Turner, Jennifer Susan (1993) "Functional analysis of the prokaryotic metallothionein locus, smt". Doctoral thesis, Durham University.
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.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||16 Nov 2012 10:55|