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.     

RecA protein from an extremely thermophilic bacterium, Thermus thermophilus HB8

The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T.th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T.th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein.     

Chromosomal changes and correspondingly altered proto-oncogene expression in human gliomas. Value of combined cytogenetic and molecular genetic analysis

The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows lambda red- gam- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.     

Biological consequences of E.coli RecA protein expression in the repair defective pso4-1 and rad51::URA3 mutants of S. cerevisiae after treatment with N-methyl-N'-nitro-N-nitrosoguanidine

RecA protein of E.coli is a multifunctional protein participating in genetic recombination, recombinational repair and mutagenesis. We used E.coli recA gene as a probe for complementation of repair defects after treatment of N-methyl-N'-nitro-N-nitrosoguanidine in the pso4-1 and rad51::URA3 mutants of S. cerevisiae. We tried to find the role of the RecA protein in S. cerevisiae mutants defective in different repair pathways. The RecA protein had no effect on survival of haploid and diploid pso4-1 mutants, but it had a significant effect on MNNG induced mutagenesis, which was increased to the wild type level. No effect of the RecA protein on survival was observed in rad51::URA3 mutant after MNNG treatment. However, in this case the RecA protein decreased the induced mutagenesis. We can suppose that the RecA protein, with its multifunctional enzymatic activity, can bind to special intermediates and initiate function of different repair pathways depending on repair defects of the mutants studied.     

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation

The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.     

The isfA mutation inhibits mutator activity and processing of UmuD protein in Escherichia coli recA730 strains

Further studies on the isfA mutation responsible for anti-SOS and antimutagenic activities in Escherichia coli are described. We have previously shown that the isfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. The isfA mutation has now been demonstrated also to suppress mutator activity in E. coli recA730 and recA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of the isfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in the recA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction

BACKGROUND: The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double-strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1. RESULTS: Recombinant XRad51.1 was expressed in E. coli. The purified XRad51.1 protein showed ssDNA-dependent ATPase activity and promoted the DNA strand exchange reaction between two 55-mer oligonucleotides. The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(d epsilonA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E. coli RecA protein. The kinetics of the fluorescence change of poly(d epsilonA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein. The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1-ssDNA complex was slower than the RecA-ssDNA complex. CONCLUSIONS: Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules. While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP.     

In vitro activity of a new oral triazole, BMS-207147 (ER-30346)

One viral strand of phi Lf, a filamentous phage of Xanthomonas campestris pv.campestris, the open reading frame (ORF440) behind gene VI was identified as gene I. This gene codes for pI protein (440 aa, 48 kDa) which was shown to be membrane-bound in the phi Lf-infected host cell by Western blot analysis using the antibody raised against the protein expressed in Escherichia coli. Its predicted amino acid sequence has a nucleotide-binding motif in the N-terminal 97 aa and a membrane-spanning domain (aa 221 to 236). These structural features are characteristic of pIs of several filamentous phages which are transmembrane proteins required for phage assembly. Thus far, nine phi Lf genes have been identified which are organized in the order GII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI, similar to the genome organization of E. coli filamentous phages.     

Characterization of a second lysine decarboxylase isolated from Escherichia coli

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.     

Isolation, characterization and sequence analysis of the scrK gene encoding fructokinase of Streptococcus mutans

A gene encoding an ATP-dependent fructokinase from Streptococcus mutans GS-5 was identified within a 2 kb DNA fragment immediately downstream from the scrA gene. The gene cloned in Escherichia coli also expressed mannokinase activity. Insertional inactivation of this gene in S. mutans markedly decreased both fructokinase and mannokinase activities. Nucleotide sequence analysis of the 2 kb fragment revealed an ORF starting 199 bp downstream from the scrA gene, preceded by potential ribosome-binding (Shine-Dalgarno) and promoter-like sequences. This ORF specified a putative protein of 293 amino acids with a calculated M(r) of 31,681. The deduced amino acid sequence of the fructokinase gene, scrK, from S. mutans exhibited no significant similarity to fructokinase genes from Klebsiella pneumoniae, E. coli plasmid pUR400 or Vibrio alginolyticus, but was similar to a comparable gene from Zymomonas mobilis.     

