Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica

Abstract

Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG^® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Keywords: Antarctica; Multilocus Sequence Analysis; P. fluorescens complex; Pseudomonas.

Fig. 1

Maximum-likelihood phylogenetic tree based on the partial 16S rRNA gene sequences of members of the Pseudomonas genus. The 16S sequence of E. coli K12 was used as an outgroup. The tree shows all bacteria of the genus Pseudomonas isolated in Antarctica to date (bold). Isolates presented in this study are shown in bold and underlined. It can be seen that the majority of the Antarctic bacteria are part of the P. fluorescens lineage, with the exception of the P. guineae isolates, which seem to be part of a separate lineage.

Fig. 2

Maximum-likelihood phylogenetic tree based on the concatenated sequences of the genes 16S-aroE-glnS-gyrB-ileS-rpoD, showing the evolutionary relationship among selected members of the Pseudomonas genus. The bacteria isolated in Antarctica are shown in bold, and isolates presented in this study are shown in bold and underlined. The tree clearly shows the lineages of P. aeruginosa and P. fluorescens. Likewise, the P. fluorescens complex and its subgroups can be clearly distinguished. All Antarctic Pseudomonas can be classified within the P. fluorescens complex. Among them, P. deceptionentis seems to form a separate subgroup, and the isolate Pseudomonas sp. 6A1 appears in a branch of the P. mandelii subgroup. Together with the species P. antarctica and P. extramaustralis, the isolates Pseudomonas sp. IB20, Pseudomonas sp. 12B3 and Pseudomonas sp. KG01 form the new subgroup P. antarctica.