SummaryThe impact of oxygen on a cell is strongly dependent on its metabolic state: survival in oxygen of free-living Lactococcus lactis , best known as a fermenting, acidifying bacterium, is generally poor. In contrast, if haem is present, L. lactis uses oxygen to switch from fermentation to respiration metabolism late in growth, resulting in spectacularly improved long-term survival. Oxygen is thus beneficial rather than detrimental for survival if haem is provided. We examined the effects of respiration on oxygen toxicity by comparing integrity of stationary phase cells after aerated growth without and with added haem. Aeration (no haem) growth caused considerable cellular protein and chromosomal DNA damage, increased spontaneous mutation frequencies and poor survival of recA mutants. These phenotypes were greatly diminished when haem was present, indicating that respiration constitutes an efficient barrier against oxidative stress. Using the green fluorescent protein as an indicator of intracellular oxidation state, we showed that aeration growth provokes significantly greater oxidation than respiration growth. Iron was identified as a main contributor to mortality and DNA degradation in aeration growth. Our results point to two features of respiration growth in lactococci that are responsible for maintaining low oxidative damage: One is a more reduced intracellular state, which is because of efficient oxygen elimination by respiration. The other is a higher pH resulting from the shift from acid-forming fermentation to respiration metabolism. These results have relevance to other bacteria whose respiration capacity depends on addition of exogenous haem.

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SummaryQuinones are essential components of the respiration chain that shuttle electrons between oxidoreductases. We characterized the quinones synthesized by Lactococcus lactis, a fermenting bacterium that activates aerobic respiration when a haem source is pro-

We recently reported that the well-studied fermenting bacterium Lactococcus lactis could grow via a respirative metabolism in the presence of oxygen when a heme source is present. Respiration induces profound changes in L. lactis metabolism, and improvement of oxygen tolerance and long-term survival. Compared to usual fermentation conditions, biomass is approximately doubled by the end of growth, acid production is reduced, and large amounts of normally minor end products accumulate. Lactococci grown via respiration survive markedly better after long-term storage than fermenting cells. We suggest that growth and survival of lactococci are optimal under respiration-permissive conditions, and not under fermentation conditions as previously supposed. Our results reveal the uniqueness of the L. lactis respiration model. The well-studied 'aerobic' bacteria express multiple terminal cytochrome oxidases, which assure respiration all throughout growth; they also synthesize their own heme. In contrast, the L. lactis cydAB genes encode a single cytochrome oxidase (bd), and heme must be provided. Furthermore, cydAB genes mediate respiration only late in growth. Thus, lactococci exit the lag phase via fermentation even if heme is present, and start respiration in late exponential phase. Our results suggest that the spectacularly improved survival is in part due to reduced intracellular oxidation during respiration. We predict that lactococcal relatives like the Enterococci, and some Lactobacilli, which have reported respiration potential, will display improved survival under respiration-permissive conditions.

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.In bacteria, double-stranded DNA breaks (DSB) are frequent events that may be provoked, for example, by pauses in the replication fork (36, 43). Such genomic disruptions are lethal in the absence of DNA repair. In Escherichia coli DSB repair requires the activity of a large enzyme complex, known as RecBCD, that has ATP-dependent helicase and exonuclease activities (see reference 32 for a review). The enzyme degrades both strands, starting from the DNA break until it reaches an octanucleotide sequence, known as Chi, that attenuates degradation and stimulates recombination (44,46). The enzyme exhibits helicase activity and residual exonuclease activity with an altered polarity after Chi (4, 16); the remaining activity provides a single-stranded DNA substrate for recombination enzymes to mediate repair.Organization of the three-subunit exonuclease/helicase (exo/hel) RecBCD. Structure-functional studies of RecBCD have revealed some of the roles of each subunit. RecB seems to possess two key activities of the enzyme. The N-terminal 929 amino acids (out of 1,180 total) have confirmed ATPase and helicase activities (13,54