Genetic systems encoding heavy metal resistance and/or transport in Achromobacter sp. strain AO22 and their applications in environmental biotechnology

Heavy metal contamination, from both natural and anthropogenic sources, is a major environmental concern in soil and marine ecosystems due to the persistence of the contaminants. While heavy metals are generally toxic to cells, certain bacteria which are exposed to heavy metal contamination have developed, or acquired, genetic systems that allow them to survive in such environments. Harnessing of these systems provides a potential route for environmental biotechnology applications. Achromobacter sp. AO22 (originally identified as Alcaligenes sp. AO22) is a multiheavy metal resistant soil bacterium isolated from a lead-contaminated battery manufacturing site in Australia. Earlier molecular studies indicated the presence of mercury and copper resistance/transport determinants in the strain. The overall aim of the present project was to characterise the mercury and copper resistance and transport determinants in Achromobacter sp. AO22 and to investigate the possibility of applying these determinants, or the bacterium, in the development of heavy metal biosensors and bioremediation systems. By introducing a broad-host-range IncP plasmid into AO22 and subsequently transferring it to Escherichia coli recipients, the mercury resistance determinants of strain AO22 were found to be located on an active transposon, designated TnAO22. It had a transposition frequency of 10−2 to 10−3 per target plasmid transferred and conferred Hg resistance in E. coli. Analysis of the 8230 bp DNA sequence of TnAO22 revealed that it has 38bp inverted repeat sequences and resolvase and transposase genes which are transcribed in the same direction, characteristics of the Tn21 subgroup of the Tn3 family of transposons. The sequence of the transposition modules of TnAO22 indicated that it is a variant of Tn5051, a mercury transposon from a strain of Pseudomonas putida isolated from water in New York. The mer operon of TnAO22 is highly similar to that of Tn501 with >99% nucleotide sequence identity. In addition, a second mer operon, merRTPA (mer2) from strain AO22 which are 68-77% identical to the corresponding genes in TnAO22, was also cloned. Further, a cluster of genes, designated copSRABGOFCDK, was identified from Achromobacter sp. AO22. copSRAB and copCD are related to the cop operons of Pseudomonas syringae and Cupriavidus metallidurans CH34, and pco of E. coli, which encode systems that sequester or extrude excess copper from the periplasm. copF encodes a putative heavy metal P-type ATPase which may have a role of cytoplasmic copper efflux, while copG, copO and copK encode putative periplasmic proteins which may function as copper chaperones. Sequencing of the flanking regions of AO22 cop locus indicated that it could be present on a genomic island (GI) (which also carries mer2). A section of DNA between copR and copA was identified as a copper responsive promoter and characterised using a lacZ reporter construct, pCOPRP, in E. coli. In an engineered strain of AO22 carrying the same plasmid, designated AO22(pCOPRP), expression of lacZ was enhanced 140-fold in the presence of 4.0 mM Cu and was minimal when exposed to cadmium, lead, mercury, silver, and zinc. Thus, AO22(pCOPRP) has the potential to be developed into a copper biosensor for the detection of bioavailable copper in environmental samples. The ability to remove copper by the live biomass of strain AO22 was investigated in closed-batch copper uptake studies. Copper removal was characterised based on the effect of pH, initial biomass concentration, initial copper concentration and fitting on adsorption isotherm models. Copper uptake capacity by AO22 biomass was 22.5 mg Cu/g and was enhanced by the addition of the biomass of a Gram positive strain, Arthrobacter sp. E11, isolated from the same source as AO22. Data obtained indicated that the live biomass of strains AO22 was a promising material to remove copper from aqueous solutions. Finally, a preliminary survey of the prokaryotic genome database for systems homologous to the AO22 cop locus revealedsequence diversity of CopA-like proteins, as well as the diversity and mosaic nature of cop loci. Phylogenetic incongruence of CopA-like proteins indicated lateral gene transfer. This work has thus made original and substantial contributions to the field of environmental microbiology through expansion of our understanding of the distribution of mer transposons and copper homeostasis/transport determinants, as well as identification of a copper responsive promoter and a soil bacterial strain with potential application as a copper biosensor and copper accumulator.