An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.     

Physical and genetic analysis of deletion mutants of plasmid R91-5 and the cloning of transfer genes in Pseudomonas aeruginosa.

We isolated deletion mutants of Pseudomonas aeruginosa plasmid R91-5 by both in vitro and in vivo means. Many of the deletion mutants selected on the basis of resistance to donor-specific phages fell into a few groups of apparently identical mutants, although the mutants were nonsibs. By analyzing plasmids with large deletions, we found that the essential replication genes of R91-5 were within a 3.85-kilobase region between coordinates 45.5 and 48.9. The origin of plasmid transfer (oriT) was mapped to a 4.5-kilobase region between coordinates 1.7 and 6.2. We indirectly determined the direction of plasmid transfer from oriT. By combining the data from our analysis of the deletions with data from complementation tests between cloned R91-5 fragments and known reference mutants, we ordered and mapped the 10 known transfer (tra) cistrons of R91-5. All of the tra cistrons mapped within the Tra2 region, and their order was as follows: traX, -Y, -T, -Q, -(V, R), -U, -(S, Z), -W (the cistrons in parentheses could not be ordered with respect to each other).     

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.     

Uptake,translocation and persistence of mycotoxins in rice seedlings

The mycotoxins citrinin, patulin and terreic acid are absorbed by rice seedling roots and translocated to shoots. Ten day analysis of toxin treated plants showed persistence of citrinin, patulin and terreic acid. All three toxins at a concentration of 100 ppm showed phytotoxic activity indicating terreic acid in addition to citrinin and patulin as phytotoxins.     

Resistance gene analogs within an introgressed chromosomal segment derived from Triticum ventricosum that confers resistance to nematode and rust pathogens in wheat

A resistance (R) gene-rich 2S chromosomal segment from Triticum ventricosum contains a cereal cyst nematode (CCN; Heterodera avenae) R gene locus CreX and a closely linked group of genes (Sr38, Yr17, and Lr37) that confer resistance to stem rust (Puccinia graminis f. sp. tritici), stripe rust (P. striiformis f. sp. tritici), and leaf rust (P. recondita f. sp. tritici) when introgressed into wheat. The 2S chromosomal segment from T. ventricosum is further delineated in translocations onto chromosome 2A of bread wheat, where the rust genes are retained but not the CreX gene. Using these critical genetic stocks, we have isolated family members of R gene analogs that are associated with either the 2S segment from T. ventricosum carrying the CreX locus or the rust genes. Derivatives of the Cre3 candidate R gene sequence and a rice (Oryza sativa) R gene analog that mapped to the 2S homologous chromosome groups in wheat were used to isolate related gene sequences from T. ventricosum that contain a nucleotide binding site-leucine rich repeat domain. The potential of these gene sequences as entry points for isolating candidate genes or gene family members of the CreX or rust genes and their further applications to plant breeding are discussed.     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

The new approaches to whole genome analysis of bacteria

A range of recombinant DNA techniques now enables whole genome analysis of any bacterium to be carried out without recourse to the classical means of bacterial genetic exchange. Using enzymes which cut infrequently, such as SpeI, combined with pulsed field gel electrophoresis, a physical map of ordered fragments can be constructed. By means of cloned fragments of known genes or oligonucleotides synthesized using data from DNA or protein sequence banks, the location of individual genes on this map can be determined. We have used these techniques to study whole genome structure in three species of Pseudomonas: P. aeruginosa, P. putida and P. solanacearum.     

Design, synthesis, and biological evaluation of a novel class of fluorescein-based N-glycosylamines

A series of fluorescein-based N-glycosylamines was synthesized from the corresponding fluorescein amine and a partially protected d-glucose. The physiochemical investigation of these compounds by spectral and morphological studies reveals their gelation potential. The exclusive localization of fluorescence in the cytoplasm through cell imaging studies reveals the anti-cancer potentials of N-glycosylamines.     

Spatial and Developmental Variation in Seed Dormancy Characteristics in the Fire-responsive Species Anigozanthos manglesii(Haemodoraceae) from Western Australia

Anigozanthos manglesii(Haemodoraceae) is a colourful, herbaceousperennial exhibiting intra- and inter-populational variationin germination in response to smoke and heat. This study investigatedthe extent and nature of this variation in A. manglesii populationscollected along a 550 km latitudinal cline from kwongan scrub(30°S) to more mesic jarrah forest habitats (34°S) insouthwestern Australia. Variation in seed germination alonga maturing inflorescence was also investigated. Germinationof this species is known to be prolific following fire, andtwo germination treatments, aerosol smoke and heat, were usedas germination cues. There was a trend of increasing responsivenessof A. manglesii to smoke with increasing latitude along a clinalrange, but there were no differences in germination of seedsalong the cline in response to control or heat treatments. Therewas no significant difference in intra-population seed germinationin response to any treatment. Lastly, a significant and increasingresponse to smoke for seeds from the apex to base of the inflorescencewas detected. This latter trend may be attributed to higherresource allocation and an accelerated after-ripening of basipetalcompared to acropetal seeds. Possible reasons for the clinalvariation are discussed.Copyright 2001 Annals of Botany Company Anigozanthos manglesii, populations, geographical cline, inflorescence level, germination, smoke, heat, fire, seed development