Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Distribution and analysis of plasmids inStreptococcus thermophilus

Summary In a survey of 35 strains ofStreptococcus thermophilus, 13 strains were found to harbor plasmid DNA. Most of these strains contained plasmid species varying in size from 2.2 to 7.15 kilobases. Only three strains had more than one plasmid species. Each of the nine distinct types of plasmid DNAs identified had two or more unique recognition sites for restriction endonucleases. The characteristics of the indigenous cryptic plasmids ofS. thermophilus may allow their development as cloning vectors useful in the genetic engineering of this species and other streptococci that are important in food production     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Involvement of a Polyethylene glycol (PEG)-oxidizing Enzyme in the Bacterial Metabolism of PEG

Eighty-seven strains of acetic acid bacteria were surveyed for plasmids by CsCl-ethidium bromide equilibrium centrifugation of cell lysates. Twenty-seven of the 33 strains of Acetobacter were found to harbor plasmid DNA and most strains contained multiple species of low-molecular-weight plasmids. On the other hand, plasmid DNAs were detected in 23 of the 36 strains of Gluconobacter and most of them had molecular weights of more than 5 megadaltons. Of the 18 strains newly isolated from a vinegar factory, some of which were examined taxonomically, 17 contained plasmid DNA. The molecular weights of plasmids detected in this study were in the range of about one to over 17 megadaltons. The plasmids with molecular weights of less than 5 megadaltons were characterized with restriction endonucleases. The physical maps of two plasmids designated as pMV1O1 and pMV102, which were found in the isolated strains, were constructed.     

Streptomyces aureofaciens strains as hosts for cloning of genes affecting antibiotic production

Summary Several mutants ofStreptomyces aureofaciens strain were used for protoplast regeneration and plasmid transformation. All tested mutants (excepting R 8/26) were transformable by number of plasmids and shuttle vectors. The transformation of the CTC production strains by plasmid containing cloned CTC resistance gene resulted in 1,1–4 times higher antibiotic production. From the restriction analysis of plasmid, phage and chromosomal DNAs it was estimated, that all tested mutants normally contain the modification system analogous toNae I (Roberts, 1987). Mutant R 8/26 expresses not only complete restriction-modification system mentioned above but also potential second system restricting several actinophages.     

Variability of endonucleolytic activity indicates high genetic diversity within the natural population ofSelenomonas ruminantium

The population of bacteria ofSelenomonas ruminantium species in the rumen of fallow-deer was analyzed using endonucleolytic activity assay and plasmid profiles. This analysis indicated a high diversity within the population ofS. ruminantium. At least 12 different restriction profiles, indicating the presence of the different specificity nucleases, have been observed. Site-specific endonucleases were detected in 17 out of 45 strains tested. In other strains a various level of nonspecific activity was detected. Plasmid DNAs ranging in size from 0.9 to more than 25 kbp were detected in 60% of strains analyzed. No or little correlation was observed between the endonuclease activity and the plasmid content. The presence of different specificity endonucleases, as well as differences of plasmid profiles of isolates possessing identical specific activity indicate that the population ofS. ruminantium in the rumen of an individual animal consists of at least 10 different clones. This work was supported byGrant Agency VEGA 3007/96.     

Enhanced Transformation Efficiency of Recalcitrant Bacillus cereus and Bacillus weihenstephanensis Isolates upon In Vitro Methylation of Plasmid DNA

Digestion patterns of chromosomal DNAs of Bacillus cereus and Bacillus weihenstephanensis strains suggest that Sau3AI-type restriction modification systems are widely present among the isolates tested. In vitro methylation of plasmid DNA was used to enhance poor plasmid transfer upon electroporation to recalcitrant strains that carry Sau3AI restriction barriers.     

Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees

Aims: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. Methods and Results: For this purpose, we established a protocol for the transformation of the plasmid pAD43‐25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re‐germination. Conclusions: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP‐tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re‐germination. Significance and Impact of the Study: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.     

Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions

Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs. Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species. Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains. Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated. The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB₂ genes, allowed fingerprinting of Ti plasmids. Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independant identification of strains and of the conjugative Ti plasmids.Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer Funded by Institut National de la Recherche Agronomique     

Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.     

