Marine Biotechnology Institute, Kamaishi Laboratories, Kamaishi City, Iwate 026, Japan1
Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK2
Author for correspondence: Satoshi Yamamoto. Tel: +81 43 203 0226. Fax: +81 43 203 0216. e-mail: yamamotost{at}nichirei.co.jp
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ABSTRACT |
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Keywords: gyrB, rpoD, Pseudomonas, phylogeny, PCR
Abbreviations: IGC, intrageneric cluster; NJ, neighbour-joining; UPGMA, unweighted pair group method with arithmetic averages
The GenBank accession numbers for the sequences determined in this work are: gyrB, D37926, D37297, D86005D86019 and AB039381AB039492; rpoD, D86020D86036 and AB039493AB039624.
a Present address: Nichirei Corporation, Research & Development Center, 9 Shinminato, Mihama-Ku, Chiba-Shi, Chiba 261-8545, Japan.
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INTRODUCTION |
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The classificatory criteria for the genus Pseudomonas have been revised along with progress in bacterial taxonomy. Today, DNADNA hybridization is the recommended standard for the delineation of a bacterial species (Wayne et al., 1987 ), but it has the drawback that it is not effective in the estimation of genetic distances between distantly related species. As a complement to DNADNA hybridization, sequence analysis of 16S rRNA or its gene (16S rDNA) is frequently used (Laguerre et al., 1994
; Moore et al., 1996
; Bennasar et al., 1998
). However, the degree of resolution obtained with 16S rRNA sequence analysis is not sufficiently discriminatory to permit resolution of intrageneric relationships because of the extremely slow rate of evolution of 16S rRNA. Due to the gap between the valid genetic ranges of the two methods, a detailed intrageneric structure of the genus Pseudomonas remains to be resolved.
In this article, we discuss the intrageneric structure of the genus Pseudomonas on the basis of the nucleotide sequences of their genes for DNA gyrase B subunit (gyrB) and 70 factor (rpoD). DNA gyrase is the enzyme responsible for introducing negative supercoils into bacterial chromosomes and plays a crucial role in the replication of chromosomes (Watt & Hickson, 1994
). The
70 factor, on the other hand, is one of the sigma factors that confer promoter-specific transcription initiation on RNA polymerase (Lonetto et al., 1992
). Both proteins are ubiquitous in bacteria and essential for their cell growth. We previously reported that these protein-encoding genes evolved much faster than rDNAs and provided higher resolution than the use of 16S rRNA sequences (Yamamoto & Harayama, 1998
). We further demonstrated that the phylogenetic clustering of Acinetobacter strains based on gyrB sequence analysis is almost equivalent to the genomic species delineated by DNADNA hybridization (Yamamoto et al., 1999
). These results suggest that a phylogenetic analysis using the gyrB and rpoD sequences may fill the resolution gap between 16S rRNA sequence analysis and DNADNA hybridization studies.
Molecular phylogeny deduced from a single locus may be unreliable due to the stochastic nature of base substitutions or to rare horizontal gene transfer events. Consequently, we decided to use a combination of the gyrB and rpoD genes to establish the phylogenetic relationships of strains in the genus Pseudomonas. This strategy allowed, first, the comparison of the gyrB-based phylogenetic tree with the rpoD-based one to confirm the consistency of the analyses, and second, the improvement of the reliability of the phylogenetic tree.
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METHODS |
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RESULTS |
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We thus conducted the phylogenetic analysis by using the combined nucleotide sequences of the gyrB and rpoD genes. A NJ tree reconstructed from the combined gyrB and rpoD nucleotide sequences (gyrB-rpoD tree) is shown in Fig. 1. The topology of trees reconstructed from the combined sequences of gyrB and rpoD by the unweighted pair group method with arithmetic averages (UPGMA) (Sokal & Michener, 1958
) and the maximum-parsimony method (Fitch, 1971
) were almost identical to that of the NJ tree. One notable difference between them was the position of P. straminea IAM 1598T. In the NJ and maximum-parsimony trees, P. straminea IAM 1598T was excluded from both of the clusters of the P. aeruginosa lineage and the P. stutzeri lineage, whereas in the UPGMA tree, P. straminea IAM 1598T was included in the cluster of the P. aeruginosa lineage. The phenotypic characteristics of P. straminea, such as its considerably lower maximum growth temperature (no growth at 37 °C) in comparison with that of the P. aeruginosa lineage and the P. stutzeri lineage (can grow at 41 °C), may account for its divergence as in the NJ tree and maximum-parsimony tree. Consequently, we chose the combined gyrB and rpoD NJ tree as the basis of Pseudomonas phylogeny in this study.
