Transcriptional and Functional Studies of Acidithiobacillus ferrooxidans Genes Related to Survival in the Presence of Copper

Abstract

The acidophilic Acidithiobacillus ferrooxidans can resist exceptionally high copper (Cu) concentrations. This property is important for its use in biomining processes, where Cu and other metal levels range usually between 15 and 100 mM. To learn about the mechanisms that allow A. ferrooxidans cells to survive in this environment, a bioinformatic search of its genome showed the presence of at least 10 genes that are possibly related to Cu homeostasis. Among them are three genes coding for putative ATPases related to the transport of Cu (A. ferrooxidans copA1 [copA1 Af ], copA2 Af , and copB Af ), three genes related to a system of the resistance nodulation cell division family involved in the extraction of Cu from the cell (cusA Af , cusB Af , and cusC Af ), and two genes coding for periplasmic chaperones for this metal (cusF Af and copC Af ). The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of Cu. The putative A. ferrooxidans Cu resistance determinants were found to be upregulated when this bacterium was exposed to Cu in the range of 5 to 25 mM. These A. ferrooxidans genes conferred to Escherichia coli a greater Cu resistance than wild-type cells, supporting their functionality. The results reported here and previously published data strongly suggest that the high resistance of the extremophilic A. ferrooxidans to Cu may be due to part or all of the following key elements: (i) a wide repertoire of Cu resistance determinants, (ii) the duplication of some of these Cu resistance determinants, (iii) the existence of novel Cu chaperones, and (iv) a polyP-based Cu resistance system.

Heavy Metal Resistance Strategies of Acidophilic Bacteria and Their Acquisition: Importance for Biomining and Bioremediation

“…The expression of most of these open reading frames (ORFs) was studied by real time RT-PCR (qRT-PCR) using A. ferrooxidans cells that were adapted to grow in the presence of high concentrations of CuSO 4 . These A. ferrooxidans copperresistance determinants were found to be upregulated when this bacterium was exposed to CuSO 4 in the range of 5 to 25 mM and conferred to E. coli a greater resistance to copper compared to wild-type cells, supporting their functionality (Navarro et al, 2009). …”

“…ferrooxidans ATCC 23270 can survive high copper concentrations by having in its genome at least ten genes that are possibly related to copper homeostasis. These include three genes coding for putative ATPases related to the transport of copper (copA1 Af , copA2 Af , and copB Af ), three genes related to a system of the RND family, involved in the extraction of copper from the cell by using the proton motive force (cusA Af , cusB Af , cusC Af ), and two genes coding for periplasmic chaperones for this metal (cusF Af and copC Af ) (Navarro et al, 2009). The expression of most of these open reading frames (ORFs) was studied by real time RT-PCR (qRT-PCR) using A. ferrooxidans cells that were adapted to grow in the presence of high concentrations of CuSO 4 .…”

“…The genomic sequence of A. ferrooxidans ATCC 53993 contains all the copper-resistance genes present in A. ferrooxidans ATCC 23270 that have been experimentally confirmed as being expressed in the presence of copper, except that strain ATCC 23270 contains a second Cus-like operon interrupted by a transposase gene which is absent in the genome of strain ATCC 53993 (Luo et al, 2008;Navarro et al, 2009;Orell et al, 2010;Orellana and Jerez, 2011). These ORFs have 100% identity between their corresponding DNA sequences.…”

“…Both strains of A. caldus contain a variety of genes coding for putative copper resistance-related proteins, since they showed a high degree of identity (between 50 and 90%) with those known for A. ferrooxidans (Navarro et al, 2009;Orellana and Jerez, 2011). These two strains of A. caldus share several of these genes in their chromosomal DNA, such as the components of an RND-type effl ux pump (CusABC), two P-type ATPases (CopB), two cytoplasmic metalochaperones (CopZ) and a copper oxidase (CueO) ( Table 2).…”

“…Some of the genes related to zinc resistance reported in A. caldus ATCC 51756 (Mangold et al, 2013) and those already mentioned for the megaplasmid of strain SM-1 that may be possible zinc transporters could also be involved in copper transport (see Table 2). Specifi cally, the RND type effl ux systems and P-type ATPases, since these genes have been previously reported as related to copper resistance in A. ferrooxidans ATCC 23270 (Navarro et al, 2009;Orellana and Jerez, 2011).…”