The superoxide dismutase-encoding gene of the obligately anaerobic bacterium Bacteroides gingivalis

The gene (sod) encoding the superoxide dismutase (SOD) of the obligately anaerobic bacterium Bacteroides gingivalis was cloned. The amino acid (aa) sequence of the SOD, deduced from the nucleotide sequence of the sod gene, basically resembled that of known Fe-SODs. However, the aa sequence of the B. gingivalis SOD was found to be intermediate between those of Fe-SOD and Mn-SOD in a limited region around the putative second ligand, where major differences between the aa sequences of Fe-SOD and Mn-SOD are known to exist.     

The iron-containing superoxide dismutase of Ralstonia metallidurans CH34

The iron-containing superoxide dismutase (Fe-SOD) of Ralstonia metallidurans CH34 was purified and characterised as a homodimer of 2 x 21500 Da containing one iron atom per monomer and exhibiting all the characteristics of the prokaryotic Fe-SODs except for a higher isoelectric point. The protein was 2-fold overexpressed in the presence of selenite, zinc or paraquat. R. metallidurans CH34 was suggested to contain a gene encoding for a manganese-containing SOD located in the inducible chromate resistance operon. Whatever the culture conditions used in this study, including the presence of chromate, only a Fe-SOD, genetically distinct from the putative Mn-SOD, was detected. This Fe-SOD seems to be the only active superoxide dismutase expressed in R. metallidurans CH34.     

Ni Uptake and Limitation in Marine Synechococcus Strains

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO₃⁻ and can grow only slowly on NH₄⁺ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.     

Isolation and in silico analysis of Fe-superoxide dismutase in the cyanobacterium Nostoc commune

Cyanobacteria are known to endure various stress conditions due to the inbuilt potential for oxidative stress alleviation owing to the presence of an array of antioxidants. The present study shows that Antarctic cyanobacterium Nostoc commune possesses two antioxidative enzymes viz., superoxide dismutase (SOD) and catalase that jointly cope with environmental stresses prevailing at its natural habitat. Native-PAGE analysis illustrates the presence of a single prominent isoform recognized as Fe-SOD and three distinct isoforms of catalase. The protein sequence of Fe-SOD in N. commune retrieved from NCBI protein sequence database was used for in silico analysis. 3D structure of N. commune was predicted by comparative modeling using MODELLER 9v11. Further, this model was validated for its quality by Ramachandran plot, ERRAT, Verify 3D and ProSA-web which revealed good structure quality of the model. Multiple sequence alignment showed high conservation in N and C-terminal domain regions along with all metal binding positions in Fe-SOD which were also found to be highly conserved in all 28 cyanobacterial species under study, including N. commune. In silico prediction of isoelectric point and molecular weight of Fe-SOD was found to be 5.48 and 22,342.98 Da respectively. The phylogenetic tree revealed that among 28 cyanobacterial species, Fe-SOD in N. commune was the closest evolutionary homolog of Fe-SOD in Nostoc punctiforme as evident by strong bootstrap value. Thus, N. commune may serve as a good biological model for studies related to survival of life under extreme conditions prevailing at the Antarctic region. Moreover cyanobacteria may be exploited for biochemical and biotechnological applications of enzymatic antioxidants.     

Copper/Zinc superoxide dismutase: How likely is gene transfer from ponyfish toPhotobacterium leiognathi?

