A Lactobacillus helveticus-Specific DNA Probe Detects Restriction Fragment Length Polymorphisms in This Species

A cloned 2-kb EcoRI fragment (fragment f) from a 34-kb plasmid of Lactobacillus helveticus CNRZ 1094 was shown by dot blot to specifically hybridize to total DNAs of 75 L. helveticus strains. No hybridization was found with L. acidophilus, L. crispatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. gasseri, or L. intestinalis strains. When Southern blots of EcoRI digests of L. helveticus strains were probed with fragment f, these strains displayed restriction fragment length polymorphisms on the basis of which they could be grouped into several clusters.     

Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration

Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration.     

Extrachromosomal plasmid-like DNA in the obligate parasitic fungus Erysiphe graminis f. sp. hordei

Summary The obligate parasitic fungus, Erysiphe graminis f. sp. hordei, was found to harbour plasmid-like extrachromosomal DNA. A 1.35-kb fragment of this 9kb plasmid was cloned into the pUC12 vector. No homology was detected to nuclear or mitochondrial DNA. As only about half of the 27 isolates examined contained plasmid-like DNA, this appears to be inessential for fungal survival. The plasmid is frequent in European isolates and is found in both newly collected isolates and in isolates kept under laboratory conditions for many years. No correlation between presence of plasmid and specific avirulence/virulence genes was found. The plasmid appear to be located in the mitochondria.     

Cloning and sequence analysis of the plasmid DNA found inRhizoctonia solani AG-2-2 LP isolate and its potential use for fungal detection

A plasmid DNA (PE-42 plasmid) obtained fromRhizoctonia solani AG-2-2 LP isolate PE-42, the causal agent of large patch disease of zoysiagrass (Zoysia spp.), was partially cloned. Sequence analyses of the 1.2-kb and 0.2-kb cloned fragments revealed that the nucleotide sequence of the 0.2-kb fragment was similar to that of the 5′ region of the 1.2-kb fragment (pSH4). Southern hybridization analysis of total DNA of a large patch isolate using the 1.2-kb fragment as a probe showed two bands differing slightly in size. These results indicated that the PE-42 plasmid consisted of at least two components having similar nucleotide sequences with different sizes. The nucleotide sequence of the pSH4 fragment showed no significant homology with known DNA sequences. The pSH4 fragment hybridized to all of the 22 large patch isolates tested, but not to other subgroup isolates in AG-2-2, other anastomosis groups ofR. solani, or other pathogens of zoysiagrass. These results indicated that the pSH4 fragment can be used as a specific probe to detect the large patch fungus. The detection limit for the large patch fungus using the pSH4 fragment as a probe was 0.1 μg of the total DNA of the fungus, which was significantly higher than those for other fungi. However, with improvement of the detection sensitivity and simplification of the detection procedure, the pSH4 fragment has potential for use in molecular diagnosis of the large patch disease of zoysiagrass. Contribution No. 140 from the Laboratory of Plant Pathology, Mie University.     

Multiple pathways for toluene degradation in Burkholderia sp. strain JS150.

Burkholderia (Pseudomonas) sp. strain JS150 uses multiple pathways for the metabolism of catechols that result from degradation of aromatic compounds. This suggests that the strain also uses multiple upstream pathways for the initial hydroxylation of aromatic substrates. Two distinct DNA fragments that allowed Pseudomonas aeruginosa PAO1c to grow with benzene as a sole carbon source were cloned from strain JS150. One of the recombinant plasmids containing the initial steps for the degradative pathway contained a 14-kb DNA insert and was designated pRO2016. We have previously shown that the DNA insert originated from a plasmid carried by strain JS150 and contained genes encoding a multicomponent toluene-2-monooxygenase (tbmABCDEF) as well as the cognate regulatory protein (tbmR) that controls expression of the 2-monooxygenase (G. R. Johnson and R. H. Olsen, Appl. Environ. Microbiol. 61:3336-3346, 1995). Subsequently, we have identified an additional region on this DNA fragment that encodes toluene-4-monooxygenase activity. The toluene-4-monooxygenase activity was also regulated by the tbmR gene product. A second DNA fragment that allowed P. aeruginosa to grow with benzene was obtained as a 20-kb insert on a recombinant plasmid designated pRO2015. The DNA insert contained genes encoding toluene-4-monooxygenase activity but no toluene-2-monooxygenase activity. The pRO2015 insert originated from the chromosome of strain JS150, unlike the region cloned in pRO2016. Southern blots and restriction map comparisons showed that the genes for the individual 4-monooxygenases were distinct from one another. Thus, strain JS150 has been shown to have at least three toluene/benzene monooxygenases to initiate toluene metabolism in addition to the toluene dioxygenase reported previously by others.     

Identification of Phytophthora citrophthora with Cloned DNA Probes

Two different DNA fragments, one of 2.9 kilobases and the other of 5.1 kilobases, were cloned from Phytophthora citrophthora and showed no homology with DNA from plants and other related fungi. These DNA probes hybridized with DNA from 12 different P. citrophthora isolates obtained from a variety of hosts but did not hybridize with DNA from 6 P. citrophthora isolates obtained from cacao. Southern blot analysis revealed that the probes contained repetitive DNA, and restriction fragment length polymorphisms were identified among several P. citrophthora isolates. Of the isolates tested, two major groups were observed whose genetic similarity correlated with geographical distribution. One of the DNA probes was used to detect P. citrophthora growing from infected citrus roots incubated on semiselective medium. P. citrophthora was not detected by a hybridization assay of total DNA extracted directly from infected roots.     

