Fernanda C. Reis

The rus Operon Genes Are Differentially Regulated When Acidithiobacillus ferrooxidans LR is Kept in Contact with Metal Sulfides

Acidithiobacillus ferrooxidans is a Gram-negative bacterium that obtains energy from the oxidation of ferrous iron or reduced sulfur compounds. In this bacterium, the proteins encoded by the rus operon are involved in electron transfer from Fe(II) to O2, and the first two proteins in this pathway also participate in the electron transfer pathway from Fe(II) to NAD(P). In this work we analyzed the expression, by real-time PCR, of the eight genes from the rus operon when A. ferrooxidans LR was grown in the presence of iron (control) and then kept in contact with chalcopyrite (CuFeS2) and covellite (CuS). A small decrease in rus operon gene expression was observed in the presence of chalcopyrite, while in the presence of covellite the expression of these genes showed a remarkable decrease. These results can be explained by the absence of ferrous iron in covellite. To explain the expression difference observed between the gene cyc1 and the gene rus, we investigated the information content presented at the Translation Initiation Site (TIS) of both genes. cyc1 showed a highly information content (8.4 bits) that can maximize translation, and rus showed a less favorable context (5.5 bits). Our hypothesis is that the energetic metabolism in A. ferrooxidans may be controlled at the transcriptional and posttranscriptional level by different mechanisms.

ribB and ribBA genes from Acidithiobacillus ferrooxidans: expression levels under different growth conditions and phylogenetic analysis.

Acidithiobacillus ferrooxidans is a Gram-negative, chemolithoautotrophic bacterium involved in metal bioleaching. Using the RNA arbitrarily primed polymerase chain reaction (RAP-PCR), we have identified several cDNAs that were differentially expressed when A. ferrooxidans LR was submitted to potassium- and phosphate-limiting conditions. One of these cDNAs showed similarity with ribB. An analysis of the A. ferrooxidans ATCC 23270 genome, made available by The Institute for Genomic Research, showed that the ribB gene was not located in the rib operon, but a ribBA gene was present in this operon instead. The ribBA gene was isolated from A. ferrooxidans LR and expression of both ribB and ribBA was investigated. Transcript levels of both genes were enhanced in cells grown in the absence of K2HPO4, in the presence of zinc and copper sulfate and in different pHs. Transcript levels decreased upon exposure to a temperature higher than the ideal 30 degrees C and at pH 1.2. A comparative genomic analysis using the A. ferrooxidans ATCC 23270 genome revealed similar putative regulatory elements for both genes. Moreover, an RFN element was identified upstream from the ribB gene. Phylogenetic analysis of the distribution of RibB and RibBA in bacteria showed six different combinations. We suggest that the presence of duplicated riboflavin synthesis genes in bacteria must provide their host with some benefit in certain stressful situations.

Chalcopyrite and bornite differentially affect protein synthesis in planktonic cells of Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans is used in bioleaching industrial operations to recover metal ions from mineral sulfides. Chalcopyrite and bornite are copper sulfides that have the same elemental composition, but differ in their susceptibility to the bioleaching process. Our objective was to identify differentially expressed proteins in A. ferrooxidans LR cells exposed to chalcopyrite or bornite, as a sole energy source, for 24 hours. Compared to the control (without minerals), proteins were induced or repressed in planktonic cells after contact with chalcopyrite or bornite by 24 hours. These results demonstrated that the time of exposure to the copper minerals was enough to trigger distinct responses in the A. ferrooxidans metabolism.

Up- and Down-Regulated Genes in Acidithiobacillus ferrooxidans Exposed to Chalcopyrite

Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe 2+ ) or reduced sulphur compounds. The economic relevance of A. ferrooxidans resides on its capacity to solubilize metal sulphides in bioleaching industrial operations. Chalcopyrite (CuFeS2) is the most abundant copper-bearing mineral, but it is very refractory to bacterial and even to chemical attack. Very little is known about the genes expressed when A. ferrooxidans is kept in contact with this copper sulphide. In order to evaluate gene expression under this condition, cells of A. ferrooxidans strain LR were grown in T&K medium with Fe 2+ (until 80% of iron oxidation), collected, washed and incubated for 24 hours in the presence of chalcopyrite (2.5%) in salts solution of T&K medium at 250 rpm, 30 o C. RNA isolated from cells grown in the presence of Fe 2+ (control) and cells exposed to chalcopyrite were used in RNA arbitrarily primed PCR experiments to identify cDNAs differentially expressed. The cDNAs were isolated, cloned and sequenced. The sequences were compared with the A. ferrooxidans ATCC 23270 genome (TIGR, http://www.tigr.org). The relative expression pattern of the identified genes was validated by real-time PCR. Genes upor down-regulated in the presence of chalcopyrite were identified. Among the down-regulated genes were those that encode proteins involved in energy metabolism, such as an aldehyde dehydrogenase, and those involved in protein degradation, such as an hflK protein. Genes encoding proteins involved in transport, such as a phosphate ABC transporter, were among the up-regulated genes. The results showed that chalcopyrite differentially modulates gene expression in A. ferrooxidans, affecting distinct biological functions such as metabolism, transport and protein synthesis.

Transporter Protein Genes are Up-Regulated by Covellite in Acidithiobacillus thiooxidans

Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans are involved in bioleaching, a process that has been successfully used in copper recovery. Apparently, A. ferrooxidans can tolerate 55 g/L of Cu 2+ in the growth medium while A. thiooxidans tolerates only 5 g/L. In bacteria many proteins involved in metal tolerance are members of a large class of membrane transporters called ATP Binding Cassette (ABC proteins). A gene that encodes for an ABC transporter protein was isolated from A. ferrooxidans LR, by RNA Arbitrarily Primed PCR (RAP-PCR), after the bacteria were kept for 24 hours in the presence of 2.5 g/ 100 mL of covellite (CuS). In comparison to ferrous iron, this gene was up-regulated in the presence of covellite. The objective of this work was to investigate the expression of transporter protein genes, in the presence of covellite, in A. thiooxidans. Three genes, two that encode for ABC transporter proteins and one that encodes for an efflux transporter protein had their relative expression pattern analyzed in this bacterium using realtime PCR. For this, A. thiooxidans FG01 was cultivated in the presence of sulphur (10 g/L) pH 1.8 at 250 rpm, 30°C, until an optical density of 0.11 was reached. The cells were collected and then kept in contact with covellite during 24 hours. The 16S rRNA gene was used as a reference standard and transcripts isolated from sulphur grown cells were used as the calibration parameter. The primers used to amplify the target genes were based on the A. ferrooxidans ATCC 23270 T complete genome sequence made available by TIGR (www.tigr.org). The real-time PCR quantification was performed twice, using RNA samples from independent cultures. In comparison to sulphur, the three genes analyzed were up-regulated in the presence of covellite. Experiments with A. ferrooxidans LR are in progress in order to compare the results among these two species. Further analyses will correlate the obtained data with bacterial colonization and adherence capability using scanning electron microscopy (SEM).