Development and Application of Random DIG-Labelled Probes Based on Total Genomic Lactobacillus DNA

Summary Nine Lactobacillus-specific and non-isotopically (digoxygenin) labelled probes were developed on the basis of Lactobacillus total chromosomal DNA. Their specificity and applicability for Lactobacillus discrimination was proven by DNA–DNA hybridization to reference strains from the American Type Culture Collection (ATCC). The DNA probes were divided into three groups depending on the ability to hybridize to DNA from the same and/or from a group of related Lactobacillus strains. They were assayed in the species-specific detection of vaginal strains from the genus Lactobacillus. Six DNA probes were successfully applied for characterization of 21 newly isolated vaginal Lactobacilli. The species affliation of some isolates was determined. The developed DNA probes were evaluated for usage as a qualitative hybridization test for detection of Lactobacillus species in mixed cultures, obtained directly from vaginal samples without strain isolation.     

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer,expression, regulation and DNA homologies to various nickel-resistant bacteria

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mm NiCl₂ in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mm NiCl₂. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mm Ni²⁺) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5–5) isolated from nickel-rich soil in New Caledonia.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Cloning and characterization of hydrogenase genes from Azotobacter chroococcum

Summary Genomic DNA from Azotobacter chroococcum was shown by DNA hybridization to contain sequences homologous to Rhizobium japonicum H₂-uptake (hup) hydrogenase genes carried on the plasmid pHU1. Two recombinant cosmid clones, pACD101 and pACD102, were isolated from a gene library of A. chroococcum by colony hybridization and physically mapped. Each contained approximately 42 kb of insert DNA with approximately 27 kb of overlapping DNA. Further hybridization studies using three fragments from pHU1 (6 kb HindIII, 6.4 kb BglII and 5 kb EcoRI) showed that the hup-specific regions of R. japonicum and A. chroococcum are probably highly conserved. Weak homology to the hydrogenase structural genes from Desulfovibrio vulgaris (Hildenborough) was also observed. A 24 kb BamHI fragment from pACD102 subcloned into a broad host-range vector restored hydrogenase activity to several Hup^- mutants of A. chroococcum.     

Detection of Two Fungal Biocontrol Agents against Root-knot Nematodes by RAPD Markers

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 μg, respectively.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Three new species ofBullera isolated from leaves in the Ogasawara Islands

Thirteen strains of ballistoconidium-forming yeasts were isolated from leaves collected in the Ogasawara Islands, Japan. They represent three different species in the genusBullera on the basis of morphological, physiological, and biochemical characteristics, analyses of the sequences of internal transcribed spacer regions and small subunit ribosomal DNA, and a nuclear DNA-DNA hybridization study. Three new species,Bullera boninensis (five strains),B. waltii (seven strains), andB. schimicola (one strain), are proposed for these 13 strains.     

Diversity of Rhizobia isolated from various Hedysarum species

Cultural and physiological properties, serology, plasmid profiles and infective traits were determined for 23 strains of rhizobia isolated from various Hedysarum species: H. coronarium (common name: sulla) (16), H. carnosum (1), H. alpinum (3), H. mackenzii (2) and H. pallens (1) from Portugal, Spain, Tunisia, Alaska and Israel. Strains isolated from H. alpinum, H. mackenzii and H. pallens have slow growth rates on yeast-extract mannitol medium and were unable to nodulate H. coronarium plants, whereas the latter were effectively nodulated by all sixteen fast growing strains from sulla. Regardless of the country of origin all H. coronarium strains fell into one serogroup and were not serologically related with strains of other Hedysarum species. The RAPD (random amplified polymorphic DNA) fingerprinting method which was carried out on five H. coronarium and three H. alpinum strains allowed distinction to be made among serologically related rhizobia. No particular plasmid profile pattern was observed in relation to the host or geographical origin of the strains.     

Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutropbus pbbB and to Rbizobium meliloti nodG

Summary A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. mehloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the pbbB gene and involved in poly--hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli ⁷⁰ consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium

Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. We tested the possible factors that may cause instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting of highly repetitive or largely single-copy DNA sequences, different Agrobacterium transformation methods and different Agrobacterium strains. The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium. All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA deficient strains. We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.Communicated by J. Dvorak     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat–, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat– information. The transformants behave in crosses as do the reference mat+ or mat– strains, thus indicating that the transgenic mat+ and mat– are fully functional even when they have integrated at ectopic sites.     

