SUMMARY Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri.

Phylogeny and polyphasic taxonomy ofThe genus Caulobacter is composed of prosthecate bacteria often specialized for oligotrophic environments. The taxonomy of Caulobacter has relied primarily upon morphological criteria: a strain that visually appeared to be a member of the Caulobacter has generally been called one without challenge. A polyphasic approach, comprising 165 rDNA sequencing, profiling restriction fragments of 165-235 rDNA interspacer regions, lipid analysis, immunological profiling and salt tolerance characterizations, was used to clarify the taxonomy of 76 strains of the genera Caulobacter, Brevundimonas, Hyphomonas and Mycoplana. The described species of the genus Caulobacter formed a paraphyletic group with Caulobacter henricii, Caulobacter fusiformis, Caulobacter vibrioides and Mycoplana segnis (Caulobacter segnis com b. nov.) belonging to Caulobacter sensu stricto. Caulobacter bacteroides (Brevundimonas bacteroides comb. nov.), C. henricii subsp. aurantiacus (Brevundimonas aurantiaca comb. nov.), Caulobacter intermedius (Brevundimonas intermedia comb. nov.), Caulobacter subvibrioides (Brevundimonas subvibrioides com b. nov.), C. subvibrioides subsp. albus (Brevundimonas alba comb. nov.), Caulobacter variabilis (Brevundimonas variabilis comb. nov.) and Mycoplana bullata belong to the genus Brevundimonas. The halophilic species Caulobacter maris and Caulobacter halobacteroides are different from these two genera and form the genus Maricaulis gen. nov. with Maricaulis maris as the type species. Caulobacter leidyia was observed to cluster with species of the genus Sphingomonas. Caulobacter crescentus is synonymous with C. vibrioides and C. halobacteroides is synonymous with Maricaulis maris as determined by these analyses and DNA-DNA hybridization. Biomarkers discerning these different genera were determined. The necessary recombinations have been proposed and a description of Maricaulis is presented.

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, were subjected to a polyphasic characterization to determine their taxonomic position. High 16S rDNA sequences similarities (99?3-100?0 %) demonstrated that they were closely related to each other. Phylogenetic evaluation of their 16S rDNA sequences revealed that they are members of the genus Sphingomonas sensu stricto, encompassing a separate branch within this genus. They shared 94?4-96?6 % 16S rDNA sequence similarity with species of this genus. All Sphingomonas-specific signature nucleotides were also detected. The presence of the major ubiquinone Q-10, sym-homospermidine as the predominant polyamine, Sphingomonadaceae-specific sphingoglycolipid in the polar lipid patterns and a fatty acid profile containing C 14 : 0 2-OH and lacking 3-OH fatty acids were in agreement with identification of these strains as members of the genus Sphingomonas sensu stricto.

The microbial content of printing paper machines, running at a temperature of 45-50 degrees C and at pH 4.5-5, was studied. Bacteria were prevalent colonizers of the machine wet end and the raw materials. A total of 390 strains of aerobic bacteria were isolated and 86% of these were identified to genus and species by biochemical, chemotaxonomic and phylogenetic methods. The most common bacteria found at the machine wet end were Bacillus coagulans and other Bacillus species, Burkholderia cepacia, Ralstonia pickettii, and in pink slimes, accumulating in the wire area and press section, species of Deinococcus, aureobacterium and Brevibacterium. Paper-making chemicals also contained species of Aureobacterium, B. cereus, B. licheniformis, B. sphaericus, Bordetella, Hydrogenophaga, Klebsiella pneumoniae, Pantoea agglomerans, Pseudomonas stutzeri, Staphylococcus and sometimes other enteric bacteria, but these did not colonize the process water. Yeasts and moulds were not present in significant numbers. A total of 131 strains were tested for their potential to degrade paper-making raw materials; 91 strains were found to have degradative activity, mainly species of Burkholderia and Ralstonia, Sphingomonas and Bacillus, and enterobacteria produced enzymes which degraded paper-making chemicals. Stainless steel adhering strains occurred in slimes and wire water and were identified as Burkholderia cepacia, B. coagulans and Deinococcus geothermalis. Coloured slimes were formed on the machine by species of Deinococcus, Acinetobacter and Methylobacterium (pink), Aureobacterium, Pantoea and Ralstonia (yellowish) and Microbulbifer-related strains (brown). The impact of the strains and species found in the printing paper machine community on the technical quality of paper, machine operation, and as a potential biohazard (Hazard Group 2 bacteria), is discussed.

