Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus.

Although cytosolic superoxide dismutases (SODs) are widely distributed among bacteria, only a small number of species contain a periplasmic SOD. One of these is Caulobacter crescentus, which has a copper-zinc SOD (CuZnSOD) in the periplasm and an iron SOD (FeSOD) in the cytosol. The function of periplasmic CuZnSOD was studied by characterizing a mutant of C. crescentus with an insertionally inactivated CuZnSOD gene. Wild-type and mutant strains showed identical tolerance to intracellular superoxide. However, in response to extracellular superoxide, the presence of periplasmic CuZnSOD increased survival by as much as 20-fold. This is the first demonstration that periplasmic SOD defends against external superoxide of environmental origin. This result has implications for those bacterial pathogens that contain a CuZnSOD. C. crescentus was shown to contain a single catalase/peroxidase which, like Escherichia coli KatG catalase/peroxidase, is present in both the periplasmic and cytoplasmic fractions. The growth stage dependence of C. crescentus catalase/peroxidase and SOD activity was studied. Although FeSOD activity was identical in exponential- and stationary-phase cultures, CuZnSOD was induced nearly 4-fold in stationary phase and the catalase/peroxidase was induced nearly 100-fold. Induction of antioxidant enzymes in the periplasm of C. crescentus appears to be an important attribute of the stationary-phase response and may be a useful tool for studying its regulation.     

Detection and quantification of superoxide formed within the periplasm of Escherichia coli

Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, including Escherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures of E. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability

Azotobacter vinelandii contains two superoxide dismutases (SODs), a cytoplasmic iron-containing enzyme (FeSOD), and a periplasmic copper/zinc-containing enzyme (CuZnSOD). In this study, the FeSOD was found to be constitutive, while the activity of CuZnSOD increased as the culture entered the stationary phase. Total SOD (units/mg protein) in stationary phase cells grown under nitrogen-fixing conditions was not significantly different from those grown under non-nitrogen-fixing conditions. The gene encoding FeSOD (sodB) was isolated from an A. vinelandii cosmid library. A 1-kb fragment containing the coding region and 400 base pairs of upstream sequence was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence had a high degree of homology with other bacterial FeSODs, particularly with P. aeruginosa. Attempts to construct a sodB mutant by recombination of a sodB::kan insertion mutation into the multicopy chromosome of A. vinelandii were unsuccessful even in the presence of SOD mimics or nutritional supplements. These results suggest that FeSOD may be essential for the growth and survival of A. vinelandii, and that the periplasmic CuZnSOD cannot replace the function of FeSOD.     

Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival.

Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

Rubrerythrin and rubredoxin oxidoreductase in Desulfovibrio vulgaris: a novel oxidative stress protection system

Evidence is presented for an alternative to the superoxide dismutase (SOD)-catalase oxidative stress defense system in Desulfovibrio vulgaris (strain Hildenborough). This alternative system consists of the nonheme iron proteins, rubrerythrin (Rbr) and rubredoxin oxidoreductase (Rbo), the product of the rbo gene (also called desulfoferrodoxin). A Deltarbo strain of D. vulgaris was found to be more sensitive to internal superoxide exposure than was the wild type. Unlike Rbo, expression of plasmid-borne Rbr failed to restore the aerobic growth of a SOD-deficient strain of Escherichia coli. Conversely, plasmid-borne expression of two different Rbrs from D. vulgaris increased the viability of a catalase-deficient strain of E. coli that had been exposed to hydrogen peroxide whereas Rbo actually decreased the viability. A previously undescribed D. vulgaris gene was found to encode a protein having 50% sequence identity to that of E. coli Fe-SOD. This gene also encoded an extended N-terminal sequence with high homologies to export signal peptides of periplasmic redox proteins. The SOD activity of D. vulgaris is not affected by the absence of Rbo and is concentrated in the periplasmic fraction of cell extracts. These results are consistent with a superoxide reductase rather than SOD activity of Rbo and with a peroxidase activity of Rbr. A joint role for Rbo and Rbr as a novel cytoplasmic oxidative stress protection system in D. vulgaris and other anaerobic microorganisms is proposed.     

The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.     

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.     

Production of superoxide dismutase in Streptococcus lactis by a combination of use of hyperbaric oxygen and fermentation with cross-flow filtration

The production conditions of superoxide dismutase (SOD) in the cells of Streptococcus lactis by using hyperbaric oxygen (O(2)) are described. The SOD activity of anaerobically grown cells was 5-6 U/mg protein. When the culture broth was pressurized by O(2) at 6 atm, the SOD activity was more than twice as high as that under anaerobic culture conditions. However, there is little or no significant increase in SOD activity by exogenous addition of catalase for detoxifying hydrogen peroxide accumulated in the broth and/or controlling the pH of the broth at 6.8 during the pressurization by O(2). The increase in SOD activity by hyperbaric O(2) was possible not only at the late-logarithmic growth phase but also at the initial time for the stationary growth phase. For improvement of SOD productivity, we tried a two-stage culture in which SOD activity in S. lactis cells was enhanced by pressurizing the culture broth using hyperbaric O(2) after achievement of a high-concentration cultivation in the anerobic fermentation system with a microfiltration module.     

Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria

Oxidative stress, through the production of reactive oxygen species, is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR and SoxR undergo conformation changes when oxidized in the presence of hydrogen peroxide and superoxide radicals, respectively, and subsequently control the expression of cognate genes. In contrast, the RpoS regulon is induced by an increase in RpoS levels. Current knowledge regarding the activation and function of these regulators and their dependent genes in E. coli during oxidative stress forms the scope of this review. Despite the enormous genomic diversity of bacteria, oxidative stress response regulators in E. coli are functionally conserved in a wide range of bacterial groups, possibly reflecting positive selection of these regulators. SoxRS and RpoS homologs are present and respond to oxidative stress in Proteobacteria, and OxyR homologs are present and function in H(2)O(2) resistance in a range of bacteria, from gammaproteobacteria to Actinobacteria. Bacteria have developed complex, adapted gene regulatory responses to oxidative stress, perhaps due to the prevalence of reactive oxygen species produced endogenously through metabolism or due to the necessity of aerotolerance mechanisms in anaerobic bacteria exposed to oxygen.     

Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and studies of KatA function

Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L. pneumophila and Escherichia coli and compared with prior studies of katB in this laboratory. katA is induced during exponential growth and is the predominant peroxidase in stationary phase. When katA is inactivated, L. pneumophila is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L. pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen.     

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.