Molecular characterization of iron-oxidizing Leptospirillum strains from around the world

[摘要] ENGLISH ABSTRACT:More than sixteen isolates of iron-oxidizing bacteria belonging to the genusLeptospirillum were included in this study, with the finding that they were clearlydivisible into two major groups. Group I leptospirilla had mol% G+C ratioswithin the range 49-52%, three copies of rrn genes and based on 16S rRNAsequence data, clustered together with the Leptospirillum ferrooxidans type strain(DSM2705or LI5). Group II leptospirilla had mol% G+C ratios of 55-58%, twocopies of rrn genes and based on 16S rRNA sequence form a separate cluster.Genome DNA-DNA hybridization experiments indicated that three similaritysubgroups were present amongst the leptospirilla tested with two DNA-DNAhybridization similarity subgroups being found within group I. The two groupscould also be distinguished based on the sizes of their 16S-23SrRNA gene spacerregions. We propose that the group II leptospirilla should be recognized as anew species with the name Leptospirillum ferriphilum sp. nov. Members of thetwo species can be rapidly distinguished from each other by amplification oftheir 16S rRNA genes and carrying out restriction enzyme digests of theproducts. Several but not all isolates of the group II leptospirilla, but none fromgroup I (L. ferrooxidans) were capable of growth at 45°C.Plasmid DNA was isolated from strain ATCC49879 (L. ferrooxidans).Restriction endonuclease mapping of what appeared to be about 60 kb ofplasmid DNA, established that two plasmids of approximately 30.0 kb and 27.0kb were present. These were named p49879.1 and p49879.2 respectively.Attempts to isolate the plasmids separately were not successful. Partialsequencing of the two plasmids was carried out and sequence analysis ofp49879.1 and p49879.2 indicated that the plasmids shared regions of homology.Total plasmid DNA was DIG-labelled and used as a probe in Southernhybridization experiments with genomic DNA from all sixteen originalleptospirilla isolates as the target DNA. All leptospirilla belonging to Group Igave a positive signal, little or no homology to Group II leptospirilla wasobtained. The region of homology present in all L. ferrooxidans strains waslocalized to an area on plasmid p49879.2 showing high amino acid identity to a transposase/putative transposase of Methanosarcina acetivorans and plasmidCPl from Deinococcus radiodurans Rl respectively. Whether these regions ofhomology indicate that complete, functional transposons are present in all L.ferrooxidans isolates still remains to be determined. Preliminary sequenceanalysis of both plasmids resulted in the identification of regions with amino acidsequence identity to the TnpA and TnpR of the Tn2l-like transposon family, andthe mobilization regions of IncQ-like plasmids (particularly that of pTFl fromAt. ferrooxidans). Another potentially interesting ORF was identified inp49879.2 with high amino acid sequence identity to an ArsR-like protein thatbelongs to a second atypical family of ArsR transcriptional regulators. Whetherthis protein is functional in the regulation of arsenic resistance genes has not yetbeen determined, nor have other arsenic resistance genes been identified. Futurework includes further sequence analysis of these plasmids to better understandtheir contribution to the isolates in which they are found.

[发布机构] Stellenbosch University