An Exported Rhodanese-Like Protein Is Induced during Growth of Acidithiobacillus ferrooxidans in Metal Sulfides and Different Sulfur Compounds

Abstract

By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of A. ferrooxidans ATCC 23270 to isolate the DNA region containing the p21 gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When p21 was cloned and overexpressed in Escherichia coli, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the Deinococcus radiodurans thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene p21 contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in A. ferrooxidans.

Common themes and variations in the rhodanese superfamily

“…The expression of the P21 rhodaneselike protein from A. ferrooxidans seems to be induced during growth on different oxidizable sulfur compounds to generate thiosulfate, which is used as electron donor by this chemolithotrophic bacterium (40). Yet, disruption of the gene encoding the rhodanese-like enzyme CysA from Saccharopolyspora erythraea results in cysteine auxotrophy (41).…”

“…It should be noted that the pathway for cysteine biosynthesis in S. erythraea differs from that established, for instance, in E. coli, in so far as only the former includes thiosulfate as an intermediate. Thus, it is possible that P21 as well as CysA have the specific function to synthesize thiosulfate (40).…”

Purification and Characterization of Sulfide:Quinone Oxidoreductase from an Acidophilic Iron-Oxidizing Bacterium,Acidithiobacillus ferrooxidans

“…Although the transfer of electrons from ferrous iron to oxygen has not been conclusively established, the electron transfer pathway is thought to involve two cytochromes c (Cyc2 and Cyc1), a rusticyanin, and an aa 3 -type cytochrome c oxidase. [4][5][6] On the other hand, although some enzyme activities thought to be involved in the aerobic oxidation of elemental sulfur by A. ferrooxidans were detected, [7][8][9][10][11][12][13][14][15][16] the oxidation pathway of elemental sulfur has not yet been clearly elucidated.…”

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“…This suggests that the Sec machinery of E. coli might recognize the secretion signals of A. ferrooxidans. The presence of another A. ferrooxidans periplasmic protein, P21, was confirmed and its signal peptide sequence could be recognized and incised during the cloning procedure in E. coli 10 . Most of the A. ferrooxidans periplasmic proteins (62.3%) possessed export signals of secretory proteins according to their sequences 11 .…”

“…www.scienceasia.org A. ferrooxidans, one of the most studied molithoautotrophic bacteria in acidic mine drainage, obtains its energy from the oxidation of ferrous iron, elemental sulphur, or partially oxidized sulphur compounds 10,13 . The thiol groups, the breaking of disulphide bonds, and the rearrangement of proteins have been considered to play a key role in the sulphur oxidation 14 .…”