UBC Theses and Dissertations
Evolution and expression of the superoxide dismutase of the archaebacterium halobacterium cutirubrum May, Bruce Pearson
Archaebacteria are a diverse group of bacteria that share a number of fundamental biochemical characteristics that distinguish them from other bacteria (eubacteria) and eucaryotes. They are believed to be an independent lineage that diverged from other organisms more than 3.5x10⁹ years ago. To study the evolution of oxygen tolerance in the archaebacteria, the superoxide dismutase (SOD) and SOD-encoding gene of the extremely halophilic archaebacterium Halobacterium cutirubrum were investigated. Purification of the SOD activity of this organism resulted in a single Mn-containing enzyme with subunits of M[sub r] 25000 that probably associate as tetramers. It has maximal activity in 2 M KC1, reflecting adaptation to the high salt concentrations within halobacterial cells. Assays of activity in the presence of SOD inhibitors (cyanide, azide, and hydrogen peroxide) gave a unique pattern of responses. The sequence of 51 of 56 amino acid residues at the amino-terminus was determined and found to be homologous to the amino-terminus of SODs of eubacteria. The gene, designated sod, that encodes the halobacterial Mn SOD was cloned by using as a probe a mixture of 20mer oligonucleotides corresponding to codons for amino acid residues 27-33. The sod gene and 5' and 3' untranslated regions were located on a Sau3AI genomic DNA fragment of 1127 nucleotides. The deduced amino acid sequence is 200 residues long and has 39 to 42% identity with Mn-containing SODs of eubacteria and mitochondria. This homology may be due to either lateral transfer of the gene between eubacteria and archaebacteria or to high amino acid sequence conservation in the enzyme during the divergent evolution of eubacteria and archaebacteria. Transcription of the gene initiates only 2-3 nucleotides upstream of the translation initiation codon. The 5' end of the transcript does not contain a purine-rich Shine-Dalgarno element and the promoter does not contain elements that closely match consensus sequences found in other archaebacterial promoters. Termination of transcription occurs at five consecutive T residues that are preceded by a GC-rich region containing short inverted repeats. The gene is basally expressed in anaerobically grown cells, and is inducible by both oxygen and paraquat, a generator of oxygen radicals. The same transcription initiation site is used in both types of expression, suggesting that one promoter is responsible. In addition to the single copy of the sod gene, the genome of H. cutirubrum contains a sequence that is very closely related to sod, but does not encode the previously purified SOD of this organism. This sod-like gene (slg) has 87% nucleotide identity with sod in the coding region and the predicted protein has 83% amino acid identity with the Mn SOD protein. It conserves the four amino acid residues that bind the prosthetic Mn atom, yet no superoxide dismutase activity corresponding to the slg product was detected in cell extracts. The 5' and 3' flanking regions of sod and slg. are unrelated and slg is not inducible by paraquat. Transcription of slg initiates 13 nucleotides upstream of the translation initiation codon and the promoter contains elements (TTCGA and TTAA) that closely match archaebacterial consensus sequences. Termination of transcription occurs in a tract of seven T residues that is preceded by short inverted repeats. The slg gene may be sufficiently divergent from sod to encode a different function. The divergence of slg and sod appears to be the product of an unusual mode of evolution.