Genotyping of heterotrophic bacteria from the central Baltic sea by use of low-molecular-weight RNA profiles

The Gotland Deep, an anoxic basin, was investigated for its heterotrophic microflora as a station representative of the central Baltic Sea and as an example of a brackish water environment. One hundred twenty-three bacterial strains were isolated along the water column by use of four different cultivation procedures. High-resolution electrophoresis of the low-molecular-weight (LMW) RNA (5S rRNA and tRNA) was used for analysis of the taxonomic position of the strains. The banding pattern of the LMW RNA generated by the electrophoresis allowed a taxonomic grouping at the species level of the 123 strains into 24 different genotypes. This grouping was confirmed by use of long-range gels with a substantially better resolution than that of standard gels; i.e., about 60% more tRNA bands were obtained on the long-range gels, and the distance between the bands was increased by about two-thirds. The majority of the strains (76%) could be identified to the species level by comparison with LMW RNA profiles from reference strains stored in an electronic database. Eighty-seven percent of the strains could be assigned to the families Vibrionaceae, Enterobacteriaceae, and Pseudomonadaceae (rRNA group I). The most abundant species among the isolates were Shewanella putrefaciens (48%) and a new Pseudomonas species (24%). The remaining fraction of 28% of the isolates was split into 22 other genotypes. Thirteen of these genotypes were represented by single isolates. This study demonstrates the utility of LMW RNA profiling for a rapid assessment of genotypic diversity of heterotrophic isolates from natural environments.