No abstract

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5 end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.

Phylogenetic analysis of the genus Pseudomonas was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas (a total of 125 strains). Pseudomonas strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the ' P. aeruginosa complex ', which included P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, P. oleovorans and P. pseudoalcaligenes, and the ' P. stutzeri complex ', which included P. balearica and P. stutzeri. IGC II was further split into three subclusters that were designated the ' P. putida complex ', the ' P. syringae complex ' and the ' P. fluorescens complex '. The ' P. putida complex ' included P. putida and P. fulva. The ' P. syringae complex ' was the cluster of phytopathogens including P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava and the pathovars of P. savastanoi and P. syringae. The ' P. fluorescens complex ' was further divided into two subpopulations, the ' P. fluorescens lineage ' and the ' P. chlororaphis lineage '. The ' P. fluorescens lineage ' contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the ' P. chlororaphis lineage ' included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the ' P. fluorescens complex '.

), which were isolated from sponges and macroalgae, are also reported. For taxonomic considerations at the species level, the resolution of gyrB sequences was superior to that of 16S rDNA sequences, and the grouping based on the gyrB phylogram was consistent with DNA-DNA hybridization results.

Variation in the adhesive protein gene sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus collected in Delaware, Kamaishi (Japan), and Alaska, respectively, was analyzed by the polymerase chain reaction (PCR) using two sets of oligonucleotide primers. The first set, Me 13 and Me 14, was designed to amplify the repetitive region. The length of the amplified fragments was highly variable, even among samples of the same species. Another set, Me 15 and Me 16, was designed to amplify a part of the nonrepetitive region. The length of the amplified fragments was uniform in each species and differed interspecifically; 180, 168, and 126 bp for M. edulis, M. trossulus, and M. galloprovincialis, respectively. The amplified sequence of M. trossulus resembled that of M. edulis. Mussels from other sites were also examined by PCR using Me 15 and Me 16. Wild mussels from Tromsö (Norway) and cultured mussels from Brittany (France) were identified as M. edulis. Cultured mussels from the Mediterranean coast of France and wild mussels from Shimizu (Japan) were identified as M. galloprovincialis. Some wild mussels from Hiura (Japan) were identified as a hybrid between M. galloprovincialis and M. trossulus. Thus, the length of this part (variable region) of the sequence is proposed as a diagnostic marker for these three morphologically similar species and their hybrids.