Physiological and genetic analysis of plasmid-mediated metal resistance in Arthrobacter sp strain AK-1
Abstract
Metals are present throughout the environment, resulting from anthropogenic input or naturally occurring metal ore deposits. Metals were likely one of the first stresses faced by early life; therefore, it is not surprising that metal resistance has been found in numerous and diverse microbial cells. The Arthrobacter sp strain AK-1 was isolated from a mixed waste contaminated site with elevated concentrations of hydrocarbons and metals including lead. AK-1 was shown to be resistant to lead, cadmium and zinc; the loss of lead resistance resulted in cadmium and zinc sensitivity as well. The metal resistance phenotype was transferable from the resistant, AK-1 strain to a sensitive Arthrobacter strain CN-2, suggesting the resistance genes were present on a mobile genetic element. An approximate 100 kb plasmid (pSI-1) was isolated from AK-1 and sequenced. An open reading frame (ORF) with similarity to the type IB family of P-type ATPase efflux pumps was identified ORF 2; these proteins mediate resistance to lead, cadmium, and zinc in other bacterial systems. Four additional ORFs were located in the same region including a putative ArsR type metalloregulatory protein (ORF 1), a lipoprotein signal peptidase (ORF 3), a cytochrome biogenesis protein (ORF 4) and a cadmium binding protein ORF 5. Reverse transcriptase PCR demonstrated ORF 1, ORF 2, ORF 3 and ORF 5 were all induced by exposure to lead, and ORF 1, ORF 2, and ORF 3 form a single transcript. ArsR regulators repress transcription in the absence of inducer; however, a basal level of ORF 2 mRNA synthesis was detected by quantitative reverse transcriptase PCR in the absence of any metal induction. The expression of cadA responded to low micromolar concentrations of lead and cadmium; the affinity of the regulatory protein for zinc appeared to be tenfold lower. A comparative analysis of pSI-1 and six other Arthrobacter plasmids identified genes related to plasmid maintenance. Genes with similarity to core functions such as conjugation and plasmid segregation were identified; however, the Arthrobacter genes generally formed separate clusters. This suggests that the Arthrobacter genes may form new branches within these previously described gene families.
Degree
Ph.D.
Advisors
Konopka, Purdue University.
Subject Area
Molecular biology|Microbiology
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