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.     

Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants.

A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.     

Periplasmic superoxide dismutase from Desulfovibrio desulfuricans 1388 is an iron protein

It is shown that the genome of the sulfate-reducing bacterium Desulfovibrio desulfuricans 1388 contains a superoxide dismutase (SOD) gene (sod). The gene encodes an export signal peptide characteristic for periplasmic redox proteins. The amino acid sequence showed high homology with iron-containing SODs from other bacteria. Electrophoretically pure SOD was isolated from the periplasmic fraction of bacterial cells by FPLC chromatography. Like other Fe-SODs, D. desulfuricans 1388 superoxide dismutase is inhibited by H2O2 and azide, but not by cyanide.     

甜菜夜蛾核多角体病毒sod基因的克隆及原核表达

甜菜夜蛾核型多角体病毒中国株(Spotoptera exigua MNPV—Z)超氧化物歧化酶基因(sod)业已被克隆及在大肠杆菌中进行了表达,证明了SeMNPV—Z的sod基因产物确有SOD活性,其活力单位约为291．19U／mL培养液。DNA测序结果表明SeMNPV-Z的sod基因编码151个氨基酸,与人的sodl基因的核苷酸的同源性为50％,与LdNPV、HaSNPV、HcNPV、AcNPV和BmNPV的sod基因的同源性分别为64％、63％、63％、65％、63％。     

美国白蛾核型多角体病毒超氧化物歧化酶基因的序列分析和表达

根据测序结果 ,HcNPVsod的核苷酸序列与BmNPVsod的完全一致 ,与AcNPVsod的核苷酸序列相比 ,同源性达到 97 2 % ;推测HcNPVsod编码 1 51个氨基酸 ,与BmNPVsod的完全一致 ,与AcNPVsod编码的氨基酸相比 ,有三个氨基酸的差别。按基酸序列分析表明 ,HcNPVSOD蛋白中含有对SOD结构和活性必需的氨基酸残基 ,在HcNPVsod中均是保守的。SOD活性测定表明酶活为 1 47 0 9U/mL菌液     

哈维氏弧菌铁超氧化物歧化酶基因表达及免疫作用研究

哈维氏弧菌(Vibrio harveyi)是鱼虾等海水动物的重要病原菌。超氧化物歧化酶(Superoxide dismutase)通过催化超氧阴离子自由基(O2-)形成O2 和H2O2, 以保持细胞自由基产生和清除之间的平衡, 在病原菌适应环境及细菌致病性方面发挥重要作用。用PCR 从哈维氏弧菌基因组扩增得到600 bp 的目的片段, 序列分析表明与弧菌Fe-SOD 的序列相似性为91%—99%。将目的基因片段克隆到原核表达载体进行表达。SDS-PAGE电泳分析显示纯化的蛋白为单一条带, 分子量为27 kD。具有典型的Fe-SOD 吸收光谱, 对氯仿-乙醇和H2O2敏感, 表明纯化的蛋白属于Fe-SOD。用邻苯三酚自氧化法测得酶的最适pH 7, 最适温度20℃。该酶在pH6—8 的范围内稳定, 当温度超过40℃时酶的活力迅速丧失。纯化的重组蛋白免疫大菱鲆, 4 周后用致病性哈维氏弧菌进行人工感染试验, 对大菱鲆的免疫保护率为80.00%。Western blot 可以检测到免疫大菱鲆的血清中的特异抗体。       

Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence

We studied superoxide dismutases (SODs) in the encapsulated yeast Cryptococcus neoformans (Cn) variety gattii to analyse the role of mitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 was cloned from a Cn cosmid library, sod2 mutant and sod2 + SOD2 reconstituted strains were constructed by homologous recombination, and two sod1sod2 double mutants were constructed by replacing SOD2 in the sod1 mutant with the sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids, with 36-66% identity with other fungal SOD2ps. SOD2 deletion rendered Cn highly growth-defective at 37 degrees C in 19-20% oxygen (normal air), and this defect was reversed by limiting oxygen to 1.3% as well in the presence of antioxidant, ascorbic acid. The sod2 mutant accumulated significantly more reactive oxygen species (ROS) at 37 degrees C as well at 30 degrees C in the presence of antimycin A, suggesting that SOD2p is the primary defence of Cn against the superoxide anion (O(2) (.-)) in the mitochondria. The sod2 was also highly susceptible to redox-cycling agents, high salt and nutrient limitations. The sod2 mutant was avirulent in intranasally infected mice and markedly attenuated in its virulence in intravenously infected mice. The virulence defect of sod2 mutant appeared related to its growth defects in high oxygen environment, but not resulting from increased sensitivity to oxidative killing by phagocytes. The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2 double mutants showed a marked reduction in the activities of other known Cn virulence factors; and they were more susceptible to PMN killing than was the sod2 single mutant. Previously, we reported that the attenuation of sod1 mutant in mice was resulting from enhanced susceptibility to phagocyte killing, combined with a reduction in the activities of a number of virulence factors. Thus, SOD1p and SOD2p play distinct roles in the biology and virulence of Cn var. gattii via independent modes of action.     

