Prokaryotic metallothionein locus and cadmium tolerance in Synechococcus PCC 6301
The aim of this study was to investigate the molecular mechanism of Cd-tolerance in the cyanobacterium Synechococcus PCC 6301 and to establish whether the prokaryotic metallothionein (MT) locus, smt, is involved. Cd-tolerant cell lines of Synechococcus PCC 6301 were developed by step-wise selection, of a culture that had undergone prolonged maintenance in liquid medium. The Cd-tolerant cell lines AO.8, A1.3 and A1.7 (tolerant to 0.8, 1.3, 1.7 µM Cd, respectively) were phenotypically different to the non-selected line AO. Genomic DNA from AO and the Cd-tolerant lines AO.8, A1.3 and A1.7 was analysed by Southern hybridisation. A ca. 4-fold increase in hybridisation to radiolabelled smtA (prokaryotic metallothionein gene), relative to AO, was observed in genomic DNA from A1.7. Equivalent amounts of DNA were loaded onto each track, and no difference in hybridisation to a control gene, psaE (photosystem I gene), was observed. Indeed, the hybridisation of DNA from Al .7 to psaE was slightly less than that observed in AO. Genomic DNA isolated from AO, AO. 8, A1.3 and A1.7 was also analysed after 2, 4, 7 and 12 subcultures in the presence of the respective Cd concentrations. An increase in hybridisation to smtA, relative to AO, was observed in DNA from all Cd-tolerant cell lines. Additionally, unique additional restriction fragments, both larger and smaller than that in AO, were observed in DNA from A1.3 and A1.7. A similar restriction pattern was observed in 3 independent restrictions of DNA from A1.3 after 2 subcultures. Cd-tolerant cell lines were also developed from a 'clonal' culture of Synechococcus PCC 6301. An increase in tolerance was marked by an increase in growth lag, which reduced upon subsequent maintenance of the Cd-tolerant line in the presence of Cd. Genomic DNA from the non-selected line CO and Cd-tolerant lines C1.4, CI.8, C2.6 and C3.2 (tolerant to 1.4, 1.8, 2.6, 3.2 µM Cd, respectively) were analysed after 1, 2, 3, 4 and 5 subcultures. In all the Cd-tolerant lines, an increase in hybridisation to smtA, and additional larger (ca. 11 kb) and smaller (ca. 5.45 kb) restriction fragments, relative to CO (ca. 5.8 kb), were observed. However, amplification and rearrangement in DNA from CI .4 were evident only after 2 subcultures. Additionally, restriction fragment equivalent in size to that observed in CO was lost in CI.8, C2.6 and C3.2, and the presence of Cd did not affect DNA restriction with Sah under in vitro and short term in vivo conditions. The rearrangement in Cd-tolerant line C3 .2 was observed on a minimal HindIII-SalI fragment (ca. 350 bp smaller than that in CO) and isolated from size-fractionated genomic libraries. The alteration was mapped by PCR to a 600 bp region in the 5' flank of smtA. Nucleotide sequence analysis of the clones identified a deletion of 352 bp within a region of 360 bp encoding the C- terminal end of smtB (repressor of smtA transcription), rendering it non-functional. Increased basal level of smtA expression (derepressed expression) and indications for complete loss of the excised fragment were observed in Cd-tolerant line C3.2. Rearrangement was detected in DNA from C3.2 even after maintenance in the absence of Cd for 3 subcultures. The clone bank pPLAN Bal-Ba7 and pPLAN B2 (carrying Bamm restriction fragments of Synechococcus PCC 7942 plasmids) were used to study the plasmid/chromosomal localisation of smtA. Weak hybridisation of pPLAN Ba2 to smtA was observed, but further Southern analysis of plasmid and genomic DNA suggested chromosomal localisation of smtA. PCR and Southern hybridisation were used to detect homologues of smtA in other cyanobacterial strains. Putative homologues were identified in Synechococcus PCC 7942, Synechococcus D562, Oscillatoria D814 and Synechocystis D840 (= PCC 6803) by heterologous probing. However, no hybridisation to smtA was observed in DNA isolated from Calothrix D184 and Microchaete D578.