Transposable elements as plant transformation vectors for long stretches of foreign DNA

The production of transgenic plants is now routine for most crops. However, using currently available transformation methods it is still difficult and time-consuming to obtain a collection of transformed individuals containing single or low-copy-number, intact transgenic inserts. Here we describe a set of broad-hostrange transformation vectors based on the Ac/Ds transposition system that improve both transformation efficiency and the quality of transgenic loci. These vectors efficiently deliver long stretches of foreign DNA into the genome, leading to transgenic strains containing an intact single-copy insert of 10kb. This type of vector could be an important additional tool for the production of transgenic plants with the well-defined, foreign DNA inserts required for biosafety approval and commercialisation.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Detection of Hyperthermophilic Archaea of the Genus Desulfurococcus by Hybridization with Oligonucleotide Probes

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a designed Crenarchaeota-specific primer pair: Cren 7F (5"-TTCCGGTTGATCCYGCCGGACC-3") and Cren 518R (5"-GCTGGTWTTACCGCGGCGGCTGA-3"). The PCR products were hybridized with Dig-11-dUTP–labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5"-CGTTAACYCCYGCCACACC-3") and its species D. mobilis (Dco_mob 198, 5"-CGTTAACCCCTGCCACACC-3") and D. amylolyticus (Dco_amy 198, 5"-CGTTAACCCCCGCCACACC-3"). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the speciesDesulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.     

Vibrio mimicus are the reservoirs of the heat-stable enterotoxin gene (nag-st) among species of the genus Vibrio

Using a 0.27 kb DNA probe specific for the heat-stable enterotoxin gene (nag-st) of Vibrio cholerae non-O1, 1109 strains representing 17 species of the genus Vibrio, isolated from clinical and environmental sources were examined. The nag-st gene was preponderantly associated with strains classified as V. mimicus; 16.8% of these strains hybridized. It was more frequent in the clinical isolates (22.6%) than in the environmental isolates (13.7%). The incidence of nag-st gene-positive strains of V. mimicus isolated from different countries was uniformly high and ranged between 8.7% (Bangladesh) and 57.1% (environmental strains from USA). The incidence of the nag-st gene was much lower among strains of V. cholerae non-O1 (3.6%). Probe-positive and-negative strains of V. mimicus and V. cholerae non-O1 were used to evaluate the performance of the conventional suckling mouse assay for detection of the NAG-ST enterotoxin. Of the 31 probe-positive strains, only five (16.1%) yielded a positive fluid accumulation ratio (FA ratio) when neat heated culture supernatant was used to perform the suckling mouse assay. All the 31 probe-positive strains gave a positive FA ratio when 20-fold concentrated and heated culture supernatants of the strains were used to perform the suckling mouse assay. The need to concentrate (by at least 20-fold) the culture supernatant of strains of V. mimicus and V. Cholerae non-O1 was identified as an important step to obtain consistent results when using the suckling mouse assay for detection of NAG-ST.P. Yuan, A. Ogawa and T. Takeda are with the Department of Infectious Disease Research, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan; P. Yuan is also with the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. T. Ramamurthy and G.B. Nair are with the National Institute of Cholera and Enteric Diseases, Calcutta, India. T. Shimada is with the National Institute of Health, Tokyo 141, Japan. S. Shinoda is with the Faculty of Pharmaceutical Sciences, Okayama University, Japan.     

Double Ds elements are involved in specific chromosome breakage

Summary The structure of the unstable Ds-induced sh-m5933 allele of the maize sucrose synthase gene was analysed and a double Ds structure found in opposite orientation on both sides of a 30 kb insert interrupting the sucrose synthase gene. The double Ds structures bordering the insert are identical over a distance of approximately 3 kb. These double Ds structures and the DNA segments beyond them are in opposite orientation and identical over a distance of approx. 5.3 kb. A hypothesis for how such a symmetrical structure could be formed is proposed. When one complete Ds element was excised from one of the double Ds structures a half Ds element was left behind. This half Ds element was found in one revertant strain which displayed an altered pattern of chromosome breakage compared to revertant strains which had not undergone Ds excision. Nine new maize strains which showed a similarly altered chromosome breakage pattern were isolated. In all nine cases we observed an indistinguishable deletion in the genomic DNA. These excisions are likely to be the result of similar excision events to that described above. We conclude that double Ds structures are responsible for Ds-induced chromosome breakage.     

The use of karyotyping in the systematics of yeasts

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungiHortaea werneckii, Filobasidiella (=Cryptococcus)neoformans, andMalassezia species.Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes ofFilobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varietiesneoformans andbacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains ofMalassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas inM. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.