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Numerous strategies allowing bacteria to detect and respond to oxidative conditions depend on the cell redox state. Here we examined the ability of Lactococcus lactis to survive aerobically in the presence of the reducing agent dithiothreitol (D-TT), which would be expected to modify the cell redox state and disable the oxidative stress response. DTTF inhibited L. lactis growth at 37 [degrees]C in aerobic conditions, but not in anaerobiosis. Mutants selected as DTT resistant all mapped to the pstFEDCBA locus, encoding a high-affinity phosphate transporter. Transcription of pstFEDCBA and a downstream putative regulator of stress response, phoU, was deregulated in a pstA strain, but amounts of major oxidative stress proteins were unchanged. As metals participate in oxygen radical formation, we compared metal sensitivity of wild-type and pstA strains. The pstA mutant showed approximately 100-fold increased resistance to copper and zinc. Furthermore, copper or zinc addition exacerbated the sensitivity of a wild-type L. lactis strain to DTT. Inactivation of pstA conferred a more general resistance to oxidative stress, alleviating the oxygen- and thermo-sensitivity of a clpP mutant. This study establishes a role for the pst locus in metal homeostasis, suggesting that pst inactivation lowers intracellular reactivity of copper and zinc, which would limit bacterial sensitivity to oxygen.

Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems. Some gram-positive bacteria, including Lactococcus lactis, do not produce glutathione, and the thioredoxin system is presumed to be essential. We constructed an L. lactis trxB1 mutant. The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT). Unexpectedly, the trxB1 mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system. Aerobic growth of the trxB1 mutant did not require glutathione, also ruling out the need for this redox maintenance system. Proteomic analyses showed that known oxidative stress defense proteins are induced in the trxB1 mutant. Two additional effects of trxB1 were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB). We determined that the two GapB isoforms in L. lactis differed by the oxidation state of catalytic-site cysteine [C.sub.152]. Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the trxB1 mutant, possibly because of proteolysis of oxidized GapB. This study showed that thioredoxin reductase is not essential in L. lactis and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels. The existence of a novel redox function that compensates for trxB1 deficiency is suggested.

Research demonstrates that facultative anaerobe lactococcus lactis, although adapted to fermentative metabolism, is capable of adapting to respiration during aerated growth. Data indicate that heme plays a major role in this adaptation physiology.

Research demonstrates that the insertion mutation in the gene encoding flavodoxin I enzyme renders Escherechia coli cells lethal under both aerobic and anaerobic conditions. Data further reveal that flavodoxin II gene is unaffected by the mutation and the two genes constitute a superoxide response regulon.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host. Author summary: The Rex family transcription repressors serve critical roles in control of cellular metabolism. Pathogenic microorganisms often use metabolic cues to regulate their metabolism and virulence functions. Here, we structurally and functionally characterized Rex in the facultative aerobic Gram-positive coccus Streptococcus agalactiae, also known as group B streptococcus (GBS). GBS is a leading cause of meningitis in newborn babies and infants. Rex interacts with both the oxidized and reduced form of the cofactor nicotinamide adenine dinucleotide (NAD). The reduced form (NADH) abrogates DNA binding whereas the oxidized form (NAD+) enhance binding of Rex to DNA. We show that the cellular concentration of NAD+ is the main effector of Rex binding to DNA. A drop in the concentration of NAD+ is triggered when cells do not find a specific sugar such as glucose in the environment. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also its own expression as well as virulence gene expression. Together our results identify GBS Rex as an important transcription regulator critical for GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to efficiently utilize nutrient resources provided by the host. [ABSTRACT FROM AUTHOR]

Results show that the the gene encoding the transcription factor SoxR protects bacteria against superoxide toxicity. Data further show that excess superoxide and NADPH depletion induce soxRS regulon.