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A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed. Type strains of the two most closely related species (P. balearica, former genomovar 6, and P. mendocina), together with P. aeruginosa, as the type species of the genus, have been included in the study. The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis. Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1, gyrB, rpoD, nosZ, catA, and nahH) confirmed the monophyletic origin of the genomovars within the Pseudomonas branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species.The genus Pseudomonas is one of the most diverse and ecologically significant groups of bacteria on the planet (28), playing an especially important role in the carbon and nitrogen cycles. Pseudomonas stutzeri is a remarkable member of this genus, with exceptional physiological capacities, being able to metabolize a wide range of organic substrates. Members of the species mineralize organic contaminants aerobically and anaerobically as denitrifiers. P. stutzeri strains are not only able to denitrify-some strains are also able to fix dinitrogen. The species is well defined phenotypically by means of few biochemical tests that discriminate P. stutzeri from other species of Pseudomonas, but additional biochemical properties are very diverse (20). P. stutzeri is ecologically relevant, occupying many niches and being commonly isolated from environmental and clinical samples (1,27).Diversity within the species is not limited to physiological traits but is also reflected at the genetic level. At least nine genomovars are distinguishable within the species (6). Two members of the same genomovar share more than 70% DNA-DNA similarity, the accepted species threshold, and these values are lower and usually less than 50% when members of different genomovars are compared (22). Genomovars are also clearly separated in most cases through phylogenetic analysis of the rrn operon (8). To date, no consistent phenotypic traits have been defined in each genomovar that could justify the splitting of P. stutzeri into several species. The extremely high genetic diversity of the species, the highest so far described in any species, was demonstrated through multilocus enzyme electrophoresis (MLEE) studies (21, 27). It has also been suggested that P. stutzeri populations have a strong clonal structure (21).Multilocus sequence typing has been proposed as a good method for population genetic analysis and to discriminate clones within a species (4). This method employs the same principles as MLEE, in that neutral genetic variation from multiple chromosomal locations is detected. This variation is identified through nucleot...

Bacteria identified and classified as Pseudomonas stutzeri, on the basis of traditional criteria, are recognized to be markedly heterogeneous, such that a systematic phenotypic characterization has not been correlated with genotypic groupings (i.e. genomovars) based upon DNA-DNA similarities. The internally transcribed 16S-23S rDNA spacer (ITS1) regions of P. stutzeri were analysed with respect to the ability of these nucleic acid regions to differentiate and identify the genomic groups (i.e. genomovars) of P. stutzeri. The ITS1s of 34 strains of P. stutzeri were amplified by PCR and the PCR product was subjected to RFLP analysis, which allowed the differentiation and identification of the strains to their respective genomovars. Sequence determination and analysis of ITS1s supported further the results obtained by RFLP, i.e. nucleotide signatures were identified in strains belonging to different genomovars. The ITS1s of all strains of P. stutzeri contained the tandem tRNA Ile /tRNA Ala genes and did not exhibit distinct sequence heterogeneity between different operons of a strain. Phylogenetically informative variable sites were located, exclusively, in non-coding regions. The results of the RFLP and sequence analysis of ITS1s supported and correlated with the phylogenetic relationships estimated from 16S rRNA gene sequence comparisons and DNA-DNA hybridizations, offering an alternative tool for genomovar and species differentiation.