Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.Heavy metals represent major environmental hazards to human health. In particular, cadmium is very toxic and probably carcinogenic at low concentrations. However, the biological effects of this metal and the mechanism of its toxicity are not yet clearly understood. It has been proposed that Cd 2ϩ ions might displace Zn 2ϩ and Fe 2ϩ in proteins (1), resulting in their inactivation and in the release of free iron, which might generate highly reactive hydroxyl radicals (OH ⅐ ) (2). In support of this hypothesis, a major effect of cadmium is oxidative stress (3), particularly lipid peroxidation (1). However, it is not known whether these effects are responsible for the extreme toxicity of the metal.Living organisms use several mechanisms to counter cadmium toxicity. In bacteria, efflux pumps are able to export toxic ions outside the cell (4). In higher eukaryotes, Cd 2ϩ is sequestered by metallothioneins through their high cysteine content (5). Cadmium can also be detoxified by chelation to GSH or to phytochelatin, a glutathione polymer of general structure (␥-Glu-Cys) n -Gly synthesized from GSH in plants and in the yeast Schizosaccharomyces pombe. Cd 2ϩ -phytochelatin and Cd 2ϩ ⅐ (GSH) 2 complexes are transported into the vacuole by ATPbinding cassette transporters (6 -8).Yap1p and Skn7p are yeast transcription factors that regulate the adaptive response to oxidative stress (9 -11). Strains lacking either transcription factor are sensitive to H 2 O 2 and are defective in the induction by H 2 O 2 of several enzymes with antioxidant properties (9). Yap1p is also important in cadmium tolerance because yap1-deleted strains are very sensitive to cadmium, and strains overexpressing YAP1 are hyper-resistant to this toxic metal (12). The contribution of Skn7p to the cadmium response is more complex, because skn7-deleted strains are hyper-resistant to cadmium (9)...

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.The toxic cellular effects of oxygen are a major factor in aging and mortality (3, 28). Oxygen toxicity is attributed to the activity of reactive oxygen species that attack proteins, lipids, and nucleic acids (15). Effects of oxygen have been extensively studied by use of bacterial models, principally with the facultatively respiring bacterium Escherichia coli (see references 7 and 13 for reviews). In this model, respiration itself is implicated as a source of oxidative damage in E. coli (8, 9, 18, 20, 27, and 36). It has been suggested that the shutdown of respiration in nutrient-limited conditions may reduce reactive oxygen species levels and thereby improve E. coli survival. Recent evidence further suggests that survival is favored by shifting cells to anaerobic conditions during entry into stationary phase (9).Current information on the effects of oxygen is mainly based on respiring organisms. As such, the question of what anaerobes do in the presence of oxidative stress has been explored little. It is presumed that these organisms cope with stress in much the same way as aerobes, except that their defense systems, which may include superoxide dismutases (SODs) and catalases, may be more limited. However, there has been no demonstration to date that responses of anaerobes to an oxidative environment are predictable from the behavior of respiring bacteria.The effects of oxygen have been examined with Lactococcus lactis, a gram-positive facultative anaerobe with a fermentative metabolism that ca...

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 hemeresponsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.

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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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.

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 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.

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.