Summary The proposed transfer of the gene for Cu/Zn superoxide dismutase from the ponyfish to its symbiotic bacteriumPhotobacterium leiognathi has been evaluated by an extensive analysis of all available Cu/Zn superoxide dismutase sequences. By the use of four different computer programs, phylogenetic trees were constructed from the sequences of the superoxide dismutases of human, ox, pig, horse, swordfish, fruit fly, yeast, andNeurospora crassa to find out whether superoxide dismutase sequences can reliably be used for the reconstruction of genealogical relationships. All programs arrived at the same most parsimonious tree (one requiring 232 amino acid replacements), the topology of which conformed to established opinions about the phylogenetic relations among these eukaryotes, except that it placed humans closer to the artiodactyls ox and pig than it placed horses. This could be corrected at the cost of two amino acid replacements. The sequence ofP. leiognathi superoxide dismutase was then connected at all possible positions to the corrected eukaryotic tree. It was slighly more parsimonious to link the bacterial sequence to the root of the tree than to the fish branch: The former required 316 (or 317) amino acid replacements, versus 319 for the latter. This relative lack of discrimination between such distinct alternative topologies may be a general complication in the comparison of prokaryotic and eukaryotic proteins: Bacterial cytochrome c sequences also were found to be connected as parsimoniously to the root of the eukaryotic tree as to any terminal or ancestral branch. It was calculated that the rate of evolution of the bacterial superoxide dismutase gene, if transfer occurred 30 million years (Myr) ago, must have amounted to 487 amino acid replacements per 100 residues per 100 Myr. This is more than 5 times the highest rate observed in any protein (that found for fibrinopeptides), and even much higher than the maximum rate of protein evolution that can be deduced from the neutral mutation rate of unconstrained DNA. Also, no significant evidence that shared derived amino acid replacements are present in swordfish andP. leiognathi superoxide dismutase, as might be expected had gene transfer occurred, was found. On the basis of the available data it seems more reasonable to ascribe the isolated occurrence of Cu/Zn superoxide dismutase inP. leiognathi (as well as inCaulobacter crescentus) to irregular patterns of gene expression and inactivation in the course of divergent evolution than to undocumented processes of gene transfer from eukaryotes to prokaryotes.     

Phylogenetic distribution of superoxide dismutase supports an endosymbiotic origin for chloroplasts and mitochondria

Three isozymes of superoxide dismutase (SOD) have been identified and characterized. The iron and manganese isozymes (Fe-SOD and Mn-SOD, respectively) show extensive primary sequence and structural homology, suggesting a common evolutionary ancestor. In contrast, the copper/zinc isozyme (CuZn-SOD) shows no homology with Fe-SOD or Mn-SOD, suggesting an independent origin for this enzyme. The three isozymes are unequally distributed throughout the biological kingdoms and are located in different subcellular compartments. Obligate anaerobes and aerobic diazotrophs contain Fe-SOD exclusively. Facultative aerobes contain either Fe-SOD or Mn-SOD or both. Fe-SOD is found in the cytosol of cyanobacteria while the thylakoid membranes of these organisms contain a tightly bound Mn-SOD. Similarly, most eukaryotic algae contain Fe-SOD in the chloroplast stroma and Mn-SOD bound to the thylakoids. Most higher plants contain a cytosol-specific and a chloroplast-specific CuZn-SOD, and possibly a thylakoid-bound Mn-SOD as well. Plants also contain Mn-SOD in their mitochondria. Likewise, animals and fungi contain a cytosolic CuZn-SOD and a mitochondrial Mn-SOD. The Mn-SOD found in the mitochondria of eukaryotes shows strong homology to the prokaryotic form of the enzyme. Taken together, the phylogenetic distribution and subcellular localization of the SOD isozymes provide strong support for the hypothesis that the chloroplasts and mitochondria of eukaryotic cells arose from prokaryotic endosymbionts.     

Mutants of cyanobacterium Synechocystis sp. PCC6803 with insertion of the sodB gene encoding Fe-superoxide dismutase

The sodB gene encoding the only superoxide dismutase (Fe-SOD) in cells of the cyanobacterium Synechocystis sp. PCC6803 was inactivated with gentamycin resistance aacC1 marker insertions located in the direct or inverted order toward the sodB gene. The corresponding delta sodB12 and delta sodB22 mutants are characterized by the complete absence of superoxide dismutase activity and the loss of viability upon standard photoautotrophic cultivation. Mutant cells can grow under conditions of a decreased illumination intensity and upon addition of NaHCO3 with catalase or bovine serum albumin in the growth medium. The delta sodB22 mutant is auxotrophic for leucine due to the polar effect of insertion into the sodB gene on the downstream leuB gene controlling leucine biosynthesis. These data suggest that Fe-SOD is very important for providing tolerance of Synechocystis cells to oxidative stress and that sodB and leuB genes are organized into a single operon.     

