High sequence diversity of Alteromonas macleodii-related cloned and cellular 16S rDNAs from a Mediterranean seawater mesocosm experiment

Small subunit rDNA clone libraries were generated from amplified DNA of bac terioplankton taken at different time points from a mesocosm containing eut rophied Mediterranean seawater and made eutrophic by the addition of N and P. Analysis of 96 partial sequences indicated that 22% of the clones formed four clusters which showed the highest sequence sirnilarity with the 16S r DNA sequence of Alteromonas macleodii DSM 6062(T). A fifth cluster, compris ing 31% of the clone sequences is moderately related to the A. macleodiiseq uence. Similarity between the almost complete sequences of two representati ves of clone clusters 1, 2 and 3 and A. macleodii ranged between 97.7 and 9 8.1%. Four oligonucleotide probes, representing four clone clusters, were d eveloped on the basis of partial clone sequences. Dot blot hybridization wi th PCR-amplified 16S rDNA from 739 clones revealed that 24% of clones belon g to one of these clusters. Dot blot hybridization between the four probes and PCR-amplified 16S rDNA from 128 strains isolated from the mesocosm iden tified 21% of the isolates possessing the probe target region. While probes GP-1 and GP-4 unambiguously identified 0.8 and 4.0% of the strains, respec tively, probes GP-2 and GP-3 showed cross hybridization with 16% of the str ains. Analysis of the probe target region of the 16S rDNA of one of the iso lates indeed demonstrated the presence of double peaks in the relevant regi on of the sequence which is indicative of microheterogeneity at the rrn ope ron level. Although some of the diversity can be attributed to intra-strain variation, the data indicate that the phylogenetic diversity of A. macleod ii is higher than represented by the type strain of this species. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Sci ence B.V. All rights reserved.