Dissertation (PhD) -- University of Stellenbosch, 2002. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52944 |
| Date | 12 1900 |
| Creators | Coram, Nicolette Joanne |
| Contributors | Rawlings, D. 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 | 202 p. : ill |
| Rights | Stellenbosch University |
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