"Functional analysis of the prokaryotic metallothionein locus, smt"

Abstract

The localisation of the prokaryotic metallothionein (MT) divergon smt (which includes the MT gene smtA and a divergently transcribed gene smtB] was examined, and smt deficient mutants of Synechococcus PCC 7942 (strain R2-PIM8) have been generated by insertional inactivation/partial gene deletion mediated by homologous recombination. The structure and homozygosity (of the smt region) of these mutants, designated R2-PIM8(smt), was confirmed by Southern analyses and plasmid recovery in Escherichia coli (involving the generation of a ca. 7.8 kb plasmid from Soil digested R2-PIM8(smt) DNA). Furthermore, smtA transcripts were not detected in R2-PIM8(smt) RNA. Viability of R2-PIM8(smt) reveals that smt performs no essential role in Synechococcus under these culture conditions. R2-PIM8(smt) has reduced tolerance to Zn(^2+) and Cd(^2+), and short term reduced resistance to Ag(^+). Restoration of Zn(^2+) tolerance was used as a phenotypic selection to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment containing an uninterrupted smt divergon. These smt-restored cells also exhibited restored Cd(^2+) tolerance. Hypersensitivity to Cu(^2+) or Hg(^2+)was not detected in R2-PIM8(smt) indicating independence of Cu(^2+) and Hg(^2+) resistance to smt-mediated metal tolerance. Sequences upstream of smtA (Including smtB and/or the smt operator-promoter) fused to a promoterless locZ, conferred metal-dependent β-galactosidase expression in R2-PIM8. At maximum permissive concentrations for growth, β-galactosidase assays revealed Zn(^2+) to be a more potent elicitor of metal-dependent expression from the smtA operator-promoter than Cd(^2+). Equivalent experiments, in R2-PIM8(smQ and R2-PIM8(smtA+/B-) (containing functional chromosomal smtA and non-functional chromosomal smtB), revealed that smtB encodes a repressor of smtA transcription. In addition, it is demonstrated that SmtB can act in trans. It is proposed that Zn(^2+) is the most potent (metal ion) inducer of SmtB mediated derepression of smtA transcription. Furthermore, β-galactosidase assays indicated that, in addition to SmtB, other regulatory elements (including a transcriptional activator) are involved in the regulation of expression from the smt operator-promoter. Restoration of Zn(^2+) tolerance was also used as a phenotypic selection to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment, containing functional smtA and non-functional smtB. The resulting transformants, R2-PIM8(smtA+/B-), exhibited increased (early) tolerance to Zn(^2+) and Cd(^2+) as compared to R2-PIM8(smt-. reintroduced ) (equivalent to R2-PIM8).The work presented in this thesis proposes a role for SmtA in Zn(^2+) homoeostasis/metabolism and Cd(^2+) detoxification. SmtB is confirmed to be a trans-acting inducer- (metal ion) responsive negative regulator of smtA. The phenotype of R2-PIM8(sm(A+/B-) (with respect to metal tolerance) has significance regarding previous work (Gupta et al., 1993. Molecular Microbiology 7, 189-195), in which analysis of the smt region of Synechococcus PCC 6301 cells selected for Cd(^2+) resistance, by stepwise adaptation, revealed the functional deletion of smtB. It was proposed that loss of smtB may be beneficial for continuously metal challenged cells. Loss of smtB, now shown to encode a repressor of smtA transcription, is shown to confer constitutive derepressed expression from the smtA operator- promoter and determine an (early) increase in metal (Zn(2+)/Cd(^2+)) tolerance.