R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa

Summary The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10^-3–10^-5 per donor cell for markers in the 0–60 min region of the chromosome. Little or no chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3–5 min after initiation of mating. These results, combined with linkage studies, indicate that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min.R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for argB ⁺ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmids of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.     

Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics.

Summary The conjugative plasmid R68.45 mobilizes the chromosome of Pseudomonas aeruginosa strain PAO from multiple sites located in different chromosome regions. In interrupted matings on the plate, selection for any single marker tested resulted in entry times of 3–5 min. When selection was imposed for two markers linked in R68.45-mediated conjugation, double recombinants appeared after a delay which corresponded approximately to the map distance between the two markers as measured by the sex factor FP2. Thus, R68.45 and FP2 appear to promote chromosome transfer at similar rates, but R68.45, unlike FP2, seems to give non-polarized transfer. R68.45 may be used to estimate map distances between linked markers located in those chromosome regions where other sex factors do not produce enough recombinants to permit accurate measurement of entry times.In R68.45 matings on the plate, most recombinants inherited short donor chromosome fragments (usually less than 10 min long) and lost the R plasmid during purification. Used like a large generalized transducing phage, R68.45 has proved valuable in construction of PAO strains with desired genotypes.     

Genetic circularity of the Pseudomonas aeruginosa PAO chromosome.

Genetic circularity of the Pseudomonas aeruginosa PAO chromosome was demonstrated by a series of two- and three-factor crosses and double-selection experiments with Cma plasmids FP2, FP5, FP110, and R68.45. A range of additional markers, including catabolic markers, were located on the chromosome map. Plasmid FP2, known to have a major origin of chromosome transfer (0 min) was shown to have at least one other minor origin from which it can transfer the chromosome in the direction opposite to that found for the major origin.     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

Analysis of flagellar genes in Pseudomonas aeruginosa by use of Rfla plasmids and conjugations.

Over 300 flagellar mutants were isolated in Pseudomonas aeruginosa PAO. R-prime plasmids carrying segments of bacterial chromosome which can complement the mutant phenotypes were isolated by means of plasmid R68.45. Among the R-prime plasmids, pMT6 complemented 167 out of 307 mutants examined, and pMT19 complemented the remaining 140 mutants. We found no mutant which was complemented by both of these plasmids. Hence, the flagellar genes were divided into two clusters by these two plasmids, namely, region I on pMT19 and region II on pMT6. By FP5- and R68.45-mediated conjugation, these two regions were located on the P. aeruginosa PAO chromosome with an order of puuF--region I--region II--oru-325.     

A comparison of three Rhizobium linkage maps

Summary Linkage maps of R. meliloti 2011 (Rm2011), R. meliloti 41 (Rm41) and R. leguminosarum 300 (R1300), all constructed by means of P1 plasmid-mediated recombination, were compared. Recombination between the two R. meliloti strains occurred at high frequency but was barely detectable in matings between R1300 and Rm41. When co-inheritance data for the three strains were transformed into additive map distances the arrangement of markers showed striking similarities. Each of eight R68.45-primes, carrying different sections of the Rm2011 chromosome, suppressed only those markers of both R1300 and Rm41 which had the same phenotype and map location. Each of these R-primes promoted polarized chromosome transfer in an anticlockwise direction in Rm41, starting from the region corresponding to that carried on the plasmid.     

Chromosome mapping in Pseudomonas aeruginosa PAT.

A linkage map of Pseudomonas aeruginosa PAT has been derived from the results of conjugation experiments using the plasmids FP2-2, R68, R91-5, and R68.45. FP2-2 and R68 each mobilize the chromosome from single, distinct transfer origins. R91-5 appears to mobilize the chromosome from two such origins, and R68.45 utilizes a number of transfer origins. R68 and R91-5 have both been shown to mobilize the chromosome with a polarity opposite to that by FP2-2. The locations of the transfer origins of these plasmids are such that it has not been possible to demonstrate chromosomal circularity by means of interrupted mating experiments. However, the available time-of-entry data combined with linkage data from plate mating experiments support the conclusion that the chromosome of P. aeruginosa is circular.     

