Establishment of a gene transfer system for Rhodothermus marinus.

Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB- mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7+/-1 and 42+/-4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3+/8-0.7x10(6) cfu/microg DNA at 22.5 kV/cm, 200 Omega and 25 microF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.

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 degrees C and it had a half-life of 2 min at 90 degrees 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 degrees C and it had a half-life of 11 min at 90 degrees C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O-helix Phe-->Tyr substitution.

Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus and Rhodothermus marinus DNA ligases to other thermophilic ligases.

We describe the characterisation of four thermo-stable NAD(+)-dependent DNA ligases, from Thermus thermophilus (Tth), Thermus scotoductus (Ts), Rhodothermus marinus (Rm) and Thermus aquaticus (Taq), by an assay which measures ligation rate and mismatch discrimination. Complete libraries of octa-, nona- and decanucleotides were used as substrates. The assay comprised the polymerisation of oligo-nucleotides initiated from a 17 base 'primer', using M13mp18 ssDNA as template. Polymers of ligation products were analysed by polyacrylamide gel electro-phoresis. Under optimum conditions, the enzymes produced polymers ranging from 8 to 16 additions; there was variation between enzymes and the length of the oligonucleotides had a strong effect. The optimal total oligonucleotide concentration for each library was approximately 4 nmol. We compared the rates of ligation between the four ligases using an octanucleotide library as substrate. By this criterion, the Ts and Rm ligases are far more active compared to the more commonly available thermostable ligases.

Cloning, sequence analysis and overexpression of a rhodothermus marinus gene encoding a thermostable thymidine kinase.

Thymidine kinase type II is an important part of the pyrimidine salvage pathway. The thymidine kinase gene from the thermophilic eubacterium Rhodothermus marinus was cloned, sequenced and overexpressed. The gene is 639 bp and encodes a protein of 213 amino acids with a calculated molecular mass of 23.6 kDa. It shows homology to other thymidine kinase proteins from eukaryotic and prokaryotic organisms. The recombinant protein is inhibited by dNTPs but not by dNDPs. It is a tetramer in its native state. Its optimum temperature of activity is 65 degrees C and it has a half life of 15 min at 90 degrees C. This is the first thymidine kinase to be described from a thermophilic bacterium.

Cloning and sequence analysis of the hemB gene of Rhodothermus marinus.

A Rhodothermus marinus gene, hemB, coding for 5-aminolevulinic acid (ALA) dehydratase (ALAD) has been cloned and sequenced. The reading frame of the hemB gene is 1020 base pairs encoding a protein of 340 amino acids with a calculated molecular mass of 37.4 kDa. The amino acid sequence shows homology with eubacterial and eukaryotic ALA dehydratases. A putative metal-binding site of the protein shows strongest homology with corresponding sites from plant ALA dehydratases that require Mg2+ for activity. It differs with respect to only one amino acid out of 20 from a corresponding site in pea ALAD.

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12.

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.

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.

Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases.

By dideoxynucleotide sequencing of a genomic clone, we have determined the complete nucleotide sequence of the gene encoding NAD(+)-dependent DNA ligase (EC 6.5.1.2) of the thermophilic bacterium Thermus scotoductus. The gene encodes a 674-amino-acid thermostable enzyme highly similar to other bacterial DNA ligases and to parts of the deduced gene product of Escherichia coli ORF f562, 5' to the spoR gene encoding 5' guanosyl kinase.

Temperature sensitivity caused by missense suppressor supH and amber suppressor supP in Escherichia coli.

The temperature-sensitive missense suppressor supH and amber suppressor supP in Escherichia coli are mutations of the serU and leuX genes, respectively. The supH tRNA, tRNA(SerCAA), is expected to recognize UUG codons, which are normally read by tRNA(LeuCAA) and tRNA(LeuUAA), coded for by the leuX gene and the leuZ gene, respectively. We show that supP and supH are incompatible and that strains carrying both supP and a restrictive rpsL allele are temperature sensitive. It is suggested that the temperature sensitivity of both supH and supP strains is caused by deficient reading of UUG codons by tRNA(LeuUAA).

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli.

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

supN ochre suppressor gene in Escherichia coli codes for tRNALys.

We describe the cloning and nucleotide sequence of a new tRNALys gene, lysV, in Escherichia coli. An ochre suppressor allele of this gene, supN, codes for a tRNALys with anticodon UUA, presumably derived by a single base change from a wild-type UUU anticodon. The sequence of the supN tRNALys is identical to the sequence of ochre suppressor tRNAs encoded by mutant alleles at the lysT locus. This locus, which contains the two previously known tRNALys genes of E. coli, is located far from the lysV locus on the chromosome.

Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene.

We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coli leuX (supP) gene. The suppressor allele codes for a tRNA with anticodon CUA, presumably derived by a single base change from a CAA anticodon. The mature coding sequence of the leuX gene is preceded by a putative Pribnow box sequence (TATAAT) and followed by a termination signal. The sequence of the leuX-coded tRNA is compared with the sequences of the four remaining tRNALeu isoacceptors of E. coli and with two tRNALeu species from bacteriophage T4 and T5. The conserved nucleotides in these seven tRNAs recognized by E. coli leucyl-tRNA synthetase are located mainly in the aminoacyl stem and in the D-stem/loop region.

Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2.

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.

Mapping of the supP (Su6+) amber suppressor gene in Escherichia coli.

The supP (Su6(+)) amber suppressor gene has been mapped on the clockwise side of the valS locus near min 95 on the Escherichia coli chromosome.

Mutations determining generalized resistance to aminoglycoside antibiotics in Escherichia coli.

Mutations conferring resistance to low levels of kanamycin in Escherichia coli have been mapped at 3 locations: the unc locus (min. 83), a locus we have designated kanA (MIN. 72), close to strA (rpsL), and a locus at min. 86.5 previously discovered by Plate (1976) that we have designated ecfB. The unc and ecfB mutations are associated with defects in energy metabolism, while mutations at kanA may be in the gene coding for ribosomal protein S12 (rpsL). The three types of mutations cause cross resistance to a number of different aminoglycoside antibiotics and the effects of the mutations are cumulative in combination.