RESUMO
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.
Assuntos
Bactérias/classificação , Água do Mar/microbiologia , Análise de Sequência de DNA , Bactérias/genética , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana , Técnicas Bacteriológicas , Sequência de Bases , Impressões Digitais de DNA , DNA Bacteriano/química , Frequência do Gene , Genes Bacterianos , Loci Gênicos , Variação Genética , Genótipo , Sedimentos Geológicos , Dados de Sequência Molecular , Mar do Norte , Oceanos e Mares , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Especificidade da Espécie , Microbiologia da ÁguaRESUMO
Rhodopirellula baltica, an attached-living marine bacterium, was so far isolated as aerobic, heterotrophic bacterium forming pink-to-red colonies on ampicillin-containing plates. But many bacteria grow in the presence of ampicillin: marine samples from European Seas contained on average 10,365 colony forming bacteria per ml water sample or sediment. Therefore we developed an improved enrichment protocol to isolate Rhodopirellula strains. To include attached-living bacteria, particles, plankton catches or sediment grains were spread on the plate surface. This was a necessity to reliably obtain new strains. Cycloserine or streptomycin served as additional selective agents. The number of colony forming cells resistant to ampicillin and cycloserine or streptomycin was low, 262 and 107 cfu/ml sample, respectively. Rhodopirellula colonies were identified with a newly developed specific PCR reaction detecting a part of the 16S rRNA gene. The phylogenetic tree of over 60 isolates was established with new primers outside of the 16S rRNA gene and revealed a diversity on the species level in European Seas.