Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex

Summary Two yeast DNA pools inserted in an hybridEscherichia coli-yeast vector pFL1 were used to transformE. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool — aBamHI yeast DNA digest — a 6.4 kbBamHI fragment was recovered that gave good complementation of theE. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool — a partialSau3A yeast DNA digest — five independent plasmids complementing eitherE. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kbBamHI fragment. One of these plasmids, which complemented the twoURA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the fullURA2 gene. A restriction map of theURA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization withURA2 messenger RNA, allowing us to estimate the limits of the gene.     

Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12

DNA from the marine bacteriumAlteromonas haloplanktis 214 was partially digested withSau 3A and inserted into theBam HI site of the cloning vector pBR322. The ligation mixture was used to transformEscherichia coli HB101. The gene bank plasmid preparation obtained was used to transformEscherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to containA. haloplanktis DNA and to be capable of complementing the appropriate mutation inE. coli EO2717. Restriction maps showed that each of the plasmids was different.     

Cloning of the gene coding for aromatic amino acid aminotransferase from anE. Coli B strain

Summary AnE. coli B strain showing high activity in the transamination of phenylpyruvate to phenylalanine was used as the DNA source for the construction of a cosmid library inE. coli DG30, a strain which is known to be defective in all three major transaminase genes (aspC, ilvE andtyrB). By complementation analysis, cosmid clones could be identified with inserts carrying atyrB gene. The DNA inserts were further subcloned into pAT153 and thetyrB gene fromE. coli B was found to be similar to the gene reported forE. coli K12. Plasmids containing theE. coli BtyrB gene were transformed into the originalE. coli B strain and the recombinant strains assayed for transaminase activity and plasmid stability.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday.     

A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells

The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process. Electronic Publication     

Genetic analysis of rec E activities in Bacillus subtilis

Summary ArecE mutant (recE6) ofBacillus subtilis was constructed by insertion of a selectable marker into therecE coding region. The insertional inactivation of therecE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination. TherecE6 allele was then introduced into a set of DNA repair-deficient strains ofB. subtilis. The removal of DNA damage by therecF,addAaddB,recH,recL andrecP gene products is strictly dependent on an activerecE gene product (recE-dependent pathway). On the other hand, the increased sensitization to purine adducts in theuvrA42recE6 andpolA5recE6 strains suggests that such lethal lesions may be removed either by therecE-dependent or by therecE-independent pathway.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Micrococcus luteus homolog of theEscherichia coli uvrA gene: Identification of a mutation in the UV-sensitive mutant DB7

Summary Restriction fragments ofMicrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in anEscherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with theE. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of theM. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of theE. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to theE. coli UvrABC excinuclease. Dedicated to the memory of Shunzo Okubo (1930–1978), who considered the possibility of a double-mutant status for the mutant DB7 most seriously     

Nitrate reductases inEscherichia coli

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on theE. coli chromosome. ThenarGHJI operon, encoding nitrate reductaseA, is located in thechlC locus at 27 minutes, along with several functionally related genes:narK, encoding a nitrate/nitrite antiporter, and thenarXL operon, encoding a nitrate-activated, two component regulatory system. ThenarZYWV operon, encoding nitrate reductase Z, is located in thechlZ locus located at 32.5 minutes, a region which includes anarK homologue,narU, but no apparent homologue to thenarXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both thenarGHJI operon and thenarK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while thenarZYWV operon and thenarU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase inE. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.Abbreviations NR nitrate reductase On leave from Department of Biochemistry and Molecular Biology, The University of Texas Medical school at Houston, Houston, Texas, 77225, USA     

Efficient construction of plant genomic libraries requires the use ofmcr host strains and packaging mixes

It has recently become apparent that many strains ofE. coli contain nucleases encoded by themcrA andmcrB loci that, recognize the modified base 5-methylcytosine in DNA. Plant DNAs have particularly high levels of this modification and the activity of these 5-methylcytosine-specific nucleases is particularly relevant to cloning plant genomic DNAs. We show here that for preparing libraries in a λ replacement vector, the use of suitablemcr hosts andmcr packaging mixes can increase the efficiency of cloning of plant genomic DNAs by at least two orders of magnitude. We also provide evidence that the activity of themcr nucleases is probably a significant source of bias in the representation of sequences in plant genomic libraries.     

A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.     

ThegrpD55 locus ofEscherichia coli appears to be an allele ofdnaB

Attempts to characterize thegrpD55 mutation ofEscherichia coli have led us to conclude that the gene had been assigned an incorrect map position. The mutation was found to cotransduce withmalF3089:: Tn10 (at 91.5 min) and adnaB-expressing plasmid was able to complement fully thegrpD55 defect in replication. These studies strongly suggest thatgrpD55 is an allele ofdnaB and is localized near 92 min on theE. coli linkage map.     

