Generation of targeted deletions in the genome of Rhodothermus marinus.

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R.

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure.

Effect of selected Ser/Ala and Xaa/Pro mutations on the stability and catalytic properties of a cold adapted subtilisin-like serine proteinase.

A subtilisin-like serine proteinase from a psychrotrophic Vibrio species (VPR) shows distinct cold adapted traits regarding stability and catalytic properties, while sharing high sequence homology with enzymes adapted to higher temperatures. Based on comparisons of sequences and examination of 3D structural models of VPR and related enzymes of higher temperature origin, five sites were chosen to be subject to site directed mutagenesis. Three serine residues were substituted with alanine and two residues in loops were substituted with proline.

Cloning and expression of heterologous genes in Rhodothermus marinus.

The construction of a shuttle cloning system for Rhodothermus marinus and Escherichia coli is described. It is based on the shuttle vector pRM3000, which contains a multiple cloning site as well as the shuttle marker trpB and TrpB(-) recipients of both species. The vector is stable and in 25 +/- 2 and 91 +/- 3 copies in R.

Rhodothermus marinus: physiology and molecular biology.

Rhodothermus marinus has been the subject of many studies in recent years. It is a thermohalophilic bacterium and is the only validly described species in the genus Rhodothermus. It is not closely related to other well-known thermophiles and is the only thermophile within the family Crenotrichaceae.

Identification and nucleotide sequence analysis of a cryptic plasmid, pRM21, from Rhodothermus marinus.

Here we report the identification and nucleotide sequence analysis of pRM21, a plasmid isolated from the thermophilic eubacterium Rhodothermus marinus. pRM21 consists of 2935 bp, has a G+C content of 58.2% and one major open reading frame whose deduced product shows significant similarities to RepA proteins from several plasmids, the highest being to the RepA of pSa from Escherichia coli.

Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.