Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The arsenic resistance operon (ars operon) of the Gram-positive, iron-oxidizing, acidophilic,
moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t.
VKM B-1269T), was isolated and characterised. The ars operon was chromosomally located
and consisted of an arsR (codes for a transcriptional regulator) and an arsB (codes for a
membrane located arsenic/antimony efflux pump). The arsRB genes were transcribed in the
same direction. An arsC (codes for an arsenate reductase), usually associated with ars
operons, was absent from this ars operon. PCR and Southern-hybridization experiments
revealed that no arsC, representative of either the Grx/GSH or Trx ArsC families was present
in the genome of Sb. t. VKM B-1269T. An interesting feature of the ars operon was the
presence of a gene encoding a 525 amino acid (60.83 kDa) kumamolisin-As precursor located
upstream of the arsRB operon. The intergenic region between the termination end of the
kumamolisin-As precursor gene and the transcriptional start of the arsR gene was only 77 bp,
suggesting that this ars operon might consist of three genes. RT-PCR analysis showed that
the ars operon of Sb. t. VKM B-1269T, was not co-transcribed with the kumamolisin-As
precursor gene in its native Sulfobacillus host.
The ars operon of Sb. t. VKM B-1269T did not complement an Escherichia coli arsenic
sensitive mutant. mRNA transcript analysis and promoter expression studies confirmed that
processes involved in the production of functional proteins from the ars operon transcript
were likely to be responsible for the inability of the arsRB operon of Sb. t. VKM B-1269T to
confer resistance to arsenic in the heterologous E. coli host.
Eight Sulfobacillus strains isolated from different geographical areas were subjected to
amplified ribosomal DNA restriction enzyme analysis (ARDREA) using the restriction
endonuclease Eco1015 (SnaBI) and revealed that they could be divided into the proposed
Sulfobacillus spp. subgroup I and subgroup II, respectively (Johnson et al., 2005). The
presence, distribution and relatedness of the ars genes among members of genus Sulfobacillus
was determined. Phylogenetic sequence comparisons revealed two clearly defined arsB
clusters within genus Sulfobacillus and showed that the arsB of a specific Sulfobacillus sub
specie is distinctive of that specific Sulfobacillus sub specie. Futhermore, sequence analysis
of the isolated arsB homologue fragments from the respective Sulfobacillus spp. showed that four distinctive profiles could be identified based on differences in the location of restriction
endonuclease recognition sites. / AFRIKAANSE OPSOMMING: Die arseen weerstandbiedendheidsoperon (ars operon) van die Gram-positiewe, ysteroksiderende,
asidofiliese, matige termofiliese bakterium, Sulfobacillus thermosulfidooxidans
VKM B-1269T (Sb. t. VKM B-1269T), was geïsoleer en gekarakteriseer. Die ars operon was
op die chromosoom geleë en het uit ‘n arsR (kodeer vir ‘n transkripsionele reguleerder) en ‘n
arsB (kodeer vir ‘n membraan geleë arseen/timien uitskeidings pomp) bestaan. Die arsRB
gene word in dieselfde rigting getranskribeer. ‘n arsC (kodeer vir ‘n arsenaat reductase), wat
gewoontlik geassosïeer word met ars operons, was afwesig van hierdie ars operon. PKR en
Southern-hibridisasie eksperimente het aangedui dat geen arsC, verteenwoordigend van
beide die Grx/GSH of Trx ArsC families, nie teenwoordig was in die genoom van Sb. t. VKM
B-1269T, nie. ‘n Interressante eienskap van hierdie ars operon was die teenwoordigheid van
‘n geen wat stroom-op van die arsRB operon geleë is en ‘n 525 amino suur (60.83 kDa)
kumamolisin-As voorloper kodeer. Die intergeniese gedeelte tussen die terminerings einde
van die kumamolisin-As voorloper en die transkriptionele begin van die arsR geen was slegs
77 bp, wat voorgestel het dat die ars operon moontlik uit drie gene bestaan. RT-PKR analiese
het bewys dat die ars operon van Sb. t. VKM B-1269T, nie geko-getranskribeer word met die
kumamolisin-As voorloper in sy oorspronklike Sulfobacillus gasheer nie.
Die ars operon van Sb. t. VKM B-1269T, het nie ‘n Escherichia coli arseen sensitiewe mutant
gekomplimenteer nie. mRNA transkrip-analiese en promoter uitdrukkings eksperimente het
bevestig dat prosesse wat betrokke is in die produksie van funksionele proteïene vanaf die ars
operon transkrip, moontlik vir die onvermoë van die arsRB operon van Sb t. VKM B-1269T
verantwoordelik was om weerstandbiedendheid teen arseen in die heteroloë E. coli gasheer te
verleen.
Agt Sulfobacillus stamme wat geïsoleer is vanuit verskillende geografiese areas, was
onderhewig aan geamplifiseerde ribosomale DNA restriksie-ensiem-analiese (ARDREA) deur
gebruik te maak van restriksie endonuklease Eco1015 (SnaBI) en het aangedui dat hulle in die
voorgestelde Sulfobacillus spp. subgroup I en subgroup II ingedeel kan word (Johnson et al.,
2005). Die aanwesigheid, verspreiding en verwantskappe van die ars gene tussen lede van
genus Sulfobacillus was bepaal. Filogenetiese DNA volgorde vergelykings het aangedui dat twee duidelik definïeerbare arsB groepe van mekaar onderskei kan word en dat die arsB van
‘n spesifieke Sulfobacillus sub spesie uniek tot daardie spesifieke Sulfobacillus subspesie is.
Bykomend, DNA volgorde analiese van die geïsoleerde arsB homoloog fragmente van die
Sulfobacillus spp. het gewys dat vier unieke profiele, op grond van verskille in die ligging van
restriksie ensiem herkenning setels, geïdentifiseer kan word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/19886 |
| Date | 03 1900 |
| Creators | Van der Merwe, Jacobus Arnoldus |
| Contributors | Rawlings, Douglas E., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xx, 149 leaves : ill. |
| Rights | Stellenbosch University |
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