In vivo generation of R68.45-pPGH1 hybrid plasmids conferring a Phl+ (meta pathway) phenotype

Summary Plasmid pPGH1 originating from Pseudomonas putida strain H carries all the genes required for the degradation of phenol (or cresols) via the meta cleavage pathway. Besides mobilization of pPGH1 by a plasmid of the incompatibility group P-1, hybrid plasmids conferring the Phl⁺ phenotype could be selected, when R68.45 was the conjugative plasmid. The hybrids contain the complete R68.45 and part of pPGH1. Integration of Phl-DNA of pPGH1 into R68.45 occurred exclusively via the IS21 region of R68.45.Dedicated to Udo Taubeneck on the occasion of his 60th birthday     

Enlarged map ofAgrobacterium tumefaciens C58 and the location of chromosomal regions which affect tumorigenicity

Summary A revised and enlarged genetic map of theAgrobacterium tumefaciens C58 chromosome has been produced with the help of plasmid R68.45. Apart from the location of several auxotrophic markers, the map also shows the position of two independent genes,ctu1 andctu2, which, when mutated, block the tumorigenesis of the bacterium. Of these two, onlyctu1 is complemented by the C58 chromosomalvir region cloned by Douglas et al. (1985). The same mutant was complemented by a chromosomal gene or genes located nearleu ofRhizobium meliloti and known to affect the nodulation properties of that bacterium. It has also been observed that C58 tryptophan auxotrophs invariably lose tumorigenicity. Prototrophic revertants and mutants supplied with extra tryptophan for about two weeks after infection produce normal tumours. These investigations suggest that for successful tumorigenesis a continuous supply of tryptophan is needed (to be converted into auxin IAA?) at least during the early stages.     

Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.     

Rough and fine linkage mapping of the Rhizobium meliloti chromosome.

Summary A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage.Three short chromosomal regions have been mapped by cotransduction. Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough. Cotransduction seems to be a useful tool for improving the linkage map of R. meliloti.     

A chromosomal linkage map of Agrobacterium tumefaciens and a comparison with the maps of Rhizobium spp

Summary Plasmid R68.45 was found to mobilize the Agrobacterium tumefaciens chromosome apolarly. With the aid of this R plasmid the linkage between 26 markers was determined, from which a circular genetic map of the A. tumefaciens chromosome could be constructed. The recombinants obtained were stable i.e. they did not segregate strains, with the parental phenotype upon purification. A system for the polar transfer of chromosomal material from a fixed origin was developed for A. tumefaciens. It was found that R plasmid pRL189, which carries a copy of transposon Tn5, is able to mobilize the chromosome polarly from chromosomal Tn5-insertion sites. A. tumefaciens phe-1 and trp-22 auxotrophs became prototrophic after the introduction of R primes pAJ21JI and pAJ73JI, respectively, which carry the corresponding phe and trp genes of Rhizobium meliloti. This result enabled a preliminary comparison of the gene orders in A. tumefaciens and Rhizobium spp. which suggested that the chromosome maps of these organisms are quite similar.     

Mapping of some genes involved in C-1 metabolism in the facultative methylotroph Methylobacterium sp strain AM1 (Pseudomonas AM1)

R68.45 mediated mobilisation of the chromosome of Methylobacterium sp strain AM1 has been investigated. High frequencies of cotransfer of four genes required for C-1 metabolism with the genes coding for streptomycin, phosphonomycin and cycloserine resistance were demonstrated. A preliminary map of this region has been constructed on the basis of the results of three and four factor crosses showing that not all the C-1 genes are contiguous.Abbreviations Str streptomycin - Pho phosphonomycin - Cyc cycloserine - Tc tetracycline - Km kanamycin - Cb carbenicillin - Ade adenine - Thi thiamine - Met methionine     

Restriction endonuclease analyses of the incompatibility group P-1 plasmids RK2, RP1, RP4, R68, and R68.45

The P-group plasmids RP1, RP4, RK2, R68 and R68.45 were analyzed by the following restriction endonucleases:BamHI,BglII,EcoRI,HindIII,PstI,PvuII,SalI, andSmaI. No differences between RP1, RP4, and RK2 were found, and the plasmid R68.45 was found to contain a direct duplication of an existing DNA sequence in R68. Our map of RK2 differs from the published map of RK2 in the corresponding region of the R68 map that is duplicated in R68.45.     