Genetic exchange between kingdoms.

Bacterial conjugation with two evolutionarily divergent yeasts has been observed in the laboratory. Whether such trans-kingdom conjugation events, other than the well known Agrobacterium-plant cell interaction, actually occur in nature is not known. However, a few putative events have recently been uncovered by gene (or protein) sequence analysis, suggesting that horizontal gene transfer between phylogenetic kingdoms may be a real phenomenon.     

Primary structure from amino acid and cDNA sequences of two Cu,Zn superoxide dismutase variants from Xenopus laevis

A mixture of two different amino acid sequences was discovered in Cu,Zn superoxide dismutase purified from the amphibian Xenopus laevis. No N-terminal post-translational modification was found. The high number of substitutions in the sequence suggested that protein heterogeneity was a product of gene duplication. This was confirmed by isolation of two different cDNA clones. Nucleotide sequence analysis allowed the primary structure of the two peptide chains to be unambiguously assigned. The observed changes (19 in 150 residues) are distributed along the peptide chain to give similar protein net charges although substitutions of the same polarity and/or charge were the exception rather than the rule. The degree of diversity between the two Xenopus variants is comparable to that between mammalian sequences and shows that the putative increase of the rate of mutation for Cu,Zn superoxide dismutase at later evolution stages (Y. M. Lee et al., 1985, Arch. Biochem. Biophys. 241, 577-589; G. J. Steffens et al., 1986, Biol. Chem. Hoppe-Seyler 367, 1017-1024) is observed in amphibians. This is the first time complete sequences for Cu,Zn superoxide dismutase variants from the same organism have been found to be products of divergent genes and not simply allelic mutations.     

Evolutionary analysis of putative olfactory receptor genes of medaka fish, Oryzias latipes

To obtain an understanding of the origin, diversification and genomic organization of vertebrate olfactory receptor genes, we have newly cloned and characterized putative olfactory receptor genes, mfOR1, mfOR2, mfOR3 and mfOR4 from the genomic DNA of medaka fish (Oryzias latipes). The four sequences contained features commonly seen in known olfactory receptor genes and were phylogenetically most closely related to those of catfish and zebrafish.Among them, mfOR1 and mfOR2 showed the highest amino acid (aa) similarity (93%) and defined a novel olfactory receptor gene family that is most divergent among all other vertebrate olfactory receptor genes. Southern hybridization analyses suggested that mfOR1 and mfOR2 are tightly linked to each other (within 24kb), although suitable marker genes were not available to locate their linkage group. Unlike observation in catfish olfactory receptor sequences, nucleotide (nt) substitutions between the two sequences did not show any evidence of positive natural selection. mfOR3 and mfOR4, however, showed a much lower aa similarity (26%) and were both mapped to a region in the medaka linkage group XX.After including these medaka fish sequences, olfactory receptors of terrestrial and aquatic animals formed significantly different clusters in the phylogenetic tree. Although the member genes of each olfactory receptor gene subfamily are less in fish than that in mammals, fish seem to have maintained more diverse olfactory receptor gene families. Our finding of a novel olfactory receptor gene family in medaka fish may provide a step towards understanding the emergence of the olfactory receptor gene in vertebrates.     

Purification of iron superoxide dismutase from the cyanobacterium Anabaena cylindrica Lemm. and localization of the enzyme in heterocysts by immunogold labeling

Iron superoxide dismutase (Fe-SOD; EC 1.15.1.1) was isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica Lemm. Polyacrylamide gel electrophoresis separated the purified protein into three closely running, enzymatically active bands. The molecular weight of the enzyme was estimated by gel filtration to be about 40 kDa. Polyclonal antibodies were produced by immunization of rabbits with the isolated enzyme, and were purified on a column of protein A-Sepharose. The Fe-SOD antibody reacted with the purified Fe-SOD and also specifically recognized the protein in extracts of A. cylindrica. In the extracts, anti-Fe-SOD did not cross-react with Mn-SOD, an enzyme which belongs to an SOD class displaying high homology of primary and three-dimensional structure with respect to Fe-SOD. Iron superoxide dismutase was localized in heterocysts by immunogold labeling and transmission electron microscopy. These results are the first in-situ evidence for the presence of SOD in the cells specialized for nitrogenase activity.Abbreviations ELISA enzyme-linked immunosorbent assay - SDS sodium dodecyl sulfate - SOD superoxide dismutase - PAGE polyacrylamide gel electrophoresis - pI isoelectric point This work was supported by a C.N.R. grant. We are grateful to Dr. A. De Martino for technical assistance.     

