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.

Genetic diversity and genetic relationships among 42 Pseudomonas stutzeri strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this P. stutzeri sample exhibited the highest mean genetic diversity (H ‫؍‬ 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (I A ) for the P. stutzeri strains analyzed was 1.10. The I A values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of P. stutzeri is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium.Pseudomonas stutzeri was first isolated by Burri and Stutzer (6) as Bacillus denitrificans II and named P. stutzeri by Van Niel and Allen (47). It has an unusual colony shape and consistency when directly isolated, being described as wrinkly, dry, and tenaciously coherent. P. stutzeri, a gram-negative rod-shaped bacterium that is mobile by means of a single polar flagellum, is a nonpigmented denitrifier that liberates nitrogen gas from nitrate, is amylase positive and gelatinase negative, and is able to grow on maltose and starch (4, 43). P. stutzeri has a wide environmental distribution but is found mainly in soil and water. Many strains have been isolated from clinical specimens (20). The members of the species share physiological characteristics that make P. stutzeri of special interest in ecological studies. This species shows high metabolic versatility (35) including the degradation of environmental pollutants (1, 37) and high-molecular-weight polyethylene glycols (30). P. stutzeri serves as a model for the study of the biochemistry and genetics of denitrification and natural transformation processes.Pseudomonas species are grouped on the basis of rRNA-DNA hybridization studies (31). P. stutzeri is a nonfluorescent denitrifying species of the genus Pseudomonas included in the rRNA group I. P. stutzeri forms a homogeneous group within the genus Pseudomonas, with phenotypic traits that permit description to the species level. However, P. stutzeri is a heterogeneous species with respect to many phenotypic characteristics and DNA composition. Several studies have demonstrated that P. stutzeri consists of a complex collection of strains that might be distributed in more than one species (2, 25, 31, 35). DNA-DNA hybridization studies (35,43) have shown the existence of at least eight genomic groups, called genomovars.Confirmation of this syste...

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A microbial consortium (AM) obtained by sequential enrichment in liquid culture with a polycyclic aromatic hydrocarbon (PAH) mixture of three- and four-ringed PAHs as a sole source of carbon and energy was examined using a triple-approach method based on various cultivation strategies, denaturing gradient gel electrophoresis (DGGE), and the screening of 16S and 18S rRNA gene clone libraries. Eleven different sequences by culture-dependent techniques and seven by both DGGE and clone libraries were obtained. The comparison of three variable regions (V3-V5) of the 16S rRNA gene between the sequences obtained yielded 19 different microbial components. Proteobacteria were the dominant group, representing 83% of the total, while the Cytophaga-Flexibacter-Bacteroides group (CFB) was 11% and the Ascomycota fungi 6%. Beta-proteobacteria were predominant in the DGGE and clone library methods, whereas they were a minority in culturable strains. The highest diversity and number of noncoincident sequences were achieved by the cultivation method that showed members of the alpha-, beta-, and gamma-Proteobacteria; CFB bacterial group; and Ascomycota fungi. Only six of the 11 strains isolated showed PAH-degrading capability. The bacterial strain (AMS7) and the fungal strain (AMF1), which were similar to Sphingomonas sp. and Fusarium sp., respectively, achieved the greatest PAH depletion. The results indicate that polyphasic assessment is necessary for a proper understanding of the composition of a microbial consortium.

A . B EN N AS AR , C. GU A SP , M . TE SA R AN D J . LA LU C AT . 1998. Detailed characterization of the genetic variability among strains belonging to Pseudomonas stutzeri was achieved using different rapid molecular typing methods based on polymerase chain reaction (PCR), Southern blot and Western blot. Consensus motifs complementary to fragments of repetitive elements dispersed throughout the genomes of bacteria were used as primers and allowed differentiation at subspecies levels. Further and simple differentiation was also achieved based on the direct amplification of spacer regions between 16S and 23S rRNA, combined with single-strand conformation polymorphism (SSCP) analysis of the generated fragments. These methods are fast, sensitive, reliable for determining relationships, and have demonstrated a great genetic diversity among the strains of Ps. stutzeri studied in agreement with the heterogeneous phenotypic traits of the species.