Cloning and biochemical characterization of Staphylococcus aureus type IA DNA topoisomerase comprised of distinct five domains

DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg²⁺-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.     

A new cell division operon in Escherichia coli

Summary At 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.     

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.     

Molecular cloning of thehom—thrC—thrB cluster fromBacillus sp. ULM1: Expression of thethrC gene inEscherichia coli and corynebacteria,and evolutionary relationships of the threonine genes

A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA ofBacillus sp. ULM1 by complementation ofEscherichia coli andBrevibacterium lactofermentum thrC auxotrophs. Complementation studies showed that thethrB gene (encoding homoserine kinase) is found downstream from thethrC gene, and analysis of nucleotide sequences indicated that thehom gene (encoding homoserine dehydrogenase) is located upstream of thethrC gene. The organization of this cluster of genes is similar to theBacillus subtilis threonine operon (hom—thrC—thrB). An 1.9 kbBclI, fragment from theBacillus sp. ULM1 DNA insert that complementedthrC mutations both inE. coli and in corynebacteria was sequenced, and an ORF encoding a protein of 351 amino acids was found corresponding to a protein of 37462 Da. ThethrC gene showed a low G+C content (39.4%) and the encoded threonine synthase is very similar to theB. subtilis enzyme. Expression of the 1.9 kbBclI DNA fragment inE. coli minicells resulted in the formation of a 37 kDa protein. The upstream region of this gene shows promoter activity inE. coli but not in corynebacteria. A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes. Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Selection againstdam methylation sites in the genomes of DNA of enterobacteriophages

Summary Postreplicative methylation of adenine inEscherichia coli DNA to produce G^6m ATC (where^6mA is 6-methyladenine) has been associated with preferential daughter-strand repair and possibly regulation of replication. An analysis was undertaken to determine if these, or other, as yet unknown roles of GATC, have had an effect on the frequency of GATC inE. coli or bacteriophage DNA. It was first ascertained that the most accurate predictions of GATC frequency were based on the observed frequencies of GAT and ATC, which would be expected since these predictors take into account preferences in codon usage. The predicted frequencies were compared with observed GATC frequencies in all available bacterial and phage nucleotide sequences. The frequency of GATC was close to the predicted frequency in most genes ofE. coli and its RNA bacteriophages and in the genes of nonenteric bacteria and their bacteriophages. However, for DNA enterobacteriophages the observed frequency of DNA enterobacteriophages the observed frequency of GATC was generally significantly lower than predicted when assessed by the chi square test. No elevation in the rate of mutation of^6mA in GATC relative to other bases was found when pairs of DNA sequences from closely related phages or pairs of homologous genes from enterobacteria were compared, nor was any preferred pathway for mutation of^6mA evident in theE. coli DNA bacteriophages. This situation contrasts with that of 5-methylcytosine, which is hypermutable, with a preferred pathway to thymine. Thus, the low level of GATC in enterobacteriophages is probably due not to^6mA hypermutability, but to selection against GATC in order to bypass a GATC-mediated host function.     

Detection and characterization of an ABC transporter in <Emphasis Type="Italic">Clostridium hathewayi</Emphasis>

An ABC transporter gene from Clostridium hathewayi is characterized. It has duplicated ATPase domains in addition to a transmembrane protein. Its deduced amino acid sequence has conserved functional domains with ATPase components of the multidrug efflux pump genes of several bacteria. Cloning this transporter gene into C. perfringens and E. coli resulted in decreased sensitivities of these bacteria to fluoroquinolones. It also decreased the accumulation and increased the efflux of ethidium bromide from cells containing the cloned gene. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) inhibited both accumulation and efflux of ethidium bromide from these cells. The ATPase mRNA was overexpressed in the fluoroquinolone-resistant strain when exposed to ciprofloxacin. This is the first report of an ABC transporter in C. hathewayi. An erratum to this article can be found at     

Low synonymous site variation at thelacY locus inEscherichia coli suggests the action of positive selection

We have determined the nucleotide sequences of sevenlacY alleles isolated from natural isolates ofEscherichia coli. Nucleotide heterozygosity estimates for this locus were compared to those obtained from previous studies of intraspecific variation at chromosomal loci, revealing thatlacY has unusually low synonymous site variation. The average pairwise heterozygosity of synonymous sites (Ks=0.0112+/-0.0100) is the second lowest reported and the lowest for loci that have an equivalent level of nonsynonymous variation. We consider several hypotheses to explain how different forces in evolution could act to create the observed pattern of polymorphism, including selection for translational efficiency and positive selection. Our analysis most strongly supports the hypothesis that positive selection has acted on thelacY locus inE. coli.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale