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

Coram, Nicolette Joanne (2002-12)

Dissertation (PhD) -- University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: More than sixteen isolates of iron-oxidizing bacteria belonging to the genus Leptospirillum were included in this study, with the finding that they were clearly divisible into two major groups. Group I leptospirilla had mol% G+C ratios within the range 49-52%, three copies of rrn genes and based on 16S rRNA sequence data, clustered together with the Leptospirillum ferrooxidans type strain (DSM2705or LI5). Group II leptospirilla had mol% G+C ratios of 55-58%, two copies of rrn genes and based on 16S rRNA sequence form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present amongst the leptospirilla tested with two DNA-DNA hybridization similarity subgroups being found within group I. The two groups could also be distinguished based on the sizes of their 16S-23SrRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a new species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and carrying out restriction enzyme digests of the products. Several but not all isolates of the group II leptospirilla, but none from group 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 of plasmid DNA, established that two plasmids of approximately 30.0 kb and 27.0 kb were present. These were named p49879.1 and p49879.2 respectively. Attempts to isolate the plasmids separately were not successful. Partial sequencing of the two plasmids was carried out and sequence analysis of p49879.1 and p49879.2 indicated that the plasmids shared regions of homology. Total plasmid DNA was DIG-labelled and used as a probe in Southern hybridization experiments with genomic DNA from all sixteen original leptospirilla isolates as the target DNA. All leptospirilla belonging to Group I gave a positive signal, little or no homology to Group II leptospirilla was obtained. The region of homology present in all L. ferrooxidans strains was localized to an area on plasmid p49879.2 showing high amino acid identity to a transposase/putative transposase of Methanosarcina acetivorans and plasmid CPl from Deinococcus radiodurans Rl respectively. Whether these regions of homology indicate that complete, functional transposons are present in all L. ferrooxidans isolates still remains to be determined. Preliminary sequence analysis of both plasmids resulted in the identification of regions with amino acid sequence identity to the TnpA and TnpR of the Tn2l-like transposon family, and the mobilization regions of IncQ-like plasmids (particularly that of pTFl from At. ferrooxidans). Another potentially interesting ORF was identified in p49879.2 with high amino acid sequence identity to an ArsR-like protein that belongs to a second atypical family of ArsR transcriptional regulators. Whether this protein is functional in the regulation of arsenic resistance genes has not yet been determined, nor have other arsenic resistance genes been identified. Future work includes further sequence analysis of these plasmids to better understand their contribution to the isolates in which they are found.

AFRIKAANSE OPSOMMING: Meer as sestien isolate van die yster-oksiderende bakterieë, wat aan die genus Leptospirillum behoort, is in die studie ingesluit en die resultate het getoon dat dié groep verder in twee hoof groepe verdeel kan word. Groep I het "n mol% G+C van tussen 49% en 52% gehad, sowel as drie kopieë van die ribosomale gene (rrn). Hiermeesaam het die 16SrRNA volgorde data getoon dat hierdie isolate groepeer saam met Leptospirillum ferrooxidans (DSM2705T en LI5). Groep II leptospirilla het "n mol% G+C van tussen 55% en 58% gehad sowel as twee kopieë van die rrn gene en saam met die 16SrRNA volgorde data het hierdie isolate "n aparte groep gevorm. Genoom DNA-DNA hibridisasie eksperimente het gewys dat daar drie subgroepe onder die Leptospirillum wat getoets was is, met twee naverwante groepe wat onder Groep I val. Daar kan ook tussen die twee hoof groepe onderskei word op grond van die grootte van hul 16S- 23SrRNA intergeniese gebiede. Ons stel dus hier voor dat die Groep II leptospirilla as "n nuwe spesie beskou word naamlik, Leptospirillum ferriphilum sp, nov. Die twee spesies kan maklik onderskei word deur die PKR amplifikasie produk van die 16SrRNA te verteer met restriksie ensieme. Vele, maar nie al van die Groep II isolate kan by 45°C groei nie, terwyl geen van die Groep I leptospirilla (L.ferrooxidans) kan nie. Plasmied DNA was geisoleer uit Leptospirillum ferrooxidans ATCC49879. Aanvanklike analise het gedui op die teenwoordigheid van een 60.0 kb plasmied. Verdere restriksie ensiem kartering het wel getoon dat hierdie, in teen deel, twee plasmiede van ongeveer 30.0 kb en 27.0 kb in grootte is: p49879.1 en p49879.2. Pogings om die twee plasmiede apart te isoleer was onsuksesvol. Totale plasmied DNA is gemerk met die Random primed DNA labelling kit (Roche diagnostics) en gebruik as peiler in Southern klad eksperimente met genoom DNA, van al sestien isolate, as teiken. Alle leptospirilla wat aan Groep I behoort het "n positiewe sein gegee terwyl geen sein teen Groep II DNA opgemerk was nie. Die area wat, tussen die plasmiede en Groep I homologie getoon het, is gelokaliseer tot "n area op plasmied p49879.2 wat hoë amino suur identiteit toon aan "n transposase geen van Methanosarcina acetivorans, en "n voorgestelde transposase geen op plasmied CPI van Deinococcus radiodurans Rl. Dit moet nog vasgestel word of hierdie area van homologie dui op die teenwoordigheid van "n volledige, funksionele transposon in alle L. ferrooxidans isolate. Gedeeltelike DNA volgorde bepalings van beide plasmiede het gelei tot die identifikasie van areas met hoë amino suur volgorde identiteit aan die TnpA en TnpR gene van die Tn21-tipe transposon familie, sowel as aan die mobilisasie gene van IncQsoortige plasmiede (veral die van pTFI uit Acidithiobacillus ferrooxidans). "n Oop lees raam van belang, wat op plasmied p49879.2 geidentifiseer was, het hoë amino suur volgorde identiteit aan "n ArsR-tipe geen getoon wat aan "n tweede atiepiese familie van ArsR transkripsionele reguleerders behoort. Op die stadium is dit nog onbekend of hierdie protein funksioneel is in die regulering van arseen weerstandbiedenheidsgene.

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