Modification of the protein expression pattern induced in the nitrogen-fixing actinomycete Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host Alnus glutinosa and cloning of the sodF gene

Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to detect proteins induced in Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host, Alnus glutinosa. The 5 most prominent proteins were purified from 2-D gels and characterized by N-terminal sequencing. All of these proteins had a high percentage of similarity with known stress proteins. One protein match was the Fe superoxide dismutase (Fe-SOD), another was a tellurite resistance protein (Ter), the third was a bacterioferritin comigratory protein (Bcp); and two matches, differing only by their isoelectric point, were the same small heat shock protein (Hsp), a major immune reactive protein found in mycobacteria. This suggests that the symbiotic microorganism Frankia, first responds with a normal stress response to toxic root products of its symbiotic host plant. To confirm its identity, the gene corresponding to the Fe-SOD protein, sodF was isolated from a genomic library by a PCR-approach and sequenced. It is the first stress response gene characterized in Frankia.     

Changes in the activity of superoxide dismutase isoforms in the course of low-temperature adaptation in potato plants of wild type and transformed with Δ12-acyl-lipid desaturase gene

We investigated the changes in the total activity of superoxide dismutase (SOD) and the role of its isoforms in hardening potato (Solanum tuberosum L., cv. Desnitsa) plants of wild type and transformed with desA gene of Δ12-acyl-lipid desaturase from Synechocystis sp. PCC 6803. Hydroponically grown 8-week-old plants were exposed for six days to hardening temperature of 5°C. Before chilling, the total SOD activity in the transformed plants was somewhat greater than in the control plants. By the first day of hardening, SOD activity in both potato genotypes rose almost 1.5 times; however, the absolute value of SOD activity was considerably greater in the transformed plants. Subsequently, the total SOD activity in both genotypes decreased and by the end of the 6th day, it almost returned to the initial level. Electrophoretic and inhibitor analyses of potato plants revealed three types of SOD with one isoform of Mn-SOD, four isoforms of Fe-SOD, and two isoforms of Cu/Zn-SOD. In both genotypes, Fe-SOD3 manifested the greatest activity before chilling and in the course of hardening. Such changes in SOD activity corresponded to the rate of generation of superoxide anion radical and elevation of the content of products of peroxide oxidation of lipids (POL). Our data suggest that in the course of hardening of cold-resistant potato plants, the total SOD activity changed mostly due to Fe-SOD3 and to some extent as a result of elevated Cu/Zn-SOD2 activity, which was particularly evident at the beginning of hardening and more pronounced in the transformed plants. We assume that such temporal pattern is related to a greater rate of superoxide anion generation in the transformed plants as compared with control plants.     

Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism. 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes. In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species. In each species additional, divergent 4CL genes were found. Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform. Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily. According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species.     

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.     

Patterns of ribosomal RNA evolution in salamanders

Sequence comparisons are presented for four segments of the large subunit of ribosomal RNA, including divergent domains D7a and D7b, portions of the large divergent domains D2, D3, and D8, and evolutionarily conservative sequences flanking divergent domains. These results resolve phylogenetic relationships among exemplars of seven families of salamanders and the three amphibian orders. Phylogenetic analysis confirms the prediction that divergent domains feature the highest relative rates of base substitution and length variation within the ribosome, but the divergent domains evolve more slowly than nuclear noncoding DNA and the silent sites of structural genes. Base substitutions demonstrate approximately twice as many transitions as transversions and an uneven distribution among sites within the divergent domains but no apparent bias in base composition. Length mutations are primarily small insertions and deletions, with deletions predominating. The divergent domains appear to be a good source of phylogenetic information for evolutionary events occurring approximately 100-200 million years ago.