J. R. Van Der Meer

Characterization of a second tfd gene cluster for chlorophenol and chlorocatechol metabolism on plasmid pJP4 in Ralstonia eutropha JMP134(pJP4).

Within the 5.9-kb DNA region between the tfdR and tfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCDEF and tfdB) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows: tfdD(II)C(II)E(II)F(II) and tfdB(II) (in short, the tfd(II) cluster), by analogy to tfdCDEF and tfdB (the tfd(I) cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdD(II), suggesting an operon-like organization for the tfd(II) genes. By expressing each ORF in Escherichia coli, we confirmed that tfdD(II) coded for a chloromuconate cycloisomerase, tfdC(II) coded for a chlorocatechol 1, 2-dioxygenase, tfdE(II) coded for a dienelactone hydrolase, tfdF(II) coded for a maleylacetate reductase, and tfdB(II) coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfd(I) and tfd(II) genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfd(II) genes seem to be redundant with respect to those of the tfd(I) cluster. One reason why the tfd(II) genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR and tfdS.

Characterization of two alternative promoters for integrase expression in the clc genomic island of Pseudomonas sp. strain B13

The clc genomic island is a 105 kb integrative and conjugative element (ICE) in Pseudomonas sp. strain B13, which encodes metabolism of 3‐chlorocatechol. The clc island is integrated in a tRNAGly gene, but can excise and form a circular intermediate in which both ends are connected. The integrase gene (intB13) of the clc genomic island is located at the right end, 202 bp from the junction site facing inwards. Fragments upstream of intB13 in the circular form and in the integrated form were fused to a promoterless gfp gene for Green Fluorescent Protein and introduced in monocopy onto the chromosome of strain B13. Quantitative GFP fluorescence measurements in individual cells of the different B13‐derivatives revealed that the circular form fragment contained a strong constitutive promoter (Pcirc) driving intB13 expression in all cells. By using primer extension Pcirc could be mapped near the left end of the clc element and Pcirc can therefore only control intB13 expression when left and right ends are connected as in the circular form. Expression from intB13 upstream fragments from the integrated clc element was weaker than that from Pcirc and only occurred in maximally 15% of individual cells in a culture. A promoter (Pint) could be roughly mapped in this region by using reverse‐transcription PCR and by successively shortening the fragment from the 5′ end. Transposon mutants in cloned left end sequences of the clc element were selected which had lost the activation potential on the Pint promoter and those which resulted in overexpression of GFP from Pint. The DNA sequence of the region of the transposon insertions pointed to a relatively well conserved area among various other genomic islands. The activator mutants mapped in an open reading frame (ORF) encoding a 175 amino acid protein without any significant similarity to functionally characterized proteins in the databases.

Characterization of a ribosomal RNA gene cluster from Clostridium tyrobutyricum : phylogenetic positioning based on the 16S and 23S nucleotide sequences

Summary A ribosomal operon of Clostridium tyrobutyricum NIZO strain 51 was cloned into plasmid vectors and characterized by DNA sequencing of the 16S rRNA, 23S rRNA and part of the 5S rRNA. The organization of this ribosomal operon of C. tyrobutyricum is similar to that of C. perfringens and Bacillus subtilis . A proposal was made for the processing stems and the sites of maturation of 16S and 23S rRNA. The phylogenetic position of C. tyrobutyricum was reconstructed by comparative 16S rRNA and 23S rRNA sequence analyses. Both analysis provided a similar position for C. tyrobutyricum . The 16S rRNA comparisons showed that C. kluyveri and C. ljungdahlii are the closest relatives of C. tyrobutyricum .