Detection and Quantification of Superoxide Formed within the Periplasm ofEscherichia coli

Abstract

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.

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

“…S. enteritidis and S. choleraesuis), sodCI is embedded in genomic regions containing sequences derived from different phages or phage remnants (16,24), suggesting a selective pressure to maintain sodCI in Salmonella. In contrast, SodCII is expressed in aerated cultures and carried by non-pathogenic as well as pathogenic bacteria, supporting the hypothesis that this enzyme protects bacteria from endogenous superoxide produced during aerobic metabolism, possibly due to electron leakage from the respiratory chain (26). Selective expression of SodCII in stationary phase corresponds to conditions in which bacterial cells are most susceptible to oxidative damage (70).…”

“…It is likely that periplasmic Cu,Zn-SOD plays additional roles that are not directly related to bacterial interaction with animal cells, as the enzyme is also present in non-pathogenic microorganisms. An interesting possibility is that Cu,Zn-SOD scavenges superoxide released in the periplasmic space during aerobic growth (26).…”

RosR (Cg1324), a Hydrogen Peroxide-sensitive MarR-type Transcriptional Regulator of Corynebacterium glutamicum*

“…Interestingly, a C. glutamicum ⌬cg1322 mutant showed an increased sensitivity to hydrogen peroxide, indicating that the Cg1322 protein is involved in the response to oxidative stress. The mechanism of this involvement is as yet unknown, but it may be related to the observation that the adventitious autoxidation of reduced menaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli (49). Superoxide is detoxified by superoxide dismutase, which leads to the formation of hydrogen peroxide.…”

The OxyR Regulon in Nontypeable Haemophilus influenzae

“…For example, in the electron transport chain, electrons can be inadvertently transferred from redox-active proteins to oxygen, producing reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ). The effects of ROS on cells can then be exacerbated by the presence of free iron which, via the Fenton reaction, can react with H 2 O 2 to produce more highly reactive hydroxyl radicals (34,40,52). Within a host, this problem is compounded by the release of extracellular ROS by phagocytes, which again, can have deleterious effects on invading pathogens (13).…”