Restriction map of the 125-kilobase plasmid of Bacillus thuringiensis subsp. israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae.

A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp. israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli. The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI. Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes. All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively). The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences. Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; without cytA, they are placed on a single BamHI fragment. This convergence enables subcloning of delta-endotoxin genes (excluding cryIVA, localized on the other linkage group) as an intact natural fragment.     

Analysis of foldback sequences and repetitive sequences in cloned segments of nuclear DNA from Physarum polycephalum

The properties of DNA segments containing foldback elements were studied after their selection from a ‘random’ recombinant library of Physarum polycephalum nuclear DNA sequences, cloned using the plasmid vector pBR322. Hybridisation of in vitro labelled recombinant plasmids to Southern blots of genomic restriction fragments demonstrated that each cloned segment contained repetitive elements located at several hundred sites in the genome. Two of the DNA clones generated hybridisation patterns which suggested that they contain repetitive elements with internal cleavage sites for the restriction endonuclease HaeIII. Cross-hybridisation of all combinations of the cloned sequences showed that most contain different arrangements of repetitive elements derived from different sequence families. The results are consistent with a model proposed previously on the basis of studies on total nuclear DNA, for the organisation of sequences closely associated with foldback elements in the Physarum genome     

Separation of Three Species of Ditylenchus and Some Host Races of D. dipsaci by Restriction Fragment Length Polymorphism

This study examined the ribosomal cistron of Ditylenchus destructor, D. myceliophagus and seven host races of D. dipsaci from different geographic locations. The three species showed restriction fragment length polymorphisms (RFLPs) in the ribosomal cistron, the 18S rDNA gene, and the ribosomal internal transcribed spacer (ITS). Southern blot analysis with a 7.5-kb ribosomal cistron probe differentiated the five host races of D. dipsaci examined. Polymerase chain reaction (PCR) amplification of the ITS, followed by digestion with some restriction endonucleases (but not others), produced restriction fragments diagnostic of the giant race. Because the PCR product from D. myceliophagus and the host races of D. dipsaci was about 900 base pairs and the ITS size in D. destructor populations was 1,200 base pairs, mixtures of populations could be detected by PCR amplification. ITS fragments differentiated between D. dipsaci and Aphelenchoides rhyntium in mixed populations. This study establishes the feasibility of differentiation of the host races of D. dipsaci by probing Southern blots with the whole ribosomal cistron.     

Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2.

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.     

Structural gene aberrations in mucopolysaccharidosis II (Hunter)

Summary A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.     

Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.     

Distribution and homology of plasmids in several strains of Cyanothece

The plasmid distribution of several clonal isolates of the unicellular, diazotrophic, cyanobacterium Cyanothece sp. has been analyzed. The Cyanothece isolates contain three to four plasmids ranging in size from 4.8 kb to 40 kb. The plasmid profiles of three Cyanothece strains (BH63, BH68, BH93) indicated that strains BH68 and BH93 were closely related and that strain BH63 may be more distantly related. A small 4.8-kb plasmid (pSE480), from the clonal isolate Cyanothece sp. strain BH68F, has been subcloned and restriction mapped. Ten restriction sites have been mapped, five of which are unique and suitable for further subcloning. Southern hybridization revealed that this plasmid was present in two out of five clonal isolates of strain BH68 and in one isolate of strain BH93. A 10-kb plasmid from strain BH68F (pSE1000) was found in all of the BH68 isolates and was absent in the BH93 isolate, Cyanothece sp. strain BH93A. No notable physiological changes were observed in the absence of either the 4.8-kb or 10-kb plasmids. Therefore, these plasmids remain cryptic. Further analysis of these plasmids may provide insight into the function of these plasmids and will allow the construction of shuttle vectors for gene transfer experiments.     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.     

Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid.

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

Genomic RFLP Analysis of Meloidogyne arenaria Race 2 Populations

Traditional morphological methods of Meloidogyne identification have been unsuccessful in distinguishing three South Carolina, USA Meloidogyne arenaria race 2 populations—Govan, Pelion, and Florence. These populations differ greatly in reproductive rate and aggressiveness on soybean hosts. Total genomic DNA from eggs of each population was digested with the restriction endonuclease Eco RI and Southern hybridization analyses were performed with single-copy and interspersed multi-copy cloned probes. Probes were isolated from a genomic library of Eco RI, M. arenaria DNA fragments cloned into pUC8. One probe, designated pE1.6A, when hybridized to Southern blots of M. arenaria genomic DNAs, displayed an interspersed repetitive pattern, and the RFLPs distinguished the Govan population from the Pelion and Florence populations. Another clone, pE6.0A, carrying moderately repeated sequences, distinguished the Pelion and Florence isolates. This communication demonstrates the utility of genomic RFLP analysis for distinguishing populations of the same race within the same species. To test the possible utility of these moderately repeated sequence probes for detecting the presence of nematode DNA in DNA samples from roots inoculated with varying numbers of nematodes, dot blot hybridization analyses were performed. It is possible to detect as few as 30 nematodes per root sample with these cloned probes.