Elena García-Valdés

Biology of Pseudomonas stutzeri

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.

Genetic characterization and evolutionary implications of a chromosomally encoded naphthalene-degradation upper pathway from Pseudomonas stutzeri AN10.

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced a total of 11514bp including the entire naphthalene-degradation upper pathway (nah) of P. stutzeri AN10. Nine open reading frames, nahAaAbAcAdBFCED, encoding the enzymes for the degradation of naphthalene to salicylate, were identified. The nah genes of P. stutzeri AN10 have been compared with genes encoding isofunctional proteins from other Pseudomonas naphthalene-degradation upper pathways. The implications of the sequence homologies to the evolution of aromatic catabolic pathways are discussed. Our findings indicate that this entire catabolic module of P. stutzeri AN10 was recruited from other microorganisms and a short period of time has elapsed after its incorporation within the P. stutzeri AN10 genome. Comparisons also suggest the coexistence of two entire nah upper pathways in a host strain, and further recombination between them. These events could accelerate the evolution of modern catabolic pathways.

Taxonomic Note: A Pragmatic Approach to the Nomenclature of Phenotypically Similar Genomic Groups

Occasionally, genomic groups (DNA groups, genomic species) that have been delimited by DNA-DNA pairing may be phenotypically so similar that they cannot be differentiated for the time being. In these situations it seems best to allow a nomenspecies to contain more than one genomic group and to refer to genomic groups of a nomenspecies as genomovars.

Genotypic and Phenotypic Diversity of Pseudomonas stutzeri

Summary A total of 49 strains of Pseudomonas stutzeri including reference strains from culture collections were phenotypically and genotypically studied. They were isolated from clinical sources, marine sediments, waste-water treatment plants and soil. DNA-DNA hybridization using the thermal melting point of the hybrids as parameter established seven genomic groups, four of them containing less than three strains. The G+C content ranged from 60.9–64,9 mol%. Phenotypical investigation using 102 characters demonstrated a considerable heterogeneity within the genomic groups. Because of the lack of useful differential tests there is still no sound basis for division of P. stutzeri at the species level. It is proposed that the term ‘genomovar’ be used to denote genomic groups of a nomenspecies.

Complete nucleotide sequence and evolutionary significance of a chromosomally encoded naphthalene-degradation lower pathway from Pseudomonas stutzeri AN10.

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced the entire naphthalene-degradation lower pathway of P. stutzeri AN10, this being, together with the upper-pathway reported previously (Bosch R. et al., 1999a. Gene 236, 149-157) the first complete DNA sequence for an entire naphthalene-catabolic pathway. Eleven open reading frames were identified. The nahGTHINLOMKJ genes encode enzymes for the metabolism of salicylate to pyruvate and acetyl-CoA, and nahR encodes the NahR regulatory protein. Our findings suggest that catabolic modules were recruited through transposition events and recombination among tnpA-like genes, and subsequent rearrangements and deletions of non-essential DNA fragments allowed the formation of the actual catabolic pathway. Our results also suggest that the genes encoding the xylene/toluene-degradation enzymes of P. putida mt-2 (pWW0) have coexisted with the nah genes of the P. stutzeri AN10 ancestral genome. This could allow the selection, via recombination events among homologous genes, for a combination of genes enabling the metabolism of a given aromatic compound in the ancestral host strain. Such events accelerate the evolution of modern catabolic pathways and provide new genetic material to the environment, ultimately resulting in improved, natural, bioremediation potential.

