Superfund Research Program

Molecular Mechanisms of Bacterial Metal Redox Transformations

Project Leader: Bradley M. Tebo (Oregon Health and Science University)
Grant Number: P42ES010337
Funding Period: 2000-2010

Project-Specific Links

Final Progress Reports

Year:   2009  2004 

Heavy metals are found as contaminants in many surface and subsurface waters as a result of industrial activity as well as natural processes.  Bacteria are able to catalyze the transformation of some toxic metals to less toxic and/or less mobile states.  Dr. Tebo and his research team are currently pursuing two strategies to harness the natural activity of bacteria to detoxify heavy metals:  1) Bacterial oxidation of Mn(II) to produce high surface area, highly reactive manganese oxides with tremendous capacity for scavenging heavy metals and degrading organic compounds, and 2) the direct microbial reduction of Cr(VI) (a toxic and soluble metal) to Cr(III) (a less toxic and insoluble metal).

1- Mn(II) oxidation

The team’s efforts this past year have focused on the purification of Mn(II)-oxidizing proteins from two organisms: a spore forming bacterium, Bacillus sp. strain PL-12, and an α-proteobacterium, strain SD-21.  They have made some good progress on the purification protocol and characterization of a Mn oxidase from SD21.  Previously, they discovered that the cofactor PQQ (pyrolloquinoline quinone) increased the Mn(II)-oxidizing activity in their assays by 7-fold.  They have now demonstrated using a quantitative quinone assay, that quinones are also enriched during the purification of the Mn oxidase, further supporting the notion that this cofactor is directly involved in Mn(II) oxidation in strain SD21.  This is another indication of the novel biochemistry involved in Mn(II) oxidation.  The researchers are currently working with the Mass Spectrometry Facility in Chemistry and Biochemistry to obtain protein sequence.
 
In addition to the biochemical approach, the researchers have also initiated a genetic approach involving transposon mutagenesis to generate Mn(II) oxidation mutants in SD21.  They have been successful in generating transposon mutants in SD21 and so far have identified 2 Mn(II) oxidation null mutants after screening about 3500 total mutants (they also have several mutants with altered Mn(II) oxidation phenotype).  The team is continuing to isolate mutants and is using inverse PCR to identify the gene(s) in which the transposon inserted.  Sequence information is not yet available.

In Bacillus PL-12 the researchers have purified the Mn(II)-oxidizing activity using size exclusion chromatography.  The active fraction was further gel purified and sent off for mass spectrometric analysis.  MS/MS analysis of trypsin digest identified several different proteins that co-migrated on the gel, none of which was a multicopper oxidase, as expected.  Subsequent analysis in the lab revealed that the Mn oxidase is not digested by trypsin and thus the team is using trypsin as a purification step for the Mn(II)-oxidizing activity.  They are currently preparing more protein for MS analysis.

2- Hexavalent chromium (Cr(VI)) reduction

Bacterial reduction of hexavalent chromium (Cr(VI)) is considered one of the promising strategies for the bioremediation of Cr(VI) contamination. In a previous study, Dr. Tebo’s team found that a limited amount of chromate could be reduced per Shewanella oneidensis MR-1 cell under both aerobic and anaerobic conditions (Middleton et al, 2003), indicating that the accumulation of reduced chromium may be deleterious to cells.  Furthermore, reduced Cr was identified both extracellularly and in the cytoplasm of cells (Middleton et al, 2003).  In that study, they hypothesized that the transport of chromate inside the cell, followed by the intracellular reduction of Cr(VI) to Cr(III) may be the mechanism of chromium toxicity.
 
The researchers have demonstrated that complexation of Cr(III) as it is produced during aerobic Cr(VI) reduction greatly enhances both cell viability and the amount of Cr(VI) reduced per cell.  Cr(III) as Cr(OH)₃(s) is not harmful to cells but soluble, uncomplexed Cr(III) is.  Quantitative Reverse Transcription Polymerase Chain Reaction was utilized to determine whether Cr(III) affects DNA transcription by comparing expression levels of house-keeping genes (alaS, dnaE, dnaN, holA, polA, proS).  These housekeeping genes are involved in basic cell functions such as protein synthesis (alaS and proS) and DNA transcription (dnaE, dnaN, holA, polA). These genes have a constant expression profile regardless of the environment and were used to find out whether Cr(III) causes a decrease in their expression.  Such a decrease would be attributable to direct interference of Cr(III) with transcription through DNA and/or protein binding. In addition, the expression of genes known to be upregulated under Cr(VI) reduction (manuscript in revision) and thought to be associated with cation efflux was also investigated in the presence of Cr(III).
 
The results show that the presence of soluble Cr(III) interferes with the transcription of house-keeping genes suggesting a general transcriptional inhibition.  Genes encoding two putative cation efflux pumps were upregulated in the presence of both Cr(III) and Cr(VI) suggesting a similarity in the mechanism of metal resistance for the two Cr oxidation states.  The research team suggests that the mechanism of inhibition of Cr(VI) is through the intracellular reduction of Cr(VI) followed by the cytoplasmic precipitation of Cr(III).  In contrast to extracellular Cr(OH)₃(s) that is not harmful to cells, Cr(III) precipitated inside the cells has the propensity to bind DNA and protein and thus inhibit metabolism.  A manuscript on this work is about to be submitted.

Two important implications emerge for the bioremediation of Cr(VI)-contaminated sites: (1) Cr(III) can persist in solution in the presence of complexing agents  and (2) Cr(VI)-reducing bacterial strains genetically modified to exclude chromate from the cytoplasm may have longer-lasting  activity.