Identification of the novel recessive gene pi55(t) conferring resistance to Magnaporthe oryzae

The elite rice cultivar Yuejingsimiao 2 (YJ2) is characterized by a high level of grain quality and yield, and resistance against Magnaporthe oryzae. YJ2 showed 100% resistance to four fungal populations collected from Guangdong, Sichuan, Liaoning, and Heilongjiang Provinces, which is a higher frequency than that shown by the well-known resistance (R) gene donor cultivars such as Sanhuangzhan 2 and 28zhan. Segregation analysis for resistance with F₂ and F₄ populations indicated the resistance of YJ2 was controlled by multiple genes that are dominant or recessive. The putative R genes of YJ2 were roughly tagged by SSR markers, located on chromosomes 2, 6, 8, and 12, in a bulked-segregant analysis using genome-wide selected SSR markers with F₄ lines that segregated into 3 resistant (R):1 susceptible (S) or 1R:3S. The recessive R gene on chromosome 8 was further mapped to an interval ≈1.9 cM/152 kb in length by linkage analysis with genomic position-ready markers in the mapping population derived from an F₄ line that segregated into 1R:3S. Given that no major R gene was mapped to this interval, the novel R gene was designated as pi55(t). Out of 26 candidate genes predicted in the region based on the reference genomic sequence of the cultivar Nipponbare, two genes that encode a leucine-rich repeat-containing protein and heavy-metal-associated domain-containing protein, respectively, were suggested as the most likely candidates for pi55(t).     

Molecular mapping of gibberellin-responsive dwarfing genes in bread wheat

Opportunities exist for replacing reduced height (Rht) genes Rht-B1b and Rht-D1b with alternative dwarfing genes for bread wheat improvement. In this study, the chromosomal locations of several height-reducing genes were determined by screening populations of recombinant inbred lines or doubled haploid lines varying for plant height with microsatellite markers. Linked markers were found for Rht5 (on chromosome 3BS), Rht12 (5AL) and Rht13 (7BS), which accounted for most of the phenotypic variance in height in the respective populations. Large height differences between genotypes (up to 43 cm) indicated linkage to major height-reducing genes. Rht4 was associated with molecular markers on chromosome 2BL, accounting for up to 30% of the variance in height. Confirming previous studies, Rht8 was linked to markers on chromosome 2DS, whereas a population varying for Rht9 revealed a region with a small but significant height effect on chromosome 5AL. The height-reducing effect of these dwarfing genes was repeatable across a range of environments. The molecular markers developed in this study will be useful for marker-assisted selection of alternative height-reducing genes, and to better understand the effects of different Rht genes on wheat growth and agronomic performance.     

Catabolic instability,plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10^-10 cell^-1 generation^-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BgIII, HindIII and SaII restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R 68.45 into Alcaligenes sp. BR60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BgIII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba⁺ mutants of Alcaligenes sp. BR60.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - HPLC high pressure liquid chromatography - ATCC American Type Culture Collection     

Isolation and characterization of an R'' plasmid in Pseudomonas aeruginosa.

An R' plasmid, R'PA1, carrying a 3- to 4-min segment of the Pseudomonas aeruginosa chromosome has been derived from the incP-1 plasmid R68.45. The chromosomal segment includes the markers argA, argB, argH, and lys-12. The plasmid retains all the properties of R68.45, including chromosome mobilization ability and wide bacterial host range. R'PA1 reverts to R68.45 in rec+ strains of P. aeruginosa, but it can be maintained in a recA strain.     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

The circling behavior of the deafblind LEW-<Emphasis Type="Italic">ci2</Emphasis> rat is linked to a segment of RNO10 containing <Emphasis Type="Italic">Myo15</Emphasis> and <Emphasis Type="Italic">Kcnj12</Emphasis>

The LEW/Ztm-ci2 rat is an autosomal recessive mutant that displays circling behavior, deafness, progressive retinopathy, locomotor hyperactivity, ataxia, and opisthotonus. We performed a genome-wide scan of a (LEW/Ztm-ci2 × BN/Ztm) F₁ × LEW/Ztm-ci2 backcross population with anonymous microsatellite markers to analyze the genetics of this mutant rat. This linkage analysis demonstrated a very strong association of RNO10 SSLP markers to the phenotype with a core region in the central part of the chromosome. The knowledge of genes mapping to this part of the rat genome and their linkage to SSLP markers is still poor. We developed SSLP markers closely linked to genes, which might be responsible for the mutant phenotype by using the growing amount of rat-specific DNA sequences available at World Wide Web databases. Application of this method facilitated the search for candidate genes for the phenotype of the LEW-ci2 rat. We were able to map Myo15 and its neighboring genes, Znf179 and Aldh3a1, to the region of interest and Myo1c to a more distal location on RNO10. Further rat BAC clones were used to create a physical map of the region of interest. This map revealed the position of further genes. Among those is Kcnj12. Owing to their localization on RNO10 and their involvement in a similar pathology in human and mouse, Myo15 and Kcnj12 can be regarded as candidate genes for the deafblind phenotype of the LEW-ci2 rat.