Sigrídur H. Thorbjarnardóttir

Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases

In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C. The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C. The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.

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. R. marinus has been isolated from several similar but distantly located geothermal habitats, many of which are subject to large fluctuations in environmental conditions. This presumably affects the physiology of R. marinus. Many of its enzymes show optimum activity at temperatures considerably higher than 65°C, the optimum for growth, and some are active over a broad temperature range. Studies have found distinguishing components in the R. marinus electron transport chain as well as in its pool of intracellular solutes, which accumulate during osmotic stress. The species hosts both bacteriophages and plasmids and a functional intein has been isolated from its chromosome. Despite these interesting features and its unknown genetics, interest in R. marinus has been mostly stimulated by its thermostable enzymes, particularly polysaccharide hydrolysing enzymes and enzymes of DNA synthesis which may be useful in industry and in the laboratory. R. marinus has not been amenable to genetic analysis until recently when a system for gene transfer was established. Here, we review the current literature on R. marinus.

Cloning, sequence analysis and functional characterization of DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus

A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified. The gene was cloned, sequenced and expressed in Escherichia coli. The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104·8 kDa. Sequence analysis showed that a generally conserved Phe residue in the O‐helix is substituted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing. The protein was purified, characterized and showed to contain specific DNA‐polymerization activity of 3100 units/mg of protein, 5′→3′ exonuclease activity and a 3′→5′ proofreading activity. Its optimum activity was at 55 °C and it had a half‐life of 2 min at 90 °C. A truncated form of the enzyme lacking the 5′→3′ exonuclease domain was also expressed in E. coli. It had a specific DNA‐polymerization activity of 5000 units/mg of protein and lacked the 5′→3′ exonuclease activity. Its optimum activity was at 65 °C and it had a half‐life of 11 min at 90 °C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O‐helix Phe→Tyr substitution.

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. A region with the characteristics of iteron-containing replicons, three 19 bp repeats, DnaA boxes, an A+T rich region and GATC sequences, was identified. Of 40 additional R. marinus strains screened for plasmids, six (15%) were found to harbour plasmids with the same size and restriction pattern as pRM21.