Synergistic effect of surfactin from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C4 and <Emphasis Type="Italic">Achyrocline satureioides</Emphasis> extracts on the viability of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>

The aim of this work was to determine the in vitro effect of the mixture between the lipopeptide surfactin, synthesized by Bacillus subtilis C4 (strain isolated from honey) and the most active vegetal extract from Achyrocline satureioides, a traditional medicinal plant, on local strains of Paenibacillus larvae, the agent of American Foulbrood in honeybees. Five P. larvae strains isolated in Córdoba, Argentina, were phenotypically characterized. These and 12 other P. larvae strains from different regions of Argentina were analysed. The antimicrobial activities of the essential oil, hexane (HE) and benzene extracts from A. satureioides were assessed against P. larvae and the HE showed the highest anti-P. larvae activity. A combination of the biosurfactant surfactin, produced by B. subtilis C4, and the HE of A. satureioides revealed a synergistic action on P. larvae. The effective surfactin concentration in the mixture decreased from 32 to 1 μg ml⁻¹ and the HE concentration from 32 to 4 μg ml⁻¹, values similar or equal to minimal inhibitory concentrations observed for oxytetracycline. The fractional inhibitory concentration index confirmed synergism in 4 strains and partial synergism in one strain. The combination of surfactin synthesized by B. subtilis C4 and the HE from A. satureioides could be a natural alternative to help beekeepers to combat the American foulbrood agent P. larvae.     

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

16S rDNA-based phylogeny of non-symbiotic bacteria of Entorno-pathogenic nematodes from infected insect cadavers

Using 16S rDNA gene sequencing technique, three different species of non-symbiotic bacteria of entomopatho-genic nematodes (EPNs) (Steinernema sp.and Heterorhabditis sp.) were isolated and identified from infected insect cadavers(Galleria mellonella larvae) after 48-hour post infections.Sequence similarity analysis revealed that the strains SRK3, SRK4 and SRK5 belong to Ochrobactrum cytisi,Schineria larvae and Ochrobactrum anthropi,respectively.The isolates O.anthropi and S.larvae were found to be associated with Heterorhabditis indica strains BDU-17 and Yer-136,respectively,whereas O.cytisi was associated with Steinernema siamkayai strain BDU-87. Phenotypically, temporal EPN bacteria were fairly related to symbiotic EPN bacteria (Photorhabdus and Xenorhabdus genera). The strains SRK3 and SRK5 were phylogeographically similar to several non-symbionts and contaminated EPN bacteria isolated in Germany(LMG3311T) and China (X-14),while the strain SRK4 was identical to the isolates of S.larvae (L1/57,L1/58, L1/68 and L2/11) from Wohlfahrtia magnifica in Hungary.The result was further confirmed by RNA secondary structure and minimum energy calculations of aligned sequences.This study suggested that the non-symbionts of these nematodes are phylogeographically diverged in some extent due to phase variation.Therefore,these strains are not host-dependent, but environment-specific isolates.     

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer. Received: 28 January 1997 / Accepted: 3 June 1997     

Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins

Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains.     

The control region of the F sex factor DNA transfer cistrons: Physical mapping by deletion analysis

Summary A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.     

A mitochondrial plasmid from the plant pathogenic fungus Cochliobolus heterostrophus

Summary Twenty-three strains of Cochliobolus heterostrophus were examined for the presence of plasmid DNA. One isolate, T40, contained a 1.9 kb sequence which occurred as a series of circular head-to-tail multimers with from 1 to 17 or more monomers per plasmid molecule. The plasmid was cloned in pBR322 to facilitate analysis. It was homologous to the mitochondrial chromosome of isolate T40 as well as to the mitochondrial DNAs of C. heterostrophus isolates that did not contain the plasmid; each isolate, including T40, had only one copy of the plasmid sequence integrated into the mitochondrial chromosome and the sequence mapped at the same location in all isolates tested. In the T40 isolate there were about 30 excised copies per chromosome in addition to the single integrated sequence. Presence of the plasmid had no apparent effect on the structural integrity of the mitochondrial chromosome. There was no detectable homology between the plasmid and either C. heterostrophus nuclear DNA or plasmids that have been isolated from mitochondria of Neurospora or Podospora. A circular map was constructed which has 6 sites for hexan-ucleotide-recognizing enzymes and the region of the splice site; no sites were detected in the plasmid for an additional 17 restriction enzymes. The plasmid functioned as an ARS (autonomously replicating sequence) in yeast, although it was highly unstable compared to other ARSs.     

Plasmids in methanotrophic bacteria: isolation,characterization and DNA hybridization analysis

Ten strains of obligate methanotrophs were screened for the presence of plasmid DNA using a variety of methods. Plasmids were detected in all strains except Methylococcus capsulatus Bath. No significant similarity between plasmids was observed with respect to size or restriction digest patterns except for three strains of Methylosinus trichosporium, which appeared to contain the same three plasmids. Nitrocellulose filter hybridization revealed that the plasmid DNA from the M. trichosporium strains shared a small region of homology with the plasmid DNA from Methylosinus sporium 5. All of the plasmids remain cryptic. As the first step in characterization, a restriction digest map of the 55 kb plasmid found in Methylomonas albus BG8 was constructed.Abbreviations kb kilobases Formerly Mary L. O'Connor     

Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature

Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.