Magdalena Mulet

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.

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.

Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp. nov.

An entomopathogenic, Gram-negative bacterium isolated from a female specimen of the fruit fly Drosophila melanogaster was taxonomically characterised. Strain L48(T) was strictly aerobic, non-fermentative, oxidase and catalase positive, rod-shaped, and motile due to a polar inserted flagellum. Phylogenetic analysis of the 16S rRNA gene and three other housekeeping genes placed strain L48 (T) in the Pseudomonas putida phylogenetic group. DNA-DNA hybridisation studies together with phenotypic metabolic tests and MALDI-TOF MS analysis justified the proposal of strain L48(T) as a representative of a novel species, for which the name Pseudomonas entomophila sp. nov. is proposed. The type strain is deposited in culture collections under accession numbers CCUG 61470(T) and CECT 7985(T).

Phylogenetic analysis and siderotyping as useful tools in the taxonomy of Pseudomonas stutzeri: description of a novel genomovar.

An examination of the results of phylogenetic analyses based on the sequences of fragments of the 16S rRNA, gyrB and rpoD genes, and the discrimination of genomovars based on siderophore diversity within the genus Pseudomonas, has added important taxonomic tools in the characterization of Pseudomonas stutzeri. Eighteen reference strains, nine newly identified hydrocarbon-degrading strains and three strains showing relevant physiological characteristics of P. stutzeri, together with the type strains of four related species, were included in the study. A novel genomovar within the species is described. A summary of the methodology used in these studies and the results of our attempts to define a solid internal subdivision of this important species within the genus Pseudomonas are presented and discussed.

Pseudomonas Diversity in Crude-Oil-Contaminated Intertidal Sand Samples Obtained after the Prestige Oil Spill

ABSTRACT The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.

PseudoMLSA: a database for multigenic sequence analysis of Pseudomonas species

BackgroundThe genus Pseudomonas comprises more than 100 species of environmental, clinical, agricultural, and biotechnological interest. Although, the recommended method for discriminating bacterial species is DNA-DNA hybridisation, alternative techniques based on multigenic sequence analysis are becoming a common practice in bacterial species discrimination studies. Since there is not a general criterion for determining which genes are more useful for species resolution; the number of strains and genes analysed is increasing continuously. As a result, sequences of different genes are dispersed throughout several databases. This sequence information needs to be collected in a common database, in order to be useful for future identification-based projects.DescriptionThe PseudoMLSA Database is a comprehensive database of multiple gene sequences from strains of Pseudomonas species. The core of the database is composed of selected gene sequences from all Pseudomonas type strains validly assigned to the genus through 2008. The database is aimed to be useful for MultiLocus Sequence Analysis (MLSA) procedures, for the identification and characterisation of any Pseudomonas bacterial isolate. The sequences are available for download via a direct connection to the National Center for Biotechnology Information (NCBI). Additionally, the database includes an online BLAST interface for flexible nucleotide queries and similarity searches with the users datasets, and provides a user-friendly output for easily parsing, navigating, and analysing BLAST results.ConclusionsThe PseudoMLSA database amasses strains and sequence information of validly described Pseudomonas species, and allows free querying of the database via a user-friendly, web-based interface available at http://www.uib.es/microbiologiaBD/Welcome.html. The web-based platform enables easy retrieval at strain or gene sequence information level; including references to published peer-reviewed articles, and direct external links to more specialized strain information databases (StrainInfo) and GeneBank (NCBI). The PseudoMLSA is intended to provide helpful strain-sequence information for a better and more comprehensive discriminative multigenic sequence based analysis of this special group of bacteria, contributing to enhance our understanding of the evolution of Pseudomonas species.

Pseudomonas aestusnigri sp. nov., isolated from crude oil-contaminated intertidal sand samples after the Prestige oil spill

Strains VGXO14(T) and Vi1 were isolated from the Atlantic intertidal shore from Galicia, Spain, after the Prestige oil spill. Both strains were Gram-negative rod-shaped bacteria with one polar inserted flagellum, strictly aerobic, and able to grow at 18-37°C, pH 6-10 and 2-10% NaCl. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus but were distinct from any known Pseudomonas species. A polyphasic taxonomic approach including phylogenetic, chemotaxonomic, phenotypic and genotypic data confirmed that the strains belonged to the Pseudomonas pertucinogena group. In a multilocus sequence analysis, the similarity of VGXO14(T) and Vi1 to the closest type strain of the group, Pseudomonas pachastrellae, was 90.4%, which was lower than the threshold of 97% established to discriminate species in the Pseudomonas genus. The DNA-DNA hybridisation similarity between strains VGXO14(T) and Vi1 was 79.6%, but below 70% with the type strains in the P. pertucinogena group. Therefore, the strains should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas aestusnigri is proposed. The type strain is VGXO14(T) (=CCUG 64165(T)=CECT 8317(T)).

Phylogenetic affiliation of Pseudomonas putida biovar A and B strains

In the genus Pseudomonas, two main lineages and 19 phylogenetic groups or subgroups have been defined based on DNA sequence studies of 16S rRNA, gyrB and rpoD genes. In the present study, 33 strains previously classified as Pseudomonas putida were phylogenetically affiliated with their closest relatives in the genus by multilocus sequence analysis. The results demonstrated that strains assigned to biovar A of the species were located in the P. putida group, though not all belonged to the species P. putida. Biovar B strains were scattered among 6 subgroups of the Pseudomonas fluorescens group and also within the P. putida group. These results were corroborated when the entire genomes of 5 strains assigned to P. putida were analyzed. The phylogenetic results show that isolates of biovars A and B are in distinct phylogenetic groups. Thus, these biotypes are not reliable taxonomic markers.

Whole-Cell MALDI-TOF Mass Spectrometry and Multilocus Sequence Analysis in the Discrimination of Pseudomonas stutzeri Populations: Three Novel Genomovars

Pseudomonas stutzeri is a widely distributed species with very high genetic diversity and metabolic capacities, occupying many diverse ecological niches. A collection of 229 P. stutzeri strains isolated from different habitats and geographical locations has been previously characterised phylogenetically by rpoD gene sequencing analysis and in the present study 172 of them phenotypically by whole-cell MALDI-TOF mass spectrometry. Fifty-five strains were further analysed by multilocus sequencing analysis to determine the phylogenetic population structure. Both methods showed coherence in strain grouping; 226 strains were allocated in the 18 genomovars known presently. The remaining three strains are proposed as references for three novel genomovars in the species. The correlation and usefulness of sequence-based phylogenetic analysis and whole-cell MALDI-TOF mass spectrometry, which are essential for autoecological studies in microbial ecology, is discussed for the differentiation of P. stutzeri populations.