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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.

On the basis of ribosomal ribonucleic acid homologies, the genus Pseudomonas can be divided into at least five distinct groups, some of which are as distantly related t o each other as they are to Escherichia coli. One of these groups contains members of the genus Xanthomonas. The data presented support and extend the previous grouping based on deoxyribonucleic acid homologies and support the current view that the portion of the genome coding for ribosomal ribonucleic acid is more conserved in the course of evolution than the bulk of the genome.For about nine years our laboratory has been largely involved in taxonomic studies of the genus Pseudomonas. Our first approach was the phenotypic characterization of a large number of strains using mainly nutritional characters. On the basis of phenotypic resemblance, we recognized several "species groups," comprised of what we regarded as recognizable species. Below the specific rank, we further recognized a number of "biotypes," some of which had been previously described as species, and many of which may deserve specific rank (14). Deoxyribonucleic acid (DNA)-DNA hybridization experiments generally supported our conclusions based on phenotypic data but showed that some of the species and biotypes were quite heterogeneous with respect to DNA homology. It was also found that some species groups were sufficiently closely related to each other t o be united in a larger DNA homology complex. For example, the "P. fluorescens complex" was found to contain not only the "fluorescent group" but also the nonfluorescent pseudomonads that had been assigned to the "alcaligenes" and "stutzeri" groups ( 10, 14). Our studies have been reviewed recently (9).To pursue further our studies on bacterial phylogeny and speciation, we have resorted to ribosomal ribonucleic acid (rRNA)-DNA hybridizations among selected members of the various DNA homology groups, and included three species of Xanthomonas in the studies. The results are reported in the present paper. MATERIALS AND METHODSBacterial strains. The bacterial strains used in the presently described experiments are numbered as in our previous publications (1,2,4,7,8,(10)(11)(12)14). Reparation of rRNA. For the preparation of unlabeled rRNA, most strains were grown in a medium containing 0.033 M K-Na phosphate buffer, pH 6.8; (NH,), SO,, 0.1%; asparagine, 0.2%; yeast extract, 0.5%; Hutner mineral base (3), 10 ml per liter. For the growth of P. rnaltophilia, DL-sodium lactate was substituted for asparagine. P. saccharophila was grown in a medium containing 0.033 M K-Na phosphate buffer, pH 6.8; NH,Cl, 0.1%; MgSO, 07 H,O, 0.05%; ferric ammonium citrate, 0.005%; CaCl, , 0.0005%; and sodium succinate, 0.2%. For the growth of P. diminuta, the same basal medium was used as for P. saccharophila except that succinate was replaced by asparagine and the essential growth factors (1) were added. The cultures were grown at 30 C on a rotary shaker. The cells were suspended in 0.02 M tris-(hydroxymethy1)aminomethane (Tris)-hydrochloride buffer, pH 7....

