Determination of the Diversity of Rhodopirellula Isolates from European Seas by Multilocus Sequence Analysis

Abstract

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of Rhodopirellula sp., an attached-living planctomycete. The development of an MLSA for Rhodopirellula baltica enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes acsA, guaA, trpE, purH, glpF, fumC, icd, glyA, and mdh indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable Rhodopirellula species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living Rhodopirellula species.The biogeography of microorganisms describes the habitat size of the species and the distribution of microorganisms on Earth. The experimental approaches depend on the focus of the studies. Habitats are often analyzed by environmental microbiologists with genetic-fingerprinting techniques, with up to 200 bands or fragments representing the whole community. Although the taxonomic resolution of these operational taxonomic units (OTUs) is limited, the studies revealed a community biogeography (22). Medical microbiologists analyze the alleles of housekeeping genes of microorganisms to gain insight into the epidemiology of pathogens, the population biogeography (2). This strain-specific, fine-scale taxonomic resolution within a species is well suited to observance of recent dispersal events. At the species level, multilocus sequence typing (MLST) and analysis (MLSA), which were developed for intraspecies and intragenus specific studies, respectively, consist of the sequences of several (at least seven) housekeeping gene fragments concatenated to an approximately 5-kilobase alignment (17). Recent MLSA studies revealed its applicability to marine isolates and the analysis of biogeographic patterns: Alteromonas macleodii isolates could be grouped in an epipelagic and an abyssal clade (6), and strains of Pseudomonas aeruginosa were genetically well separated into groups of coastal and oceanic origin (8). However, for Salinibacter ruber strains, biogeographical distinctness was not resolved in an MLSA study but showed allopatry in a metabolic analysis (31). Several studies used MLSA together with DNA-DNA hybridization (DDH) for the delineation of new species, e.g., for Vibrio and Ensifer spp. (20,36).In the biogeography of microorganisms, the experimental proof of a local genetic evolution was first revealed at sample sites...

Isolation and characterization of Planctomycetes from the sediments of a fish farm wastewater treatment tank

“…collected in Foz, Porto (Lage and Bondoso 2011). R. baltica is a cosmopolitan species of Planctomycetes as confirmed by 16S rRNA gene sequence analysis, DNA-DNA hybridization, and MLSA analysis (Schlesner et al 2004;Winkelmann et al 2010). It was found for the first time as a free-living organism in the water column in the Baltic Sea (Schlesner 1994) and, posteriorly, in the tissue of the Mediterranean sponge Intraspecies variability was accessed with the ERIC-PCR fingerprinting (Fig.…”

Geographic distribution at subspecies resolution level: closely related Rhodopirellula species in European coastal sediments

“…A systematic isolation resulted in a strain collection of the genus Rhodopirellula from European coastal seas and sediments, with many strains affiliating according to 16S rRNA gene sequence identity of 99.5-100% to R. baltica SH1 T (Winkelmann and Harder, 2009). However, a multilocus sequence analysis and DNA-DNA hybridization experiments revealed the presence of several species (Winkelmann et al, 2010). This observation was confirmed by draft genome sequences: the average nucleotide identity of shared genes between the strains revealed three species; R. baltica (strains SH1 T , SH28, WH47, SWK14 ), 'R.…”

“…europaea' and 'R. islandica' were found only in the samples from the regions where Winkelmann et al (2010) had isolated the strains and which had been suggested as a preferable habitat. However, as we found these species in almost all samples it is likely that upon favorable change in the environment they could become abundant in other regions.…”

“…Primer design and amplification of acsA gene Amplicon sequences (Winkelmann et al, 2010) and genomes of nine Rhodopirellula strains and six other planctomycetes (Blastopirellula marina, Gemmata obscuriglobus, Planctomyces maris, Planctomyces limnophilus, Planctomyces staleyi and Candidatus Kuenenia stuttgartiensis) were used to design degenerated nested PCR primers. Partial gene amplicons of acsA, carB, fumC, glyA, guaA and icd were in silico specific for R. baltica, 'R.…”

“…These amplicons were designed to cover the whole genus. In this study with a phylogenetically more closely related target group of organisms, we analyzed the genomes, designed primers for a nested PCR and amplified a part of the acetyl CoA synthetase gene (acsA) to investigate the hypothesis of Winkelmann et al (2010) that Rhodopirellula species have a limited geographic distribution in European coastal areas, with R. baltica limited to parts of the Baltic Sea and the east coast of the North Sea, 'R. europaea' to southern North Sea to the Mediterranean Sea and 'R.…”