Analysis of the role of recA in phenotypic switching of Pseudomonas tolaasii

Switching between the pathogenic smooth (1116S) and nonpathogenic rough (1116R) forms of Pseudomonas tolaasii occurs due to the reversible duplication of a 661-bp element within the pheN locus. Disruption of the chromosomal recA locus of 1116S and 1116R produced strains 1116SrecA and 1116RrecA, respectively, which showed typical loss of UV resistance. Switching from the smooth to the rough form was virtually eliminated in the 1116SrecA strain, whereas the extent of switching from the rough to the smooth form was almost identical in 1116R and 1116RrecA. It is concluded that phenotypic switching from 1116S to 1116R is recA dependent whereas that from 1116R to 1116S is recA independent.     

Genomic rearrangement at 10q24 in non-syndromic split-hand/split-foot malformation

Split-hand/split-foot malformation (SHFM) is a congenital limb malformation characterized by a median cleft of hand and/or foot due to the absence of central rays. Five loci for syndromic and non-syndromic SHFM, termed SHFM1-5, have been mapped to date. Recently, a 0.5 Mb tandem genomic duplication was found at chromosome 10q24 in SHFM3 families. To refine the minimum duplicated region and to further characterize the SHFM3 locus, we screened 28 non-syndromic SHFM families for tandem genomic duplication of 10q24 by Southern blot and sequence analysis of the dactylin gene. Of 28 families, only two showed genomic rearrangements. Representative patients from the two families exhibit typical SHFM, with symmetrically affected hands and feet. One patient is a familial case with a 511,661 bp tandem duplication, whereas the second is a sporadic case arising from a de novo, 447,338 bp duplication of maternal origin. The smaller duplication in the second patient contained the LBX1, BTRC, POLL, and DPCD genes and a disrupted extra copy of the dactylin gene, and was nearly identical to the smallest known duplicated region of SHFM3. Our results indicate that genomic rearrangement of SHFM3 is rare among non-syndromic SHFM patients and emphasize the importance of screening for genomic rearrangements even in sporadic cases of SHFM.     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

The Sinorhizobium meliloti Insertion Sequence (IS) Element ISRm14 Is Related to a Previously Unrecognized IS Element Located Adjacent to the Escherichia coli Locus of Enterocyte Effacement (LEE) Pathogenicity Island

ISRm14 is 2695 basepairs (bp) in size and bordered by 22 bp imperfect inverted repeats (IRs). A 9-bp target sequence is duplicated upon ISRm14 transposition. The DNA strand that putatively encodes the transposase enzyme carries three open reading frames (ORFs) designated ORFs1 to 3, which specify putative proteins of 15.9 kDa, 13.1 kDa, and 61.1 kDa, respectively. According to its structural characteristics, ISRm14 belongs to the recently proposed IS66 family of IS elements. The ORFs1 to 3 encoded putative proteins displayed significant similarities to ORFs of the previously unrecognized IS element ISEc8, which is inserted adjacent to the locus of enterocyte effacement (LEE) pathogenicity island of Escherichia coli EDL933. Analyses of the distribution of ISRm14 in a natural S. meliloti population showed its widespread occurrence in 66% of the strains tested with a copy number ranging from 1 to 6. Received: 13 May 1999 / Accepted: 14 June 1999     

High genetic variability of the Streptococcus thermophilus cse central part, a repeat rich region required for full cell segregation activity

The cse gene of Streptococcus thermophilus encodes an extracytoplasmic protein involved in cell segregation. The Cse protein consists of two putative domains: a cell wall attachment LysM domain and a catalytic CHAP domain. These two domains are spaced by an interdomain linker, known as Var-Cse, previously reported to be highly divergent between two S. thermophilus strains. The aim of this study was to assess the extent of this intraspecific variability and the functional involvement of the var-cse region in cell segregation. Analysis of the var-cse sequence of 19 different strains allowed detection of 11 different alleles, varying from 390 bp to 543 bp, all containing interspersed and tandem nucleotides repeats. Overall, 11 different repeat units were identified and some series of these small repeats, named supermotifs, form large repeats. Results suggested that var-cse evolved by deletion of all or part of the repeats and by duplication of repeats or supermotifs. Moreover, sequence analysis of the whole cse locus revealed that the cse ORF is mosaic suggesting that var-cse polymorphism resulted from horizontal transfer. The partial deletion of the var-cse region of the S. thermophilus strain CNRZ368 led to the lengthening of the number of cells per streptococcal chain, indicating that this region is required for full cell segregation in S. thermophilus strain CNRZ368.     