Characteristics of the genus Xknthomonas. Following a suggestion by Burkholder (2), Dowson (4) created the name Xanthomonas for a well-defined subgroup of yellow-pigmented phytopathogenic pseudomonads which had been placed together with many other unrelated bacteria in the genus Phytomonas. Dowson (4) defined Xanthomonas as follows: "non-sporing, rod-shaped bacteria. Gram-negative, motile by one polar flagellum (rarely 2 present), or nonmotile, yellow in the mass on nutrient agar and on sterile potato, on both of which abundant slimy growths are formed. Most species digest starch and produce acid in lactose; none produces acid in salicin. " Two corrections were made later by Dowson: deletion of nonmotility and a change from nutrient agar to nutrient glucose agar (5).In subsequent definitions of the genus, the following characteristics were added: absence of poly-P-hydroxybutyrate inclusions, production of highly characteristic brominated aryl-polyene yellow pigments (xanthomonadins), infrequent production of a diffusible, brown color in beef extract agar, digestion of proteins, production of hydrogen sulfide, inability to use asparagine as the sole source of carbon and nitrogen, absence of denitrification and of nitrate reduction, negative or weak oxidase reaction, inhibition of growth by 0.1% (and frequently by 0.02%) triphenyltetrazolium chloride, requirement for growth factors (usually methionine, glutamic acid, nicotinic acid, or a combination of these), optimal growth temperature of 25 to 27"C, DNA base composition (63 to 71 mol% G+C), and plant pathogenicity. Thus far, presence of pili has not been reported for any of the species (1). The slimes produced by various * Corresponding author. species and pathovars were identified as anionic exopolysaccharides, to which the common name "xanthan gums" has been applied. These compounds have very interesting properties and may play a role in the interactions of the pathogens with their respective plant hosts (17). As pointed out by Dye (6), these organisms "form a well-defined remarkably uniform group from which other yellow-pigmented, Gramnegative, monotrichous, aerobic, non-sporing organisms can be distinguished in the laboratory without difficulty."In Bergey's Manual of Systematic Bacteriology (l), five well-characterized species (X. campestris, X. fragariae, X. albilineans, X. axonopodis, and X. ampelina) are described; one of these, X. campestris, is subdivided into many pathovars that are named according to the host plant of origin. The five species accepted by Bradbury (I) were those already included in the 8th edition of Beeey ' s Manual of Determinative Bacteriology (8), but a sixth species of doubtful affiliation (X. populi) was added. For the list of pathovars, Bradbury accepted the one given by Dye et al. (7) and added only two.A review of the genus Xanthomonas was included in the first edition of the treatise The Prokalyotes: a Handbook on Habitats, Isolation and Identification of Bacteria (26). Unfortunately, aside from a summary of the general prope...

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A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and welldefined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.

S U M M A R YStrains of Pseudomonas stutzeri and related denitrifying bacteria were compared in their phenotypic properties and mean deoxyribonucleic acid (DNA) base composition. On the basis of this comparison and of in vitro DNA hybridization experiments, it was concluded that, using practical diagnostic tests, no more than two nomenspecies can be recognized within the group. One, P. stutzeri, was extremely variable in phenotypic characteristics and in DNA base composition; we included in it the strains previously assigned to P. stanieri. The other was a new species, P. rnendocina Palleroni, which was more homogeneous in phenotypic characters, and in DNA base composition and homology. The comparative properties of known denitrifying pseudomonads are tabulated. I N T R O D U C T I O NPseudomonas stutzeri is a non-fluorescent, denitrifying pseudomonad, widely distributed in soil and water. It was discovered and described as Bacillus denitrijkans I1 by Burri & Stutzer in 1895, and renamed Bacterium stutzeri by Lehmann & Neumann in I 896. Thereafter, the taxonomic and nomenclatural status of this well-characterized species became confused, and was cleared up by van Niel & Allen only in 1952, when they retraced the history of the species, and redescribed it. P. stutzeri can be distinguished from other Pseudomonas spp. by many properties, of which the salient ones are: vigorous denitrifying ability; use of starch and maltose as carbon sources; and a characteristic and unusual colony structure ; colonies of freshly isolated strains are wrinkled, tough and coherent, and have a light-brown colour which reflects the unusually high cytochrome c content of the cells.

Denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. The strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. The isolates obtained on 2-fluorobenzoate could use 2-fluorobenzoate under both aerobic and denitrifying conditions, but did not degrade other halobenzoates. In contrast, the 4-fluorobenzoate isolates degraded 4-fluorobenzoate under denitrifying conditions only, but utilized 2-fluorobenzoate under both aerobic and denitrifying conditions. The strains isolated on either 3-chlorobenzoate or 3-bromobenzoate could use 3-chlorobenzoate, 3-bromobenzoate, and 2-and 4-fluorobenzoates under denitrifying conditions. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the ␣-, ␤-, or ␥-branch of the Proteobacteria, namely, Acidovorax, Azoarcus, Bradyrhizobium, Ochrobactrum, Paracoccus, Pseudomonas, Mesorhizobium, Ensifer, and Thauera. These results indicate that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in the Proteobacteria and that these bacteria are widely distributed in soils and sediments.