Phylogenomics and systematics in Pseudomonas

The genus Pseudomonas currently contains 144 species, making it the genus of Gram-negative bacteria that contains the largest number of species. Currently, multilocus sequence analysis (MLSA) is the preferred method for establishing the phylogeny between species and genera. Four partial gene sequences of housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) were obtained from 112 complete or draft genomes of strains related to the genus Pseudomonas that were available in databases. These genes were analyzed together with the corresponding sequences of 133 Pseudomonas type strains of validly published species to assess their correct phylogenetic assignations. We confirmed that 30% of the sequenced genomes of non-type strains were not correctly assigned at the species level in the accepted taxonomy of the genus and that 20% of the strains were not identified at the species level. Most of these strains had been isolated and classified several years ago, and their taxonomic status has not been updated by modern techniques. MLSA was also compared with indices based on the analysis of whole-genome sequences that have been proposed for species delineation, such as tetranucleotide usage patterns (TETRA), average nucleotide identity (ANIm, based on MUMmer and ANIb, based on BLAST) and genome-to-genome distance (GGDC). TETRA was useful for discriminating Pseudomonas from other genera, whereas ANIb and GGDC clearly separated strains of different species. ANIb showed the strongest correlation with MLSA. The correct species classification is a prerequisite for most diversity and evolutionary studies. This work highlights the necessity for complete genomic sequences of type strains to build a phylogenomic taxonomy and that all new genome sequences submitted to databases should be correctly assigned to species to avoid taxonomic inconsistencies.

An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples.

A polymerase chain reaction-based approach was developed for species identification of Pseudomonas strains and for the direct detection of Pseudomonas populations in their natural environment. A highly selective set of primers (PsEG30F and PsEG790R), giving an amplicon of 760 nucleotides in length, was designed based on the internal conserved sequences of 33 selected rpoD gene sequences (the sigma 70 factor subunit of the DNA polymerase) of Pseudomonas type strains, representing the entire intrageneric phylogenetic clusters described in the genus. The utility of the primer set was verified on 96 Pseudomonas type strains and on another 112 recognised Pseudomonas strains. The specificity of the primer set was also tested against strains from species not belonging to the genus Pseudomonas. These primers were also shown to be useful for the direct detection of Pseudomonas species in environmental DNA after a cloning procedure. These results were compared in parallel with other cloning procedures described previously, based on the analysis of other genes (16S rDNA and ITS1) and also by using primers designed for rpoD on sequences from gamma-proteobacteria. All of the cultured Pseudomonas strains tested could be amplified with these novel primers, indicating that this method is also a useful tool for the specific analysis of Pseudomonas populations from environmental samples without the need for cultivation.

Comparative Genetic Diversity of Pseudomonas stutzeri Genomovars, Clonal Structure, and Phylogeny of the Species

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.

Coexistence of Two Distinct Copies of Naphthalene Degradation Genes in Pseudomonas Strains Isolated from the Western Mediterranean Region

ABSTRACT We analyzed the occurrence of the naphthalene degradation upper-pathway (nah) genes in the western Mediterranean region. The amplification, restriction, and sequence analysis of internal fragments for several nah genes (nahAc, nahB, nahC, and nahE) from naphthalene-degrading strains isolated from this geographical area proved the coexistence of two distinct sets of nah genes.

Concordance between whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and multilocus sequence analysis approaches in species discrimination within the genus Pseudomonas.

Multilocus sequence analysis (MLSA) is one of the most accepted methods for the phylogenetic assignation of Pseudomonas strains to their corresponding species. Furthermore, updated databases are essential for correct bacterial identification and the number of Pseudomonas species is increasing continuously. Currently, 127 species are validly described in Euzébys List of Species with Standing in Nomenclature, and 29 novel species have been described since the publication of the last comprehensive MLSA phylogenetic study based on the sequences of the 16S rDNA, gyrB, rpoB and rpoD genes. Therefore, an update of the sequence database is presented, together with the analysis of the phylogeny of the genus Pseudomonas. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight (WC-MALDI-TOF) mass spectrometry (MS) analysis has been applied very recently to the identification of bacteria and is considered to be a fast and reliable method. A total of 133 type strains of the recognized species and subspecies in the genus Pseudomonas, together with other representative strains, were analyzed using this new technique, and the congruence between the WC-MALDI-TOF MS and MLSA techniques was assessed for the discrimination and correct species identification of the strains. The utility of both methods in the identification of environmental and clinical strains is discussed.