Impact of aeration and heme-activated respiration on Lactococcus lactis gene expression: identification of a heme-responsive operon.

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O(2) and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.

Roles of thioredoxin reductase during the aerobic life of Lactococcus lactis.

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 C152. 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.

Respiration metabolism reduces oxidative and acid stress to improve long-term survival of Lactococcus lactis.

The 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.

Proteome analyses of heme-dependent respiration in Lactococcus lactis: involvement of the proteolytic system.

Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

Respiration capacity and consequences in Lactococcus lactis.

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.