Characterization of the 450-kb Linear Plasmid in a Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

ABSTRACT A strong polychlorinated biphenyl (PCB) degrader,Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 × 10−5 transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.

Analysis of the 5′ Portion of the Type 19A Capsule Locus Identifies Two Classes of cpsC, cpsD, andcpsE Genes in Streptococcus pneumoniae

ABSTRACT Analysis of the sequence data obtained from the 5′ portion of theStreptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designatedcps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniaeserotypes with probes specific for the cps19aC,cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported forcps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or thecps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify thecps regions between cpsB and aliAfrom a variety of pneumococcal serotypes. Direct sequencing of the 5′ end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes ofcpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.

A New Ac-Like Transposon of Arabidopsis Is Associated With a Deletion of the RPS5 Disease Resistance Gene

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4–11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA endjoining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.

Detection and Isolation of Novel Rhizopine-Catabolizing Bacteria from the Environment

ABSTRACT Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 106 and 107 catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA,mocB, and mocC genes of Sinorhizobium meliloti L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium,Pseudomonas, Aeromonas, andAlcaligenes) were represented in this set. Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti. However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to thenodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.

Recognition of adenovirus types in faecal samples by Southern hybridization in South Australia

SUMMARYThe distribution of adenovirus types in faecal samples of patients with suspected viral gastroenteritis from South Australia was determined during the 12-month period, July 1991–June 1992. There were 3299 samples tested and 226 (6·9%) were positive for adenovirus by enzyme immunoassay. Of these 226 samples. 154 (68%) were typed directly using virus DNA extracted from the faecal samples according to theSmaI,HindIII andBstEII restriction patterns and Southern hybridization analysis with pooled viral genomic DNA probes. In this group, 86% of the samples were from patients who were < 3 years of age. Enteric adenovirus types 40 and 41 accounted for 20 and 40% respectively, of these samples, and types 1, 2, 3, 5, 6, 7 and 31 comprised the remainder. Type 40 was detected mainly in the winter and spring periods, and type 41 predominated in the autumn period. The majority of the non-enteric types were found during the late winter and spring periods.

In situ localization of a mammalian protamine gene: parameters affecting specificity of hybridization

Southern hybridization analysis of Indian muntjac genomic DNA with the Eco-Taq bovine genomic protamine probe revealed a simple banding pattern. The pattern of hybridization was identical with that previously observed in the genus Bos. This suggested that the bovine probe specifically hybridized to the Indian muntjac protamine gene. The opportunity was thus provided to assign the chromosomal location of the protamine gene in a comparatively simple system. Accordingly, this probe and the corresponding cDNA probe were used for in situ chromosome hybridization and localization. Various parameters affecting specificity and the resolution of hybridization were examined. Subsequent to optimization, the Indian muntjac gene was shown to be autosomal and distally located in the telomeric region of the p arm of chromosome 1.Key words: chromosomal localization, muntjac, protamine, specificity, hybridization.