Antonio Bennasar

Phylogeny and polyphasic taxonomy of Caulobacter species. Proposal of Maricaulis gen. nov. with Maricaulis maris (Poindexter) comb. nov. as the type species, and emended description of the genera Brevundimonas and Caulobacter.

The 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 16S rDNA sequencing, profiling restriction fragments of 16S-23S 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 comb. 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 comb. 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.

16S rRNA gene sequence analysis relative to genomovars of Pseudomonas stutzeri and proposal of Pseudomonas balearica sp. nov.

We compared the 16S rRNA gene sequences of 14 strains of Pseudomonas stutzeri, including type strain CCUG 11256 and strain ZoBell (= ATCC 14405), which represented the seven P. stutzeri genomovars (DNA-DNA similarity groups) that have been described. Our sequence analysis revealed clusters which were highly correlated with genomovar clusters derived from DNA-DNA hybridization data. In addition, we identified signature nucleotide positions for each genomovar. We found that the 16S rRNA gene sequences of genomovar 6 strains SP1402T (T = type strain) and LS401 were different enough from the sequence of the type strain of P. stutzeri that these organisms should be placed in a new species, Pseudomonas balearica. The type strain of P. balearica is strain SP1402 (= DSM 6083).

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.

Utility of internally transcribed 16S-23S rDNA spacer regions for the definition of Pseudomonas stutzeri genomovars and other Pseudomonas species.

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.

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.

Molecular Methods for the Detection and Identification of Pseudomonas stutzeri in Pure Culture and Environmental Samples

A bstractA polymerase chain reaction and an oligonucleotide probe method were developed for the specific detection and identification of the Gram-negative bacterium Pseudomonas stutzeri. This is an important species in environmental studies and can also be present in clinical samples. The primers for the amplification and the probe were designed from the analysis of 16S rRNA sequences. The PCR/oligonucleotide probe method, in combination with a direct lysis DNA extraction and an effective purification method, is an alternative to viable methods for the detection of P. stutzeri in environmental samples.

Achromobacter marplatensis sp. nov., isolated from a pentachlorophenol-contaminated soil

A polyphasic taxonomic approach was applied to the study of a Gram-negative bacterium (B2(T)) isolated from soil by selective enrichment with pentachlorophenol. 16S rRNA gene sequence analysis of strain B2(T) showed that the strain belongs to the genus Achromobacter within the Betaproteobacteria. The 16S rRNA gene sequence displayed more than 99 % similarity to the sequences of the type strains of all species of Achromobacter, with the highest sequence similarity to those of Achromobacter spanius CCM 7183(T) and A. piechaudii CCM 2986(T) (99.8 %). On the basis of phylogenetic analysis, genomic DNA-DNA relatedness and phenotypic characteristics, including chemotaxonomic (cellular fatty acid profile) analysis, a novel species is proposed, Achromobacter marplatensis sp. nov., with the type strain B2(T) ( = CCM 7608(T)  = CCUG 56371(T)  = CECT 7342(T)).

Environmental microbiota represents a natural reservoir for dissemination of clinically relevant metallo-beta-lactamases.

ABSTRACT A total of 10 metallo-β-lactamase-producing isolates of six different species, including Brevundimonas diminuta (n = 3), Rhizobium radiobacter (n = 2), Pseudomonas monteilii (n = 1), Pseudomonas aeruginosa (n = 2), Ochrobactrum anthropi (n = 1), and Enterobacter ludwigii (n = 1), were detected in the sewage water of a hospital. The presence of blaVIM-13 associated with a Tn1721-class 1 integron structure was detected in all but one of the isolates (E. ludwigii, which produced VIM-2), and in two of them (R. radiobacter), this structure was located on a plasmid, suggesting that environmental bacteria represent a reservoir for the dissemination of clinically relevant metallo-β-lactamase genes.

Draft Genome of Pseudomonas stutzeri Strain ZoBell (CCUG 16156), a Marine Isolate and Model Organism for Denitrification Studies

Pseudomonas stutzeri strain ZoBell, formerly a strain of Pseudomonas perfectomarina (CCUG 16156 = ATCC 14405), is a model organism for denitrification. It was isolated by ZoBell in 1944 from a marine sample, and here we report the first genome draft of a strain assigned to genomovar 2 of the species P. stutzeri.

Complete genome sequence of the naphthalene-degrading bacterium Pseudomonas stutzeri AN10 (CCUG 29243).

Pseudomonas stutzeri AN10 (CCUG 29243) can be considered a model strain for aerobic naphthalene degradation. We report the complete genome sequence of this bacterium. Its 4.71-Mb chromosome provides insights into other biodegradative capabilities of strain AN10 (i.e., benzoate catabolism) and suggests a high number of horizontal gene transfer events.