Downloaded from orbit.dtu.dk on: Oct 01, 2021 Defining the Pseudomonas Genus: Where Do We Draw the Line with Azotobacter? Özen, Asli Ismihan; Ussery, David Published in: Microbial Ecology in Health & Disease Link to article, DOI: 10.1007/s00248-011-9914-8 Publication date: 2012 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Özen, A. I., & Ussery, D. (2012). Defining the Pseudomonas Genus: Where Do We Draw the Line with Azotobacter? Microbial Ecology in Health & Disease, 63(2), 239-248. https://doi.org/10.1007/s00248-011-9914-8 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. 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This article is published with open access at Springerlink.com Abstract The genus Pseudomonas has gone through many nas syringae has been linked to the environmental cycle of taxonomic revisions over the past 100 years, going from a water as an ice nucleus in the clouds and is found in rain, very large and diverse group of bacteria to a smaller, more snow, lakes, and plants [31]. Because of its abundance in refined and ordered list having specific properties. The the environment, the Pseudomonas genus was first charac- relationship of the Pseudomonas genus to Azotobacter terized long ago, and over the past hundred years, it has vinelandii is examined using three genomic sequence-based gone through many taxonomic revisions. The number of methods. First, using 16S rRNA trees, it is shown that A. organisms placed in the Pseudomonas group grew steadily vinelandii groups within the Pseudomonas close to Pseu- over a period of 60 years. However, through refinement of domonas aeruginosa. Genomes from other related organ- defining criteria, many bacteria were moved to other genera isms (Acinetobacter, Psychrobacter, and Cellvibrio) are over the next 50 [24, 36, 42, 47]. outside the Pseudomonas cluster. Second, pan genome EarlystudiesbasedonrRNA–DNA hybridization family trees based on conserved gene families also show A. postulated five RNA subdivisions in the genus, where vinelandii to be more closely related to Pseudomonas than rRNA group I, including the type species Pseudomonas other related organisms. Third, exhaustive BLAST compar- aeruginosa, was named after the genus as Pseudomonas isons demonstrate that the fraction of shared genes between [34]. Studies on the determination and comparison of 16S A. vinelandii and Pseudomonas genomes is similar to that rRNA sequences of Pseudomonas species resulted in the of Pseudomonas species with each other. The results of clustering of Pseudomonas into two groups: P. aeruginosa these different methods point to a high similarity between and Pseudomonas fluorescens [32]. Later on, the extensive A. vinelandii and the Pseudomonas genus, suggesting that study of Anzai and collaborators on more than 100 Azotobacter might actually be a Pseudomonas. Pseudomonas species based on 16S rRNA sequence comparison suggests seven clusters from the group of species of Pseudomonas sensu stricto, which also agreed in Introduction some parts with Palleroni’s report in 1973 [3]. Although it is still a widely accepted method, debates on the poor Pseudomonas bacteria are naturally widespread in the resolution of the phylogeny analysis with rrs gene environment. For example, the plant pathogen, Pseudomo- sequences lead to the idea of using other marker genes to characterize and classify Pseudomonas,suchasgryB, Electronic supplementary material The online version of this article rpoD, oprI, oprF,andrpoB sequences [2, 8, 13, 57]. In (doi:10.1007/s00248-011-9914-8) contains supplementary material, another study, ten housekeeping genes were used to assess which is available to authorized users. the phylogeny of 2,4-diacetylphloroglucinol-producing : A. I. Özen D. W. Ussery (*) fluorescent Pseudomonas spp. [16]. Other phenotypic Center for Biological Sequence Analysis, methods, such as siderotyping, were also suggested for Department of Systems Biology, the classification of plant-associated Pseudomonas [30]. The Technical University of Denmark, Lyngby, Denmark Pseudomonas sensu stricto (rRNA similarity group I) e-mail: [email protected] could be further divided into subgroups due to its 240 A. I. Özen, D. W. Ussery considerable heterogeneity based on pathogenicity or nitrogen and that their genes related to this machinery pigment production [35]. closely resemble that of A. vinelandii [39, 58, 59]. Another The current status of the Pseudomonas genus today similarity is the alginate production in A. vinelandii, which shows 202 species assigned to Pseudomonas on the is also a by-product in pathogenic P. aeruginosa infections Approved Lists of Bacterial Names, where the classification in the lungs of cystic fibrosis patients [20]. However, other method depends on a combination of 16S rRNA, the phenotypic characteristics of Pseudomonas have been analysis of the cellular fatty acids, and differentiating shown to be different from Azotobacter species, such as classical physiological and biochemical tests [52]. The cell morphology and motility [35]. This suggests that the genus consists of a group of medically and biotechnolog- diversity in some phenotypic characteristics might be the ically important bacteria that are inhabitants of a wide outcome of their adaptive properties since the two genera range of niches including soil and water environments, share the same set of core housekeeping genes or other in addition to plant and animal associations. Hence, they conserved genes [59]. In this context, analysis of 16S are well known for having enormous metabolic versa- rRNA gene sequences by Rediers and collaborators tility [17, 18, 47]. They are non-sporulating, aerobic showed that A. vinelandii was in the P. aeruginosa clade, Gram-negative rods that are found in biofilms or in sharing 96% identity with P. aeruginosa PAO1 strain. planktonic forms. Most of the pathogenic members are Owing to the low resolution in 16S rRNA sequences for related to plants, whereas several strains are pathogenic to these genomes, they conducted a phylogenetic analysis of animals [35]. 25 protein-coding genes, some of which were housekeep- Azotobacter vinelandii, in this context, is interesting ing genes. Phylogenetic trees generated with their dataset because of its common metabolic characteristics with again revealed that A. vinelandii homologues are clustered Pseudomonas. A nitrogen-fixing member of Gammapro- within or close to the Pseudomonas group. The consensus teobacteria, A. vinelandii is found mostly in soil environ- tree out of these 25 topologies showed A. vinelandii ments where its nitrogen and energy metabolism is phylogeny being closest to P. aeruginosa PAO1, conclud- significant to agriculture. Many years ago, this organism ing A. vinelandii to belong to the Pseudomonas genus was often used in biochemistry experiments for isolating [39]. Young and Park [59] use a broader approach to this enzymes during the kinetics studies which resulted in idea, taking into account all the morphological differences, surprising yields and qualities [29]. It is a free-living concluding that Azotobacter species can be transferred to obligate aerobe known for having the highest respiratory Pseudomonas alongwithachangeinthecriteriausedfor characteristics, but it can still fix atmospheric nitrogen classification. using a respiratory protection mechanism [23]. It also has In this article, we analyze the evolutionary relationships distinct properties, such as dramatic increase in chromo- of the Pseudomonas genus to A. vinelandii, discussing some numbers when reached at a stationary phase, whether or not this species is actually a Pseudomonas, formation of cysts under carbon depletion that helps the using comparative genomic methods such as phylogeny bacteria to resist dehydration [41], where alginate is a trees, pan–core genome analysis, and protein BLAST across structural component, and accumulation of poly-beta- the whole genomes. Genomes from related genera— hydroxybutyrate at the end of the exponential growth as a Acinetobacter, Psychrobacter, and Cellvibrio—were also carbon and energy source storage [48]. Although the used for the analysis to provide a better resolution. All Azotobacter genus has been studied over 100 years in genomes are therefore members of Pseudomonadales order, various experiments, currently, there is only one complete where Pseudomonas, Azotobacter, and Cellvibrio belong to genome sequence available on NCBI GenBank database— the Pseudomonadaceae family and Acinetobacter and A. vinelandii DJ [43]. There are