Isolation of a genetic locus associated with metronidazole resistance in Helicobacter pylori

We examined the molecular mechanism of metronidazole resistance by constructing a lambda-Zap II phagemid expression library with genomic DNA from a metronidazole-resistance strain of Helicobacter pylori. Twenty-two clones were found to have elevated MTZ resistances in XLOLR strain of E. coli. Phagemids belonging to the twenty two clones were extracted and then retransformed into the XLOLR strain of E. coli. After MTZ selection, five clones could confer metronidazole resistance consistently. According to Southern hybridization and DNA sequencing, the five clones contained a same locus, recA. In addition, transforming the five clones into BL21 strain of E. coli produced a higher resistance to MTZ. Interestingly, electroporation of one of the five phagemid clones into two MTZ sensitive H. pylori yielded MTZ resistant strains. Comparing amino acid sequence in MTZ resistant with sensitive isolates revealed two point mutations at this locus. Above results suggest that mutation in recA may be associated with metronidazole resistance of H. pylori.     

Cloning and sequence analysis of the gbpC gene encoding a novel glucan-binding protein of Streptococcus mutans

We have isolated dextran-aggregation-negative mutants of Streptococcus mutans following random mutagenesis with plasmid pVA891 clone banks. A chromosomal region responsible for this phenotype was characterized in one of the mutants. A 2.2-kb fragment from the region was cloned in Escherichia coli and sequenced. A gene specifying a putative protein of 583 amino acid residues with a calculated molecular weight of 63,478 was identified. The amino acid sequence deduced from the gene exhibited no similarity to the previously identified S. mutans 74-kDa glucan-binding protein or to glucan-binding domains of glucosyltransferases but exhibited similarity to surface protein antigen (Spa)-family proteins from streptococci. Extract from an E. coli clone of the gene exhibited glucan-binding activity. Therefore, the gene encoded a novel glucan-binding protein.     

Percutaneous biopsy of the thoracic spine

The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80 degrees C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.     

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.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.     

Chromosomally encoded ampC-type beta-lactamase in a clinical isolate of Proteus mirabilis

A clinical strain of Proteus mirabilis (CF09) isolated from urine specimens of a patient displayed resistance to amoxicillin (MIC >4,096 microg/ml), ticarcillin (4,096 microg/ml), cefoxitin (64 microg/ml), cefotaxime (256 microg/ml), and ceftazidime (128 microg/ml) and required an elevated MIC of aztreonam (4 microg/ml). Clavulanic acid did not act synergistically with cephalosporins. Two beta-lactamases with apparent pIs of 5.6 and 9.0 were identified by isoelectric focusing on a gel. Substrate and inhibition profiles were characteristic of an AmpC-type beta-lactamase with a pI of 9.0. Amplification by PCR with primers for ampC genes (Escherichia coli, Enterobacter cloacae, and Citrobacter freundii) of a 756-bp DNA fragment from strain CF09 was obtained only with C. freundii-specific primers. Hybridization results showed that the ampC gene is only chromosomally located while the TEM gene is plasmid located. After cloning of the gene, analysis of the complete nucleotide sequence (1,146 bp) showed that this ampC gene is close to blaCMY-2, from which it differs by three point mutations leading to amino acid substitutions Glu --> Gly at position 22, Trp --> Arg at position 201, and Ser --> Asn at position 343. AmpC beta-lactamases derived from that of C. freundii (LAT-1, LAT-2, BIL-1, and CMY-2) have been found in Klebsiella pneumoniae, E. coli, and Enterobacter aerogenes and have been reported to be plasmid borne. This is the first example of a chromosomally encoded AmpC-type beta-lactamase observed in P. mirabilis. We suggest that it be designated CMY-3.