Polyphosphates, polyphosphate kinase activity and ppk gene in the extremophilic bacterium Acidithiobacillus ferrooxidans ATCC 19859

摘要

Acidithiobacillus ferrooxidans (former Thiobacillus ferrooxidans) is one of the main acidophilic chemolithotrophic bacteria involved in the bioleaching of metal sulfide ores. The biomining process is subject to varying environmental conditions, which induce changes in the bacterial physiological state and, consequently, in the bioprocess efficiency. Therefore, understanding the molecular mechanisms that At. ferrooxidans uses to respond and adapt to its environment is of a special interest. Recently, inorganic polyphosphate (polyP) has been implicated in the bacterial response to environmental changes and stress adaptation. PolyP is a linear polymer of many tens or hundreds of orthophosphate residues linked by phosphoanhydride bonds through the action of a polyphosphate kinase enzyme (PPK). There is a wide spread of polyP in various microorganisms and, frequently, it forms cytoplasmic granules in bacteria. We have initiated the study of polyP metabolism in At. ferrooxidans. When cells were analyzed by transmission electron microscopy, we observed the presence of abundant polyP granules in the cytoplasm. We searched for an in vitro PPK activity by following the conversion of γ[~(33)P]ATP into polyP. We found a PPK activity associated with the membrane protein fraction. The ppk gene was studied by reverse genetics. Based on the known PPK amino acid sequences and the available incomplete genomic sequence of At. ferrooxidans ATCC 23270 strain, we designed degenerate primers which were employed successfully for the isolation of the ppk gene by means of CODEHOP-PCR.