Analysis of an 18kb accessory region of plasmid pTcM1 from Acidithiobacillus caldus MNG

Louw, Lilly-Ann (2009-03)

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009.


Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.

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