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IGC I was further split into at least 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. P. aeruginosa strains were monophyletic within the P. aeruginosa complex. The type strains of P. oleovorans and P. pseudoalcaligenes, IFO 13583T and IFO 14167T, respectively, were genetically almost identical. As we discussed previously, the phylogenetic position of P. straminea in IGC I was uncertain as its bootstrap probability was 46%. P. straminea possibly represents an independent third subcluster.
IGC II
The general characteristics of IGC II are: more than one polar flagellum; a lower range of G+C contents (59·063·6 mol%) than IGC I (60·666·3 mol%); inability to grow at 41 °C and ability of some strains to grow at 4 °C; production of fluorescent pigments by most strains (Palleroni, 1984 ). IGC II is more species-rich than IGC I despite the comparable extent of genetic diversity of the two clusters. Within IGC II, at least three distinct monophyletic groups were recognized. We designated these subclusters the P. putida complex, the P. syringae complex and the P. fluorescens complex.
The P. putida complex included P. putida and P. fulva. All of the P. putida strains characterized as biovar A belonged in this group; however, all of the P. putida biovar B strains were included in the P. fluorescens complex. Strains of P. putida biovar B have been distinguished from biovar A mainly by their ability to grow on L-tryptophan and L-kynurenine. Collectively, the phenotypes of the P. putida biovar B strains resemble more closely those of P. fluorescens strains than those of biovar A. For example, P. putida biovar B strains can grow at 4 °C and can utilize a broader range of sugars than biovar A strains (Stanier et al., 1966 ). These physiological differences support the phylogenetic separation of biovar B strains from the clade of P. putida including the type strain and biovar A strains.
The P. syringae complex was the clade of phytopathogenic Pseudomonas, representing a huge pathogenicity island. The P. syringae complex included P. amygdali, P. caricapapayae, P. cichorii, P. ficuserectae, P. viridiflava, P. savastanoi pathovars (pv. savastanoi, pv. phaseolicola and pv. glycinea) and P. syringae pathovars (pv. antirrhini, pv. coriandricola, pv. coronafaciens, pv. lachrymans, pv. maculicola, pv. morsprunorum, pv. pisi and pv. syringae).
The P. fluorescens complex was the most species-rich subcluster and at least 16 validly described species belonged in this clade. The majority of the constituents of the P. fluorescens complex were fluorescent, with the exception of P. corrugata. Many are saprophytic and/or psychrophilic (growth at 4 °C), and are frequently associated with food spoilage, especially at chilling temperatures (110 °C). Some members are also important as pathogens of plants and fungi. This cluster 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, whilst 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, especially those of biotype B, were positioned either in the P. fluorescens lineage or in the P. chlororaphis lineage. It is clear that a reclassification of the P. fluorescens strains is required. On the other hand, P. agarici, P. asplenii, P. chlororaphis, P. corrugata and P. tolaasii formed tight monophyletic branches and can be considered as solid species.
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DISCUSSION |
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The extent of genetic diversity among the Pseudomonas species was quite variable. The P. stutzeri complex and the P. putida complex consisted of single species, P. stutzeri and P. putida, respectively. In contrast, the P. fluorescens complex, which is genetically less divergent than either the P. stutzeri complex or the P. putida complex, contained no less than 16 species.
Three validly described, but as yet unconfirmed, Pseudomonas species (Kersters et al., 1996 ), P. azotoformans, P. fulva and P. straminea, were newly confirmed as genuine members of the genus Pseudomonas in this study. Conversely, type strains of two valid Pseudomonas species, P. stanieri ATCC 27130T and P. iners IAM 1419T, were excluded from the genus.
