Molecular characterization of Sulfobacillus and related organisms

Schutte, Mart-Alet (Martha Aletta) (2004-03)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: Thirteen Sulfobacillus strains from different geographical locations and two Alicyclobacillus strains were included in this study. These organisms proved to be moderately thermophillic (two different sets of optimal temperatures of 45°C and 55°C were found), Gram-positive, endospore forming bacteria. The pH optima of the strains tested was pH 2.5 and the pH range lay between pH 1.5 and pH 5.0. It was established that some strains of Sulfobacillus had the capacity for anaerobic growth when using ferric iron as an electron donor. It was determined that S. thermosuljidooxidans was the species found within South African biooxidation plants. Plasm ids were identified within strain 611 (S. thermosuljidooxidans) isolated from a Billiton commercial plant. The sample of Sulfobacillus strains used in this study could clearly be divided into two groups based on the analysis of their 16S rRNA gene sequences as well as the number of ribosomal (rm) operons present as determined by Southern hybridization. A system for the convenient identification of Sulfobacillus species was developed using several of the techniques employed in this study. Preliminary identifications can be obtained by restriction enzyme digestion of the PCR amplified 16S rRNA gene. Confirmation of this placement can be done by comparison of the 16S - 23S rRNA spacer region amplification band sizes. Once the preliminary identification has been completed it is possible to place the isolate in the correct species by making use of the differences in sugar utilization that the species exhibit. The more laborious method of 16S rRNA sequence comparisons can be undertaken if there is still any uncertainty as to which species an isolate belongs to. Phylogenetic results obtained from the 16S rRNA gene sequence indicates that the genus Sulfobacillus should probably be divided into two individual genera. Further information gathered from the phylogenetic comparisons indicates that strain Riv-14 previously assigned to S. ambivalens is more closely related to S. montseratensis. Data obtained from 16S - 23S rRNA spacer region analysis confirms this result. Future work includes the use of DNA-DNA hybridization studies and mol% G+C ratio's in order verify the presence of two distinct genera as well as placing Riv-14 within the correct species.

AFRIKAANSE OPSOMMING: Dertien isolate van die genus Sulfobacillus afkomstig van geografies verskillende areas en twee isolate van die genus Alicyclobacillus is in die studie ingesluit. Hierdie organismes het gewys dat hulle gematigde termofiele (twee verskillende groepe met optimale temperature van 45°C en 50°C elk was waargeneem), Gram-positiewe, endospoorvorrnende organismes is. Die pH optima van die isolate was pH 2.5 en die reeks van pH waar groei moontlik was het tussen pH l.5 en pH 5.0 gelê. Dit was bewys dat sekere van die Sulfobacillus isolate oor die vermoë beskik het om anaerobies te respireer deur ferri yster (Fe3+) as elektron akseptor te gebuik. Dit was bepaal dat S. thermosulfidooxidans die spesies is wat teenwoordig was in die bio-oksidasie reaktors in Suid Afrika. Plasmiede vanuit die isolaat 611 (s. thermosulfidooxidans) afkomstig vanuit 'n Billiton komersieële reaktor, is geidentifiseer. Die toetsmonster van Sulfobacillus isolate gebruik in hierdie studie het duidelik daarop gewys dat daar twee groepe binne Sulfobacillus is. Hierdie stelling is gebaseer op data afkomstig van die analiese van die 16S rRNA volgorde asook die aantal ribosomale (rm) kopieë teenwoordig soos bepaal deur Southern klad eksperimente. 'n Sisteem vir die maklike identifikasie van Sulfobacillus spesies is ontwerp deur van verskeie tegnieke, soos in hierdie studie toegepas, gebruik te maak. Aanvanklike identifikasie kan verkry word deur gebruik te maak van restriksie ensiem vertering van PKR geamplifiseerde 16S rRNA geen. Hierdie plasing van die isolaat kan bevestig word deur die grootte van die 16S - 23S rRNA intergeniese amplifikasie produkte te vergelyk. Sodra die aanvanklike plasing van die isolaat voltrek is, kan daar van die verskille in die vermoëns van die spesies om sekere suikers the benut, gebruik gemaak word om die isolaat binne die regte spesies te plaas. Die meer werksintensiewe metode van 16S rRNA volgorde vergelyking kan gebruik word indien daar enige onsekerheid is oor by watter spesies die isolaat hoort. Filogenetiese resultate verkry van die vergelyking van die 16S rRNA geen volgorde dui daarop aan dat die genus Sulfobacillus waarskynlik uit meer as een genus bestaan. Die filogenetiese data dui verder daarop dat die isolaat Riv-14 wat as 'n S. ambivalens geklassifiseer is, nader verwant is aan die spesies S. montseratensis. Data verkry vanaf die 16S - 23S intergeniese gebied analiese bevestig hierdie resultaat. Toekomstige werk sluit DNA-DNA hibridisasie en mol% Gte ratio eksperimente in om sodoende die teenwoordigheid van meer as een genus sowel as die plasing van Riv-14 in die korrekte spesies te bevestig.

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