Tandem arrangement of photolyase and superoxide dismutase genes in Halobacterium halobium

Abstract

A DNA fragment containing the photolyase gene was cloned from Halobacterium halobium. The deduced amino acid sequence is highly similar to those of four known photolyases from eubacteria and a eucaryote. The cloned gene expressed in Escherichia coli cells increased the survival of UV-irradiated host cells by photoreactivation. These results indicate that photolyases of eucaryotes, eubacteria, and archaebacteria are derived from a common origin. In this cloned DNA fragment, two additional open reading frames (ORFs), ORF 151 and ORF 200, were found in the 5' and 3' adjacent flanking regions of the photolyase gene. ORF 200 shows unequivocal amino acid sequence homology to all known manganese and iron superoxide dismutases. Northern (RNA) hybridization analysis of H. halobium RNA revealed the existence of three transcripts, one of which covered all three ORFs, indicating that photolyase and superoxide dismutase are partly cotranscribed in this bacterium.Photoreactivation repair (PHR) is a DNA repair mechanism in which photolyase (EC 4.1.99.3) monomerizes UVinduced pyrimidine dimers by utilizing near-UV or visiblelight energy. All photolyases analyzed to date contain a reduced flavin adenine dinucleotide and a second cofactor which determines the optimal wavelength of the absorption and action spectra of the enzymes. Photolyases from Escherichia coli and Saccharomyces cerevisiae contain a folate derivative as the second cofactor (folate-type photolyases) and show maximum activity at about 380 nm (16), while photolyases from Anacystis nidulans (A. P. M. Eker and A. Yasui, manuscript in preparation), Streptomyces griseus (8), and Scenedesmus actus (9) contain a 7,8-didemethyl-8-hydroxy-5-deazaflavin (HDF-type photolyases) and are most active around 440 nm (for a review, see reference 28).Halobacterium halobium is an archaebacterium, according to the system of classification in which all organisms are divided into three kingdoms (32). The photolyase of this bacterium seems to be of the HDF type (7, 15) because its in vivo action spectrum peaks at 440 nm. We cloned the photolyase gene of this bacterium in order to compare it with those of organisms in the other kingdoms. In the 3'-flanking region of the H. halobium photolyase gene, we found a superoxide dismutase (EC 1.15.1.1) (SOD) gene. We present here the complete nucleotide sequences of both genes and evidence that these two genes are cotranscribed in this bacterium. MATERIALS AND METHODSEnzymes and sequencing kits. Restriction enzymes and modifying enzymes were purchased from Boehringer Mannheim Biochemicals, New England BioLabs, Inc., TAKARA, and TOYOBO. The multiprime labeling kit, the 7-deaza sequencing kit, and the Taq polymerase sequencing kit were obtained from Amersham, Corp., TOYOBO, and Promega, Biotec, respectively. * Corresponding author. Preparation of total DNA and RNA. H. halobium R1M1 was cultured under standard conditions (23). Cells were collected, washed once with basal solution (culture medium without peptone), and lysed in 10 mM EDTA containing 1...

Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ

“…The enzyme from M. thermoautotrophicum is one of the Fe containing SODs exhibiting a high resistance to H 2 O 2 . It retains its activity after 24 h of incubation in the presence of 0.5 mM H 2 O 2 [13]. Thus, the Fe containing SODs from methanogenic Archaea differ from the known bacterial Fe containing SODs in the character of their inhibition and inactivation.…”

“…Fe and Mn containing SODs exhibit significant similarity in their amino acid sequences and tertiary structures [12]. However, the bacterial Mn and Fe containing SODs are more similar to each other than to the corresponding enzymes of Archaea, which suggests that the common features in the amino acid sequences are rather connect ed with evolutionary relations than with the presence of a specific metal as the cofactor [13]. However, SODs belonging to these two classes differ in their sensitivity to sodium azide: 1 mM NaN 3 inhibits Fe containing SODs by 70 90%, while Mn containing SODs lose only 50% of their activity in the presence of 20 mM NaN 3 [14].…”

“…The preparation of SOD purified from M. arbo riphilus strain AZ exhibited a rather high specific activity (3970 U/mg) compared to other methanogens: the spe cific activity of SOD from M. bryantii constituted 2060 U/mg [16], the activity of SOD from M. barkeri strain Fusaro was 1500 U/mg [17], and SOD from M. thermoautotrophicum exhibited 855 U/mg [13]. The described SODs from methanogenic Archaea are also localized in the soluble cell fraction and stable under aer obic conditions.…”

“…Superoxide dismutases from M. bryantii [16] and M. thermoautotroph icum [13] are also homotetramers, but they are composed of the subunits of 26 and 24 kD, respectively. The molec ular weight of the SOD from M. barkeri strain Fusaro is 25 kD [17].…”

“…Investigation of SOD in these microorganisms extremely sensitive to the presence of oxygen was further continued. Now SOD has been isolated from M. bryantii [16], M. thermoau totrophicum [13], and M. barkeri strain Fusaro [17]. It was assumed that the SOD from M. bryantii is an evolutionary ancestor of the related Fe and Mn containing prokary otic SODs [16].…”