Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked. Received: 4 December 1996 / Accepted: 21 January 1997     

Specificity of recognition sequence forEscherichia coli primase

Summary We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of theEscherichia coli genome. We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites. Based on the examination of various reports of the priming reaction catalyzed byE. coli primase in vivo and in vitro, we propose that (i)E. coli primase itself recognizes a 3′GTC 5′ sequence on the template strand, (ii) DnaB helicase releases the specificity ofE. coli primase and, (iii) the consensus recognition sequence forE. coli primase associated with DnaB helicase is 3′PuPyPy 5′.     

Homologous recombination involving cox2 is responsible for a mutation in the CMS-specific mitochondrial locus of Petunia

We have characterized the only mutation detected so far in S-Pcf, the mitochondrial cytoplasmic male sterility (CMS)-specific locus of petunia. This locus consists of three open reading frames (ORFs): the first contains part of atp9, an intron-less cox2 pseudogene (which does not contain the original cox2 ATG) and the unidentified reading frame urf-s; the second and third ORFs correspond to the only copies of nad3 and rps12 genes in the genome, respectively. In the cell line R13-138, which was generated from a male-sterile somatic hybrid (line SH13-138), a change in the first ORF of the S-Pcf locus has been characterized: the atp9 sequence has been lost, while exon1 of the normal copy of the cox2 gene (including the original ATG sequence) and the adjacent 5′ sequence of the petunia recombination repeat, have been introduced. The data suggest that this reorganization of mtDNA is the consequence of a homologous recombination event involving part of the cox2 coding region, and that the cox2 coding region may serve as an active site for inter- or intra-mtDNA homologous recombination. The results further suggest that in line SH13-138 (or during its maintenance in tissue culture), segregation of the S-Pcf-containing mtDNA molecules has occurred, and the mutant mtDNA is now predominant in the population. Received: 9 September 1996 / Accepted: 27 January 1997     

Cloning and characterisation of the Neisseria gonorrhoeaearoB gene

The gene coding for the 3-dehydroquinate synthetase (aroB) of Neisseria gonorrhoeae has been cloned by functional complementation of an Escherichia coli aroB mutant. The aroB gene isolated from a gonococcal plasmid library encodes a 359 amino acid protein with a molecular mass of 38.6 kDa. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 33 to 55%. An open reading frame coding for an aroK homologue is located immediately upstream of aroB. Downstream of aroB a region of inverted repeats and a gene showing high homology to yafJ of E. coli has been identified. Disruption of aroB generates a gonococcal mutant that is unable to grow in the absence of aromatic compounds. Complementation of the mutant with the intact aroB gene intrans indicates that the gene is responsible for the auxotrophic phenotype. In infection assays with AroB-deficient gonococcal strains, binding, entry and short-term survival in epithelial cells is not affected. The aroB gene might be useful as a selectable marker and target for attenuation of a gonococcal live vaccine strain or as a biosafe laboratory strain. Received: 23 September 1997 / Accepted: 19 November 1997     

Unidentified Open Reading Frames in the Genome of Methanococcus jannaschii Are Similar in Sequence to an Archaebacterial Gene for tRNA Nucleotidyltransferase

The protein sequence of ATP/CTP:tRNA nucleotidyltransferase (cca) from Sulfolobus shibatae was used to search open reading frames in the genome of Methanococcus jannaschii. Translations of two unidentified open reading frames showed significant sequence similarity to portions of the Sulfolobus cca protein. When the two open reading frames were joined together, the expanded open reading frame was similar in sequence to the entire Sulfolobus cca protein and displayed features of the active site signature sequence proposed for members of class I enzymes within the superfamily of nucleotidyltransferases (Yue et al., 1996, RNA 2, 895–908). A possible UUG start codon was identified based on significant sequence similarity of the resulting amino-terminal region to that of Sulfolobus, and on a six-base complementarity between an adjacent upstream sequence and Methanococcus 16S rRNA. Received: 10 February 1997     

The regulatory protein MucR binds to a short DNA region located upstream of the muc R coding region in Rhizobium meliloti

The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding. Received: 9 October 1996 / Accepted: 20 December 1996     

Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

The region ofBacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance inEscherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted M_r, of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent M_r of approximately 8–10 kDa.     

Genetic organization and transposition properties of IS 511

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobactercrescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISRI, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5′ end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family. Received: 18 August 1996 / Accepted: 17 September 1996     

Nucleotide sequence of the Vibrio alginolyticus glnA region

The nucleotide sequence of a 4 kb fragment containing the Vibrio alginolyticus glnA, ntrB and ntrC genes was determined. The upstream region of the glnA gene contained tandem promoters. The upstream promoter resembled the consensus sequence for Escherichia coli ⁷⁰ promoters whereas the presumptive downstream promoter showed homology with nitrogen regulated promoters. Four putative NR_I binding sites were located between the tandem promoters. The ntrB gene was preceded by a single presumptive NR_I binding site. The ntrC gene was located 45 base pairs downstream from the ntrB gene. The V. alginolyticus ntrB and ntrC genes were able to complement ntrB, ntrC deletions in E. coli.Abbreviations bp base pair(s) - CAP catabolite-activating protein - GS glutamine synthetase - kb kilobase(s) - ORF open reading frame - SD Shine-Dalgarno     

An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK

Summary A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation. Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114. The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R. meliloti FixK. The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK. A similar protein domain is also present in E. coli Fnr and is essential for the oxygen-regulated activity of this protein. Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence. A Tn5-generated mutant in orf240 lost the ability to induce the R. meliloti fixN-lacZ fusion. Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.     

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression

Summary Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium mehloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nod[ gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible -galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.     

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     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined –16 to –25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.