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We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coileuX (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 kuX-coded tRNA is compared with the sequences of the four remaining tRNALUN isoacceptors of E. coli and with two tRNALU

Rhodothermus marinus, a gram-negative heterotrophic marine thermophile, has been the subject of several recent studies. Isolation, sequencing, and analyses of a 16S rRNA gene have shown that R. marinus diverges sharply from major bacterial phyla and is most closely allied to the Flxibacter-Cytophaga-Bacteroides group.Further analyses revealed that the R. marinus chromosome contains a single rRNA operon with a 16S-23S intergenic region coding for tRNAIle and tRNA"'".Thermophilic bacteria have been the subject of rising interest because of their biological adaptations and potential in biotechnology. A number of novel bacteria have been isolated at submarine hot springs and vents. Among these are eubacteria of the genus Rhodothermus which have been isolated in Iceland and the Azores (3, 15). Rhodothermus marinus is an aerobic heterotroph with optimum growth at 65 to 750C (11) and may therefore be well suited for genetic engineering of thermostable enzymes. Plasmid vectors are being developed for this purpose (5), and genes coding for DNA ligase (23a), P-glucanase, and cellulase have been isolated (18a).The genus Rhodothermus is not affiliated with the wellknown thermophilic genus Thermus (19), and to clarify the phylogenetic status of the genus Rhodothermus, we isolated and sequenced a 16S rRNA gene and the 16S-23S intergenic spacer region coding for two tRNAs. Comparisons with other available 16S rRNA sequences utilizing several methods for reconstruction of phylogenies place the genus Rhodothermus close to the root of the Flexibacter-Cytophaga-Bacteroides (F-C-B) group with affinities to green sulfur bacteria, fibrobacteria, and spirochetes. MATERIALS AND METHODS DNA was extracted from R marinus R-10 (DSM 4252, ATCC 43812) and used to construct a genomic library in phage EMBL4 (20). This library was screened with a 3_Plabeled probe made by reverse transcription of total R marinus RNA primed with three universal 16S rRNA-specific oligonucleotides (13). DNA fragments generated from recombinant phage by restriction enzymes EcoRI and XhoI were cloned in phages M13mpl8 and M13mpl9 and screened with a 16S rRNAspecific probe as described above. Single-stranded M13 DNA was sequenced by the dideoxy method (21) by using modified T7 DNA polymerase (Sequenase; United States Biochemical) and a standard sequencing primer, 16S rRNA universal primers, or custom-made primers. DNA fragments generated by amplification with AmpliTaq (Perkin-Elmer) and labeled with digoxigenin (Boehringer Mannheim) were used as hybridization probes. 16S-23S intergenic sequences were amplified from * Corresponding

An assay based on reverse transcription and nested PCR amplification of hypervariable regions within the 16S rRNA sequence was used to specifically detect Renibacterium salmoninarum, the slowly growing causative agent of bacterial kidney disease in salmonid fish. This assay detected 1 to 10 bacteria per sample and took 1 to 2 days to perform. The assay was used to detect R. salmoninarum in ovarian fluid obtained from naturally infected fish. The assay was unreliable when it was used to examine kidney tissue.

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.