节杆菌A．pascens DMDC12中SOD基因的克隆和序列分析

根据基因库中细菌SOD的基因保守序列和Arthrobacter pascens DMDC12的N-末端氨基酸序列设计引物,采用PCR技术,以A.pascens DMDC12基因组为模板,克隆了A.pascens DMDC12中Mn-SOD的567 bp的基因片段;再结合该基因片段和A.pascens DMDC12中SOD的N-末端氨基酸序列的有关信息,设计包含该sod完整ORF区域的引物,成功扩增了A.pascens DMDC12中Mn-SOD的基因序列,新sod序列(sodAP)已提交Gen-Bank(收录号DQ779150)。利用生物学软件对该序列进行分析表明,该sodAP序列的ORF区域全长651 bp,编码216个氨基酸;该序列和Arthrobacter sp.FB24的SOD基因序列同源性为86%。     

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.     

Cloning and expression of superoxide dismutase from Mycobacterium bovis BCG

We have previously purified the superoxide dismutase (SOD) of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and there is no signal peptide necessary for protein exportation [S.K. Kang, Y.J. Jung, C.H. Kim, C.Y. Song, Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG, Clin. Diagn. Lab. Immunol. 5 (1998) 784-789]. In the present study, SOD gene of M. bovis BCG was cloned and expressed in Escherichia coli, and its complete nucleotide sequence and deduced amino acid composition were determined. The open reading frame from the GTG initiation codon was 621 base pair (bp) in length for the SOD structural gene. The ribosomal-binding sequences (GGAAGG) were 6-12 bp upstream from the initiation codon. The amino acid sequence, deduced from the nucleotide sequence, revealed that the SOD consists of 207 amino acids residues with a molecular weight of 22.8 kDa. The N-terminal amino acid sequence predicted from the nucleotide sequence showed that the structural gene of the SOD is not preceded by leader sequences. There were no cysteine residues in the deduced amino acid composition, indicating that the SOD does not consist of disulfide bonds. Analyses of both nucleotide and amino acid sequences of the SOD showed significant similarity to other pathogenic mycobacterial SODs. Furthermore, the results of fractionation and two-dimensional electrophoresis showed that SOD is also associated with cell membrane, suggesting that there might be a specific mechanism for exportation of SOD in M. bovis BCG as well as other pathogenic mycobacteria. Overexpressed SOD in E. coli was purified from the inclusion bodies, and the histidine tag was removed from the protein using enterokinase. Enzyme activity was then determined by gel staining analysis.     

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.     

Unusual evolution of a superoxide dismutase-like gene from the extremely halophilic archaebacterium Halobacterium cutirubrum.

The archaebacterium Halobacterium cutirubrum contains a single detectable, Mn-containing superoxide dismutase, which is encoded by the sod gene (B. P. May and P. P. Dennis, J. Biol. Chem. 264:12253-12258, 1989). The genome of H. cutirubrum also contains a closely related sod-like gene (slg) of unknown function that has a pattern of expression different from that of sod. The four amino acid residues that bind the Mn atom are conserved, but the flanking regions of the two genes are unrelated. Although the genes have 87% nucleotide sequence identity, the proteins they encode have only 83% amino acid sequence identity. Mutations occur randomly at the first, second, and third codon positions, and transversions outnumber transitions. Most of the mutational differences between the two genes are confined to two limited regions; other regions totally lack differences. These two gene sequences are apparently in the initial stage of divergent evolution. Presumably, this divergence is being driven by strong selection at the molecular level for either acquisition of new functions or partition and refinement of ancestral functions in one or both of the respective gene products.     

Rapid viability loss on exposure to air in a superoxide dismutase-deficient mutant of Porphyromonas gingivalis.

Porphyromonas gingivalis, an obligate anaerobe, exhibits a relatively high degree of aerotolerance and possesses superoxide dismutase (SOD) which is induced by exposure to air. To clarify roles for SOD in this organism, the gene encoding SOD (sod) on the P. gingivalis chromosome was disrupted in a gene-directed way by use of a suicide plasmid containing a mutated sod. A sod mutant thus obtained showed no SOD activity in crude extracts and exhibited a rapid viability loss immediately after exposure to air, whereas the wild-type parent showed no decrease in viability for at least 5 h under aerobic conditions. These results clearly indicate that SOD is essential for aerotolerance in P. gingivalis.     

陆地棉叶绿体铜锌超氧化物歧化酶基因的克隆与表达

以陆地棉‘CRI36＇的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1 043 bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%～74%之间。基因的表达谱分析显示：棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实：该基因主要在苗期表达,以后表达逐渐减少。用pET-21a（＋）构建了原核表达载体,在大肠杆菌BL21（DE3）的表达结果显示：表达后得到一个29.0 kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。