Ecological Interactions Between Metals and Microbes

Konopka, Allan E.

Purdue University, West Lafayette, IN

关键词: Genetics;    Plasmids;    Genes;    Physiology;    Transcription;   

DOI  :  10.2172/893456
RP-ID  :  NABIR-1011901-2005
RP-ID  :  None
RP-ID  :  893456

美国|英语

来源: UNT Digital Library

【 摘 要 】

Analysis of Lead Resistant Arthrobacter sp. SI-1 Arthrobacter sp. SI-1 was isolated from contaminated soils at the Seymour site, and was found to be resistant to Pb at concentrations near its solubility limit (150 micromolar). The genetic region that confers lead resistance is located on a plasmid (PSI-1)has been cloned. We have continued to analyze the sub-clones from the pSI-1 region. Initially we had predicted that ORF1-ORF5 were involved in lead resistance because their organization suggest a potential operon. In addition these same five genes have been found in a similar organization on a plasmid from Arthrobacter FB24, while the pAA1 plasmid from A. aurescens TC1 contains three of the five genes. In order to determine the minimum genes required for lead resistance a series of deletion mutants were constructed from the 14.7 kb clone pKJ60. Deletion of ORFs 3-5 did not have any measurable effect on the ability of the cloned fragment to rescue the lead resistance phenotype in a lead sensitive strain of E. coli (RW3110). The construct pKJ65 was generated by removing approximately 200 bp from the center region of ORF2, which codes for the P-Type ATPase; as expected this deletion resulted in a lead sensitive phenotype. While the genes downstream of ORF 2 do not appear to play a significant role in lead resistance the same cannot be said for ORF1 which is upstream. Based on amino acid sequence homology a BLAST search indicates ORF1 is likely a regulatory protein from the ArsR family. When ORF1 is removed (pKJ64, pKJ67), a lead sensitive phenotype occurs. Approximately 100 bp from the sequence of ORF1 was deleted (pKJ70) in order to test if ORF1 is required for lead resistance, or if the cells require something in the upstream non-coding region (binding site, promoter). Cells with pKJ70 show some limited growth in the presence lead, but it is generally much slower than the lead resistant constructs where ORF1 is present. These results suggest that ORF1 has a positive effect on lead resistance, perhaps acting as an activator of transcription. We are currently working to repeat this same set of experiments using cadmium. Previous work on the physiology of lead resistance was done in a MES buffered minimal media at pH6.5, the concentration of PbNO3 in these experiments ranged from 0 to 200 micromolar.