Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

The yeast PCK1 gene coding for phosphoenolpyruvate carboxykinase (PEPCK) was isolated by functional complementation of pck1 strains from S. cerevisiae. Only one copy of the gene was found per haploid yeast genome. An RNA of about 2 kb which hybridized with a DNA probe internal to the PCK1 gene was found only in cells growing in non-fermentable carbon sources. Yeast strains carrying multiple copies of the PCK1 gene showed normal catabolite repression of PEPCK except those carrying the shortest insertion complementing the mutation (2.2 kb) that presented an altered kinetics of derepression. Catabolite inactivation was decreased in strains transformed with multicopy plasmids carrying the PCK1 gene.     

The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites

We present the sequence of a 2 kb fragment of the Bacillus subtilis Marburg genome containing sacB, the structural gene of levansucrase, a secreted enzyme inducible by sucrose. The peptide sequence deduced for the secreted enzyme is very similar to that directly determined by Delfour (1981) for levansucrase of the non-Marburg strain BS5. The peptide sequence is preceded by a 29 amino acid signal peptide. Codon usage in sacB is rather different from that in the sequenced genes of other secreted enzymes in B. subtilis, especially alpha-amylase. Genetic evidence has shown that the sacB promotor is rather far from the beginning of sacB (200 bp or more). The 200 bp region preceding sacB shows some of the features of an attenuator. A preliminary discussion of the putative workings and roles of this attenuator-like structure is proposed. sacRc mutations, which allow constitutive expression of levansucrase, have been located within the 450 bp upstream of sacB. It is shown that sacRc and sacR+ alleles control in cis the expression of the adjacent sacB gene.     

Genes required for cellulose synthesis in Agrobacterium tumefaciens.

A region of the chromosome of Agrobacterium tumefaciens 11 kb long containing two operons required for cellulose synthesis and a part of a gene homologous to the fixR gene of Bradyrhizobium japonicum has been sequenced. One of the cellulose synthesis operons contained a gene (celA) homologous to the cellulose synthase (bscA) gene of Acetobacter xylinum. The same operon also contained a gene (celC) homologous to endoglucanase genes from A. xylinum, Cellulomonas uda, and Erwinia chrysanthemi. The middle gene of this operon (celB) and both the genes of the other operon required for cellulose synthesis (celDE) showed no significant homology to genes contained in the databases. Transposon insertions showed that at least the last gene of each of these operons (celC and celE) was required for cellulose synthesis in A. tumefaciens.