Some of the phenotypic traits of the strains in the genus Pseudomonas did not reflect their phylogenetic relationships. The fluorescent strains were polyphyletic and belonged to both IGC I and IGC II. Within IGC II, non-fluorescent pseudomonads formed several independent clusters (e.g. P. corrugata in the P. fluorescens complex and P. amygdali in the P. syringae complex). We presume that the common ancestor of the genus Pseudomonas was originally fluorescent and that non-fluorescent species have appeared by losing their ability to produce a fluorescent pigment.
Phytopathogenic Pseudomonas species were also distributed throughout the genus Pseudomonas, not only in the P. syringae complex. The polyphyletic relationship of phytopathogenic pseudomonads suggests that pathogens in separate clades have independently acquired their phytopathogenicity, presumably by the intergeneric transmission of virulence factors. It is likely that the common ancestor of the P. syringae complex had acquired a set of factors that determine pathogenicity to plants, after the divergence of the P. syringae complex and the P. fluorescens complex. More recently, such genes would probably be horizontally acquired via plasmid transfer and involve virulence and/or avirulence genes such as avrPpiB described in P. syringae pv. pisi race 3 (Cournoyer et al., 1995 ) or the pathogenicity island in P. savastanoi pv. phaseolicola (Jackson et al., 1999
). If so, virulence/avirulence genes would be expected to evolve along with the evolutionary course of the P. fluorescens complex. However, correlation was not necessarily observed between the phylogenetic relationships of phytopathogens and their host range. For example, strains of P. viridiflava (pathogenic on bean) were scattered at various phylogenetic positions in the P. syringae complex, whilst those of P. caricapapayae (pathogenic on pawpaw) formed a tight cluster. Polyphyly of host range may be due to the horizontal transfer of virulence and/or avirulence genes.
Within the P. syringae complex, P. cichorii strains formed an independent monophyletic cluster with two strains of P. syringae pv. syringae. P. cichorii strains are oxidase-positive, as are the majority of pseudomonads, whilst exceptionally, the P. syringae strains were oxidase-negative. This phenotypic difference corroborates the hypothesis that the P. cichorii lineage branched at an early stage after the acquisition of phytopathogenicity by the common ancestor, and subsequently the ancestor of the P. syringae lineage lost the ability to produce oxidase. The detached P. syringae cluster in the P. cichorii lineage might also have lost the ability to produce oxidase by an independent event.
As stated above, reclassification of P. fluorescens biotype strains, the major constituents of the P. fluorescens complex, is required since they were polyphyletic. Thus, a DNADNA hybridization study accompanied by a phenotypic analysis should be carried out to propose new classificatory criteria for the P. fluorescens complex. Experiments with an optimal combination of strains for DNADNA hybridization should be designed, guided by data from the gyrB and rpoD sequence analysis rather than that from 16S rDNA analysis (Yamamoto & Harayama, 1998 ). The close relationship between P. savastanoi pathovars was clearly demonstrated in the gyrB and rpoD sequence analyses presented here. The new species P. savastanoi was created to include the P. syringae pathovars glycinea, phaseolicola and savastanoi, on the basis of their DNA relatedness obtained by DNADNA hybridization experiments (Gardan et al., 1992
).
Because of the high ratios of evolution in the gyrB and/or rpoD nucleotide sequences, it was very easy to design PCR primers or probes having specificity for clusters of species, subspecies or higher classes such as the lineages and complexes designated in this paper (data not shown). On the other hand, the design of such primers or probes based on the variable regions of 16S rRNA presents a problem because sequence similarity among the variable regions does not always guarantee a close phylogenetic relationship (Yamamoto & Harayama, 1998 ). In conclusion, classification, identification and detection systems for pseudomonads based on gyrB and/or rpoD sequences can be very useful in microbial ecology and other fields of bacteriology.
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ACKNOWLEDGEMENTS |
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Received 4 April 2000;
revised 17 July 2000;
accepted 21 July 2000.