Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean

Glycine max (soybean) plants can be nodulated by fast-growing rhizobial strains of the genus Sinorhizobium as well as by slow-growing strains clustered in the genus Bradyrhizobium. Fast-growing rhizobia strains with different soybean cultivar specificities have been isolated from Chinese soils and from other geographical regions. Most of these strains have been clustered into the species Sinorhizobium fredii. The S. fredii strain HH103 was isolated from soils of Hubei province, Central China and was first described in 1985. This strain is capable to nodulate American and Asiatic soybean cultivars and many other different legumes and is so far the best studied fast-growing soybean-nodulating strain. Additionally to the chromosome S. fredii HH103 carries five indigenous plasmids. The largest plasmid (pSfrHH103e) harbours genes for the production of diverse surface polysaccharides, such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS). The second largest plasmid (pSfrHH103d) is a typical symbiotic plasmid (pSym), carrying nodulation and nitrogen fixation genes. The present mini review focuses on symbiotic properties of S. fredii HH103, in particular on nodulation and surface polysaccharides aspects. The model strain S. fredii HH103 was chosen for genomic sequencing, which is currently in progress. First analyses of the draft genome sequence revealed an extensive synteny between the chromosomes of S. fredii HH103 and Rhizobium sp. NGR234.     

Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector

Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker. Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

Development of cloning vehicles from the Streptomyces plasmid pFJ103

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies     

Characterization of pKU701, a 2.5-kb plasmid,in a Japanese Helicobacter pylori isolate

A cryptic plasmid of Helicobacter pylori, pKU701 (accession number AB078638), was isolated and the complete nucleotide sequence was determined. No drug resistance properties were mediated by pKU701. The 2454b pKU701 sequence, which had a 38% content of G-C residues, generated one polypeptide from a single open reading frame (ORF1). Extensive sequence homology was evident between pKU701 and ORF1 of H. pylori plasmid pHPO100 (repA, 88.8% identity) as well as ORF3 of plasmid pHPS1 (repB, 80.2% identity), but pKU701 showed only 46.3% homology with ORF1 of plasmid pHPK255 (repA). Tandem direct repeats of a 33-bp segment were found in pKU701 outside ORF1, but there were no inverted repeat ends such as those found in typical insertion sequences. The ability of drug resistance plasmids to replicate in H. pylori is probably limited, so chromosomal mutation may be a more likely cause of resistance.     

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.     

Attaching and effacing lesions in vivo and adhesion to tissue culture cells of Vero-cytotoxin-producing Escherichia coli belonging to serogroups O5 and O103

Certain isolates of Escherichia coli from humans and animals with enteric disease attach to enterocytes and cause 'attaching and effacing' (AE) lesions. E. coli strain S22-1, serotype O103:H2, isolated from a child with diarrhoea, contained two plasmids; one of these (pDEP12) hybridized with the CVD419 DNA probe derived from a plasmid found in E. coli O157:H7 and associated with expression of fimbriae and ability to adhere to Intestine 407 cells. Strain S102-9, serotype O5:H-, isolated from a calf with dysentery, contained six plasmids, one of which also hybridized with the CVD419 probe. Loss of pDEP12 coincided with reduced adhesion to HEp-2 or Intestine 407 cells cultured in vitro; reintroduction of this plasmid restored adhesiveness. Loss of the plasmid in strain S102-9 that hybridized with the CVD419 probe did not cause a decrease in adhesion. Accumulations of actin were seen in vitro in the fluorescence actin staining (FAS) test of strains S22-1, S102-9 and their derivatives, irrespective of the plasmid content of these strains or the prevalence of attached bacteria. Strain S22-1 and its plasmidless derivative caused AE lesions of equal severity in experimentally infected gnotobiotic piglets; piglets inoculated with an isolate from a healthy human or pig did not develop these lesions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X₂ even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X₂ has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5–100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA–DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.     

Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species

A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis.     

Isolation and Genetic Analysis of an Agrobacterium tumefaciens Avirulent Mutant with a Chromosomal Mutation Produced by Transposon Mutagenesis

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harbouring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent- mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3′-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tu?efaciens.     

Identification and characteristics of plasmids of the strains of erythromycin-producing Streptomyces erythreus

Presence of plasmid DNA was investigated in laboratory strains 2 and 4 (NRRL 2338) of S. erythreus, as well as in strains 1 and 3 of S. erythreus subjected to improvement with respect to erythromycin production. Families of plasmids close by their molecular weights were identified in S. erythreus strains 3 and 4 (NRRL 2338). A plasmid DNA fraction of S. erythreus strain 3 was studied with electron microscopy. It enabled to identify 5 plasmids: pSE11, pSE12, pSE13, pSE14 and pSE15 with length of 5.3, 12.4, 16.3, 29.6 and 86.9 kb respectively. Using of various procedures for isolation of extrachromosomal DNA did not provide its detection in S. erythreus strains 1 and 2. At least a part of the plasmids detected in S. erythreus strains 3 and 4 (NRRL 2338) was conjugative. 32R-Labeled plasmid DNA of S. erythreus strain 3 was subjected to hydridization according to Sauthern with total DNA of the 4 strains treated with restrictases BamHI, PstI and BgIII. The studies showed that the genome of S. erythreus strain 2 was not homologous with the probe while S. erythreus strain 1 contained one of the plasmids or its part in chromosome-integrated state. In strains 3 and 4 (NRRL 2338) of S. erythreus certain plasmid DNAs were present in both autonomous and chromosome-inserted states. 32P-Labeled gene of erythromycin resistance (ermE) was subjected to hybridization according to Southern with total DNA of the 4 strains and with DNA plasmid fraction of S. erythreus strain 3. The signal was positive only in hydridization of the probe with total DNA of S. erythreus strains 1, 3, and 4 (NRRL 2338).(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid deoxyribonucleic acid and translucent-to-opaque variation in Mycobacterium intracellulare 103.

After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium intracellulare strain 103, gave rise to variants forming opaque colonies. These opaque variants were more sensitive streptomycin, kanamycin, viomycin, and rifampin than were the wild-type translucent variants. Plasmid deoxyribonucleic acids taken from translucent strain cells and from cells of certain opaque variants were analyzed by agarose gel electrophoresis. Two plasmids of molecular weights of approximately 2 x 10(6) and 50 x 10(6), respectively, were found in the wild-type translucent cells; one of them, the 2 x 10(6)-molecular-weight plasmid, was always missing from deoxyribonucleic acids of the opaque variant cells. The results suggested that translucent colonial appearance and antibiotic resistance of the strain are plasmid-determined functions.     

The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE(C103S) and wild-type penDE genes in P. chrysogenum (Wis54-DE(C103S) strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (beta subunit formation) were observed in the Wis54-DE(C103S) strain, suggesting a regulatory role of the unprocessed IAT(C103S) in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT(C103S) was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing.