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.

FIG. 1.

Phylogenetic tree of different clusters with concatenated partial sequences of the housekeeping genes mdh and glpF, calculated in ARB with RAxML and a 50% base frequency filter. OTU groups are indicated by letters in parentheses.

FIG. 2.

Phylogenetic tree of clusters A, B, and I with concatenated partial sequences of 9 different housekeeping genes, calculated in ARB with RaxML. OTU groups are indicated by letters in parentheses.

FIG. 3.

BOX-PCR analysis of strains affiliated with OTUs A and B. The patterns were obtained by gel electrophoreses and analyzed with Totallab TL120 software (Nonlinear Dynamics Inc., Newcastle upon Tyne, United Kingdom). The numbers in parentheses show the original lane numbers in the gel. Missing lane numbers are due to marker lanes or insufficient resolution of band patterns.

FIG. 4.

Micrographs of R. baltica SH1^T (c), Rhodopirellula sp. strain 6C (a, d, and e), and strain SM1 (b and f). Images a and b show Pt-shadowed cells, c and d show freeze-etch preparations, and e and f show ultrathin sections of embedded cells. Bars, 0.5 μm.