The complete mitochondrial genome of Calicogorgia granulosa (Anthozoa: Octocorallia): potential gene novelty in unidentified ORFs formed by repeat expansion and segmental duplication

Mitochondrial genomes of many nonbilaterian animals show high diversity of genome size and gene content, revealing many intergenic regions (IGRs), diverse repeats and additional genes. Here we present a new complete mitogenome of the cnidarian sea fan species, Calicogorgia granulosa (Anthozoa: Octocorallia) and its novel genomic features. The 20,246 bp of the complete mitogenome, which is the largest among the nine octocorals sequenced to date, contains 13 protein coding genes, 2 rRNAs and a tRNA within its circular form of mitochondrial DNA. We found an identical segmental duplication (S1 and S2, 913 bp) composed of an ORF (672 bp) coding for a hypothetical protein within which Direct Variant Repeat (DVR) expansions reside in-frame to the coding sequence. Additionally, the duplicated segmental DNA showed no variation in nucleotide sequences both between S1 and S2 and across multiple individual samples. Upon these observations, we discuss plausible causes for the intramitochondrial segmental duplication and the absence of sequence variation, and a need for further investigation of the novel ORF as well. In conclusion the present mitogenome of C. granulosa adds more information to our understanding of the diversity and evolution of mitogenomes of nonbilaterian animals.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus.

Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii.     

IS431mec-mediated integration of a bleomycin-resistance gene into the chromosome of a methicillin-resistant Staphylococcus aureus strain isolated in Japan

A methicillin-resistant Staphylococcus aureus (MRSA) strain B-26, isolated clinically in Hiroshima University Hospital, is resistant to bleomycin together with kanamycin. In the present study, we analysed the nucleotide sequence of the 5.1-kb HindIII fragment containing the bleomycin- and kanamycin-resistance genes, which were previously cloned [Bhuiyan et al. (1995) Appl Microbiol Biotechnol 43: 65–69] from the chromosomal DNA of MRSA B-26. The present study found that the DNA sequence contains the duplicated target sequence (GATTAGAT) consisting of 8 bp for transposase and the entire nucleotide sequence of the plasmid pUB110, together with the sequence of inverted repeats (16 bp), designated IR-r and IR-l in IS431mec. The 8-bp duplication sequence, produced by the transposable element, was first found by us. We proposed that bleomycin resistance in MRSA B-26 is attributed to the IS431mec-mediated integration of pUB110 into the chromosome. Received: 13 September 1995/Received last revision: 20 February 1996/Accepted: 30 March 1996     

Transformation by integration in Podospora anserina. III. Replacement of a chromosome segment by a two-step process

Summary We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.     

Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae

Summary A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn²⁺, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.     

Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins

Neisseria meningitidis FAM20 has recently been shown to produce two Fe-regulated proteins (FrpA and FrpC) related to the RTX family of cytotoxins. Here we report the cloning and DNA sequence of the locus containing the gene encoding the larger meningococcal RTX protein FrpC. FrpC was highly similar to FrpA throughout much of the predicted protein, with two main differences. Whereas the FrpA protein had 13 copies of the nine-amino-acid repeat units typical of RTX proteins, FrpC had 43 copies. The additional copies in FrpC apparently arose from a threefold tandem amplification of a 600bp DNA fragment encoding the repeats. In addition, the frpC gene lacked good promoter consensus sequences. An open reading frame (0RF1) of unknown function was found immediately upstream of frpC, suggesting the possibility that frpC was cotranscribed with ORF1. A probable promoter was found 300 bp upstream of ORF1, and it contained a Fur protein-binding sequence found in the promoters of Fe-regulated Escherichia coli genes. DNA upstream of the ORF 1/frpC promoter was homologous to IStO76-like elements surrounding capsulation loci of strains of Haemophilus influenzae. A FrpC-like protein (reactive in immunoblots with monoclonal antibody 9D4; multiple reactive bands of about 200 to 120kDa) was found in five out of eight meningococcal strains but only in one out of 14 other Neisseria, suggesting that FrpC may participate in the pathogenesis of meningococcal disease.     

Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form

Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura⁺ transformants with 1 μg pRPPyr4 DNA and 1 × 10⁷ viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man_0∼1GlcNAc₂ at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains. Received: 1 February 1999 / Received revision: 26 February 1999 / Accepted: 20 March 1999     

Gene Cloning,Expression, and Substrate Specificity of an Imidase from the Strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> YZ-26

A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni²⁺–NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.     

Sequence Analysis of Small Cryptic Plasmids Isolated from Selenomonas ruminantium S20

Two small cryptic plasmids designated pONE429 and pONE430 were isolated from a rumen bacterium, Selenomonas ruminantium S20. The complete sequence of pONE429 was 2100 bp and contained one open reading frame (ORF) of 201 amino acids. The sequence of pONE430 had 1527 bp and one ORF of 171 amino acids with the similarity of replication protein (Rep protein) of pOM1, pSN2, and pIM13 isolated from Butyrivibrio fibrisolvens, Staphylococcus aureus, and Bacillus subtilis, respectively. In these plasmids, the upstream nucleotide sequence of Rep protein had the conserved nucleotides which could be double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The plasmids of pONE429, pONE430, pJJMI, pJDB21, and pS23 were isolated from S. ruminantium strains and had similar regions that were located within a <450-bp nucleotide. These similar regions may be the location that was recognized by the host strain, S. ruminantium. Received: 10 July 1998 / Accepted: 11 September 1998     

Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit

A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.