Shotgun Proteomics Strategy Employed in Acidithiobacillus ferr…

The bacterium Acidithiobacillus ferrooxidans ATCC 23270 can grow under aerobic conditions using ferrous iron, sulphide, and pyrite substrates.¹ The ability to grow under these conditions is made possible by the formation of biofilm. During this formation, an extracellular polymeric substance (EPS) is produced. EPS is made up of sugars, fatty acids, and lipids that aid in the organization of larger colonies. EPS is increased in pyrite-grown cells compared with those grown with iron. These abilities have been capitalized in low-grade copper sulphide biomining.²

According to Vera et al.,³ the process of biofilm formation in At. ferrooxidans is not well understood: in particular, the early formation, and proteins involved in attachment and colonization. In their publication, shotgun proteomics was employed in the study of biofilm formation. At. ferrooxidans was cultivated in Mackintosh media in contact with pyrate for 24 hours. Planktonic and sessile subpopulations were separated and proteins extracted, with four samples analyzed from each subpopulation.

The proteins were purified for SDS-PAGE. Gel lanes were cut, and an in-gel trypsin digest was performed prior to mass spectrometry (MS) on an LTQ-XL Orbitrap (Thermo Scientific) mass spectrometer.

The SEQUEST algorithm in Bioworks (rev. 3.3.1; Thermo Scientific) aided database searches in the At. ferrooxidans ATCC 23270 database (GenBank ID: NC_011761.1). Tryptic peptides with more than two missed cleavages were not included in the experimental results. For protein quantification, mass spectra points were normalized to account for proteins found in three of four samples, and proteins not present in three or more samples were thrown out.

Results of LC/MS/MS revealed 1319 proteins detected in sessile and planktonic groups, which accounted for 42% of proteins within the predicted proteome. The sessile biofilm samples contained an increase of 62 proteins. These proteins functioned in information storage and posttranslational modifications, cell wall or EPS biosynthesis, membrane transport, signal transduction, and metabolic processes. Other proteins were implicated in sulfur assimilation and glutathione metabolism, cofactor and coenzyme biosynthesis, chaperone functions, and stress responses.

Twenty-five proteins were decreased in biofim samples. Decreased proteins included those related to stress responses, respiratory chains, and fatty acid synthesis pathways. Interestingly, two transcription factors, MerR and IclR, were increased, and one factor decreased AbrB in the biofilm subpopulations. These transcription factors may be useful in future studies involving At. ferrooxidans biofilm formation.

References

1. Kelly, D.P., and Wood, A.P. (2000) ‘Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov.‘, International Journal of Systematic and Evolutionary Microbiolgy, 50 (2), (pp. 511–516)

2. Valdes, J., et al. (2008) ‘Acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications‘, BMC Genomics, 9, (p. 597)

3. Vera, M., et al. (2013) ‘Shotgun proteomics study of early biofilm formation process of Acidithiobacillus ferrooxidans ATCC 23270 on pyrite‘, Proteomics, published online January 14, 2013. doi: /10.1002/pmic.201200386