis of adhesion molecule expression in kidneys infected with murine malaria

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P(ow) 2.8) but they could grow in medium overlaid with organic solvents having a log P(ow) value higher than that of toluene such as p-xylene (log P(ow) 3.1), cyclohexane (log P(ow) 3.4) and n-hexane (log P(ow) 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3.7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.     

Long-term interferon-alpha therapy induces autoantibodies against epidermis

Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.     

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.     

Characterization of the glnK-amtB operon of Azotobacter vinelandii

To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized. Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was contranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote. glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.     

The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N6-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C5-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5'-CGATCG-3', corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation M.Ssp6803I [corrected] (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.     

Imaging of en bloc renal transplants: normal and abnormal postoperative findings

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.     

Cloning of a recA-like gene from the diazotroph Herbaspirillum seropedicae strain Z78

A recombinant plasmid, pBMR5, carrying a recA-like gene of Herbaspirillum seropedicae, was isolated from a H. seropedicae genomic library by intergeneric complementation of Escherichia coli recA mutant strain HB101. Quantitative survival experiments showed that pBMR5 restored the ultraviolet radiation and methyl methanesulfonate resistances and recombinational proficiency of this strain. Hybridization studies showed that there is DNA sequence homology between the recA gene of E. coli K12 and that of H. seropedicae. Restriction sites for EcoRI, HindIII, BamHI, and Bg/II were found in the DNA insert derived from H. seropedicae in pBMR5. A Tn5 insertional mutant of pBMR5, called pBMR26.2, failed to restore recombination proficiency and methyl methanesulfonate and ultraviolet resistance to recA mutants of E. coli.     

Trimethoprim resistance plasmids: transferability and incompatibility groups (author's transl)

Over a three year period, 119 strains of enterobacteria isolated from patients have been found resistant to trimethoprim (TMP) and sulfonamides (Su); 11 strains were resistant to TMP only. MIC of TMP were between 32 and 2048 microng/ml. Three groups of strains are described: (1) thymineless variants (2 strains); (2) TMP resistance non-transferable into Escherichia coli K12 (95 strains); (3) TMP resistance transferable into E. coli K12 (33 strains). TMP marker and Su marker have been transferred independantly from 13 strains; they were cotransferred from 20 strains. The incompatibility group of 31 plasmids has been determined: 10 belong to the fi+ type, group FII; 21 belong to the fi--type, group 6, group 7, group 10, group N and group I1. Epidemiological implications of such a wide range of incompatibility groups among a small number of plasmids specifying TMP resistance are discussed.     

Comparison of loss of serum resistance by defined lipopolysaccharide mutants and an acapsular mutant of uropathogenic Escherichia coli O75:K5

In order to determine the importance of the O75 O antigen versus the K5 capsular antigen and the bimodal distribution of lipopolysaccharides (LPSs) in protection from complement-mediated lysis, mutants were made by insertion of a cat or an aphA gene in or in place of genes necessary for the synthesis of LPS and/or the K antigen of an O75(+) K5(+) uropathogenic Escherichia coli strain, GR-12. Mutations were made in the following genes: the rfbD gene (required for the synthesis of TDP-rhamnose), the rfbKM genes (necessary for the synthesis of GDP-mannose), the rol gene (regulating O-antigen length), the kfiC gene (encoding a putative glycosyltransferase), and the kfiC-rfbD genes. The resulting phenotypes were rough (O75(-)), core plus one partial O-antigen subunit, random distribution of O-antigen chain lengths, acapsular (K5(-)), and O75(-) K5(-), respectively. All five mutants and GR-12 were analyzed for survival in 80% serum. The GR-12 parent was resistant, exhibiting a 500% increase in numbers. The rol, rfbKM, rfbD, and kfiC-rfbD mutants were sensitive, experiencing 99%, 99.9%, 99.9%, and at least 99.999% killing, respectively, in the first hour. The kfiC mutant, however, increased in numbers in the first hour but experienced delayed sensitivity, decreasing in viability by 80% in the third hour. Single mutants were complemented with the wild-type gene in trans, showing restoration of the wild-type phenotype and serum resistance. Therefore, the O75 antigen is more important for survival in serum than the K5 antigen, and regulation of the O75 O-antigen chain length is crucial for protection of the bacteria from complement-mediated lysis.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh8Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh8Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh8Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh8Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.     

Characterization of mutations that allow p-aminobenzoyl-glutamate utilization by Escherichia coli

An Escherichia coli strain deficient in p-aminobenzoate synthesis was mutagenized, and derivatives were selected for growth on folic acid. Supplementation was shown to be due to p-aminobenzoyl-glutamate present as a breakdown product in commercial folic acid preparations. Two classes of mutations characterized by the minimum concentration of p-aminobenzoyl-glutamate that could support growth were obtained. Both classes of mutations were genetically and physically mapped to about 30 min on the E. coli chromosome. A cloned wild-type gene from this region, abgT (formerly ydaH) could confer a similar p-aminobenzoyl-glutamate utilization phenotype on the parental strain. Interruption of abgT on the plasmid or on the chromosome of the mutant strain resulted in a loss of the phenotype. abgT was the third gene in an apparent operon containing abgA, abgB, abgT, and possibly ogt and might be regulated by a divergently transcribed LysR-type regulator encoded by abgR. Two different single-base-pair mutations that gave rise to the p-aminobenzoyl-glutamate utilization phenotype lay in the abgR-abgA intercistronic region and appeared to allow the expression of abgT. The second class of mutation was due to a tandem duplication of abgB and abgT fused to fnr. The abgA and abgB gene products were homologous to one another and to a family of aminoacyl aminohydrolases. p-Aminobenzoyl-glutamate hydrolysis could be detected in extracts from several of the mutant strains, but intact abgA and abgB were not essential for p-aminobenzoyl-glutamate utilization when abgT was supplied in trans.     

The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.     

The merG gene product is involved in phenylmercury resistance in Pseudomonas strain K-62

The physiological function of a new gene, hereby designated merG, located between merA and merB on the broad-spectrum mer operon of Pseudomonas strain K-62 plasmid pMR26 was investigated. The 654-bp merG gene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of the merG gene from the broad-spectrum mer operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain. Escherichia coli cells bearing pMU29, which carries a deletion of the merG gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the merG gene in a compatible plasmid was transformed into the E. coli strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the merG gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.     

phnE and glpT genes enhance utilization of organophosphates in Escherichia coli K-12

Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (< or = 1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified as phnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coli K-12 phnE gene that has been shown previously to result in phnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with the glpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnE and multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnE gene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-Pi). This result indicates that in E. coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.