Susan J. Assinder

The TOL plasmids: determinants of the catabolism of toluene and the xylenes.

Publisher Summary The chapter introduces TOL plasmids and discusses pathways for toluene and xylenes catabolisms, regulation of catabolic genes, and evolution of catabolic pathways. The TOL pathway is one of three metabolic routes described by which toluene can be catabolized. The TOL plasmid encodes enzymes for conversion of the aromatic hydrocarbons to the corresponding carboxylic acids as well as the meta-pathway enzymes. The structure of the promoter sequences in pseudomonads involved in degradation of toluene to central metabolites encompasses four main elements. These are upper- and meta-pathway operon promoters and promoters of the two regulatory genes xylS and xy/R . The growth of TOL strains on benzoate (“Benzoate Curing”) is described. Uses of TOL plasmid genes in construction of novel strains and vectors are also discussed.

Inheritance of mitochondrial DNA in Phytophthora infestans

Mature (three-week) and immature (five-day) sexual progeny were raised from two matings between isolates of Phytophthora infestans with 2C-DNA content (and therefore presumed diploids) but which differed for isozyme genotype and restriction fragment length polymorphisms (RFLPs) in their mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Approximately 90% of the progeny which were derived from mature oospores, but approximately 50% of the progeny from immature oospores, were classed as hybrids on the basis of their Gpi 1 genotype. All single-oospore cultures, whether derived from a mature or an immature oospore, contained a single mtDNA type from either the A1 or the A2 parent. All single-zoospore lines derived from a single germ sporangium of a germinating hybrid oospore also had the same mtDNA type, Gpi 1 genotype and mating type. These data suggest that mtDNA is inherited uniparentally in P. infestans with no evidence for segregation, elimination or recombination of types. Mating type segregated differently in hybrid offspring; those with type II mtDNA (from the A1 parent) had a preponderance of A1 mating type. Some nRFLPs also segregated unexpectedly in the population with type II mtDNA, suggesting that any bias in the segregation of mating type is caused by viability disturbance rather than linkage between mating type gene(s) and mtDNA.

Comparison of the meta pathway operons on NAH plasmid pWW60-22 and TOL plasmid pWW53-4 and its evolutionary significance.

The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17.5 kbp BamHI fragment. The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130. A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P. putida hosts. The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate. DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4. The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.

AFLP and RFLP (RG57) fingerprints can give conflicting evidence about the relatedness of isolates of Phytophthora infestans

Selected isolates of Phytophthora infestans from around England and Wales were fingerprinted using both RG57, a multi-locus RFLP probe, and Amplified Fragment Length Polymorphisms (AFLPs). The larger number of polymorphisms detectable with the AFLP method allowed resolution of several similar AFLP genotypes among isolates with identical RG57 fingerprints. However, some isolates with the same RG57 genotype had remarkably dissimilar AFLP genotypes, suggesting that there has been convergent evolution of some RG57 fingerprints. Also, some isolates with dissimilar RG57 fingerprints had similar or identical AFLP fingerprints. Both techniques distinguished isolates of mitochondrial DNA haplotype la from those of haplotype IIa. However, with AFLPs only, most of the isolates of A2 mating type were very similar and were distinguished from those of A1 mating type, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual recombination is rare.

Inheritance of streptomycin and chloramphenicol resistance in Phytophthora infestans: evidence for co-segregation of mitochondrial DNA and streptomycin resistance

Spontaneous mutant strains of Phytophthora infestans resistant to either streptomycin or chloramphenicol were selected by prolonged exposure of colonies to drug-amended media. Each strain was then crossed with a sensitive strain having different RFLP markers in its mitochondrial (mt) DNA and nuclear (n) DNA. A majority (56 out of 65) of the sexual progeny from the streptomycin mating inherited at least one nRFLP band from each parent. Fifty-two of these hybrids were streptomycin resistant and inherited their mtDNA from the resistant parent, while the four streptomycin sensitive isolates inherited their mtDNA from the sensitive parent. These data are consistent with streptomycin resistance being cytoplasmically inherited, possibly on the mitochondrial genome. The inheritance of chloramphenicol resistance in hybrids did not correlate well with that of mtDNA type but neither was it obviously Mendelian.

The Aspergillus nidulans hfa mutations affect genomic stability and cause diverse defects in cell cycle progression and cellular morphogenesis

The hfa (high frequency of aneuploidy) mutants of Aspergillus nidulans carry conditional lethal (temperature-sensitive) defects which cause an increased frequency of aneuploids to be produced amongst their asexual progeny. When examined microscopically, most of the mutants grew and divided their nuclei at restrictive temperature, albeit more slowly than the wild-type, and aneuploidy was not attributable to an obvious cell cycle lesion. Exceptions were hfaB3 and hfaL1 which exhibited defects in nuclear division, although neither mutant arrested at a specific point in the cell cycle. Cells carrying hfaB3 contained only a single enlarged nucleus which was often transected (’cut’) by the first septum and temperature-shift experiments showed that the mutation triggers aneuploidy by causing failure to properly exit mitosis. Although the hfaD1 mutant underwent nuclear division, it differed morphologically from wild-type by exhibiting a hyper-branching phenotype. The original hfaD1 isolate was shown also to carry a second unlinked mutation (designated hurA1 ) which confers resistance to hydroxyurea and partly alleviates the growth defects imposed by hfaD1 .