pBFK, a new thermosensitive shuttle vector for Streptococcus pyogenes gene deletion by homologous recombination.

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a major human pathogen for which genetic manipulation remains an ongoing challenge. We created a new temperature-sensitive plasmid pBFK expressing spectinomycin resistance adapted to homologous recombination procedure to perform a complete gene deletion in GAS. Herein the mutagenesis strategy with pBFK was performed in a highly virulent GAS emm3 genotype.

Respiration capacity of the fermenting bacterium Lactococcus lactis and its positive effects on growth and survival.

Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe Lactococcus lactis, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to L. lactis if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of L. lactis to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The cydA gene, encoding cytochrome d oxidase, is required for respiration and plays a direct role in oxygen utilization. cydA expression is induced late in growth under respiration conditions. (ii) The hemZ gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in L. lactis and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.

Flavodoxin mutants of Escherichia coli K-12.

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.

Activation of SoxR by overproduction of desulfoferrodoxin: multiple ways to induce the soxRS regulon.

The soxRS response, which protects cells against superoxide toxicity, is triggered by the oxidation of SoxR, a transcription factor. Superoxide excess and NADPH depletion induce the regulon. Unexpectedly, we found that the overproduction of desulfoferrodoxin, a superoxide reductase from sulfate-reducing bacteria, also induced this response. We suggest that desulfoferrodoxin interferes with the reducing pathway that keeps SoxR in its inactive form.

Regulation of the soxRS oxidative stress regulon. Reversible oxidation of the Fe-S centers of SoxR in vivo.

SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable [2Fe-2S] centers that are presumed to serve as redox sensors. In vitro transcription experiments previously demonstrated that only the oxidized form is active. Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells. Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements. In uninduced cells, 90% of the SoxR was in the reduced form. Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers. Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state. The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters.

SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form.

SoxR protein is known to function both as a sensor and as a transcriptional activator for a superoxide response regulon in Escherichia coli. The activity of SoxR was tested by its ability to enable the transcription of its target gene, soxS, in vitro. The activity of the oxidized form was lost when its [2Fe-2S] clusters were reduced by dithionite under anaerobic conditions, and it was rapidly restored by autooxidation. This result is consistent with the hypothesis that induction of the regulon is effected by the univalent oxidation of the Fe-S centers of SoxR. In vivo, this oxidation may be caused by an alteration of the redox balance of electron chain intermediates that normally maintains soxR in an inactive, reduced state. Oxidized SoxR was about twice as effective as reduced SoxR in protecting the soxS operator from endonucleolytic cleavage. However, this difference could not account for a greater than 50-fold difference in their activities and therefore could not support a model in which oxidation activates SoxR by enabling it to bind to DNA. NADPH, ferredoxin, flavodoxin, or ferredoxin (flavodoxin):NADP+ reductase could not reduce SoxR directly in vitro at a measurable rate. The midpoint potential for SoxR was measured at -283 mV.

The irreversible inactivation of ribonucleotide reductase from Escherichia coli by superoxide radicals.

The expression of superoxide dismutase in all aerobic living organisms supports the concept that superoxide radicals are toxic species. However, because of the limited chemical reactivity of superoxide, the mechanisms of this toxicity are still uncertain. Protein R2, the small component of ribonucleotide reductase, a key enzyme for DNA synthesis, is shown here to be irreversibly inactivated during incubation with an enzymatic generator of superoxide radicals, at neutral pH. During inactivation the essential tyrosyl radical of protein R2 is irreversibly destroyed. Full protection is afforded by superoxide dismutase. It is proposed that coupling between superoxide radicals and the radical protein R2 generates oxidized forms of tyrosine, tyrosine peroxide and 3,4-dihydroxyphenylalanine.

The NADPH: sulfite reductase of Escherichia coli is a paraquat reductase.

The NADPH:sulfite reductase of Escherichia coli is a soluble enzyme that has a subunit structure alpha 8 beta 4, where the alpha subunit is a flavoprotein and the beta subunit is a metalloprotein. Overexpression of the holoenzyme in E. coli has greatly simplified the purification of this enzyme. Under aerobic conditions, recombinant sulfite reductase catalyzes the reduction of 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat) by NADPH, with Km values for paraquat and NADPH of approximately 70 microM and 80 microM, respectively. Since pure flavoprotein alpha subunit, encoded by the cysJ gene, has similar catalytic activities, it is suggested that paraquat receives electrons directly from the alpha subunit. A mutant strain lacking an active cysJ gene is resistant to paraquat. The NADPH:ferredoxin reductase of E. coli is also a paraquat reductase but with much higher Km values for paraquat and lower enzyme activities. These results suggest that the sulfite reductase is a major paraquat reductase in E. coli and is responsible for the toxic activation of the drug.

The NAD(P)H:flavin oxidoreductase from Escherichia coli as a source of superoxide radicals.

The NAD(P)H:flavin oxidoreductase (encoded by the fre gene) of Escherichia coli is a soluble enzyme which, under aerobic conditions and together with NAD(P)H and flavins, generates superoxide radicals selectively. This was demonstrated from spin trapping experiments and from the ability of the flavin reductase to achieve a superoxide dismutase (SOD)-sensitive reduction of cytochrome c. The participation of the flavin reductase to O2-. generation in E. coli cells has been studied. Superoxide production in dialyzed cytosolic fraction of SOD-deficient E. coli was stimulated by the addition of flavins. There was no stimulation in soluble extracts of flavin reductase-deficient strains. Moreover, using fusions of sodA promoter to lacZ, we showed that sodA transcription was diminished in flavin reductase-deficient E. coli and that the induction of MnSOD by flavin reductase was SoxRS-independent. These results suggest that the flavin reductase might: (i) in vivo, be an important cytosolic site of O2-. generation; (ii) in vitro, serve as a simple, efficient, and selective O2-. generator.