Williams, Timothy David (1995) Aspects of lithoautotrophy in iron-oxidizing thermoacidophilic archaea. PhD thesis, University of Warwick.

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Abstract

The phylogeny and proteins of chemolithoautotrophic growth of thermoacidophilic Sulfolobus-like archaea were investigated.

Sulfolobus-like strain HT is a high growth temperature chemolithotrophic isolate with potential application in mineral sulphide bioleaching. The gene encoding its 16S rRNA was cloned and sequenced. Phylogenetic analysis of this sequence showed strain HT segregating within the genus Sulfolobus. It showed greater similarity to the sequence of Sulfolobus shihatae, a facultative chemolithotroph, than to the sequence of Sulfolobus acidocaldarius, an obligate heterotroph. Flanking regions of the 16S rRNA gene were also sequenced, showing secondary structure similarity to those of S. acidocaldarius, implying a similar excision and processing pathway.

A protein of 330 kDa, consisting of 59 kDa and 19 kDa subunits, was overexpressed during CO2-limited autotrophic growth of Sulfolohus strain LM and had previously been shown to co-purify with ATP and acetyl-CoA dependent CO2 uptake. The 59 kDa subunit was partially purified and its N-terminal amino acid sequence obtained. The gene encoding this polypeptide was cloned and sequenced. An open reading frame likely to encode the 19 kDa subunit was adjacent to this gene, forming a possible operon. Homology searches revealed that the predicted amino acid sequence of the 59 kDa subunit was similar to those of ATP-dependent biotin carboxylase enzymes, predicted active site residues being conserved. Homology searches of the predicted amino acid sequence of the ORF likely to encode the 19 kDa subunit revealed similarity to biotin carboxyl carrier proteins, with a biotin binding motif being conserved.

In Sulfolobus LM, a polypeptide of 27 kDa molecular weight was overexpressed during autotrophic growth on ferrous iron in comparison with autotrophic growth on tetrathionate. This polypeptide was partially purified and its N-terminal amino acid sequence obtained. After the cloning and sequencing of the gene encoding this protein by a co-worker, homology searches were carried out. It showed homology to the alkyl hydroperoxide reductase / thiol specific antioxidant (AbpCrrSA) family of proteins, members of which are thought to play a role in protection against oxidative stress. The predicted amino acid sequence was phylogenetically analysed, segregating within a group of sequences derived from eukaryotes and archaea, which possess one conserved cysteine residue, as opposed to a group consisting of eukaryotes and eubacteria, possessing two conserved cysteine residues.

A membrane bound cytochrome showing a difference spectrum alpha absorbance peak at 572 nm had previously been found to be present only during ferrous iron oxidation in thermoacidophilic archaea. This novel cytochrome was partially purified from the membrane fraction of Sulfolohus strain LM autotrophically grown on ferrous iron. It was shown to retain haem staining activity after SDS treatment, thus allowing its identification as a polypeptide of approximately 66 kDa. A procedure which may allow the N-terminal sequencing of this protein and the initiation of its molecular biological study was identified.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Archaebacteria -- Genetics
Official Date:	1995
Dates:	Date Event 1995 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Norris, David
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain)
Extent:	xv, 201 leaves
Language:	eng

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