Oxidative stress in Lactococcus lactis

INTRODUCTION

Lactic acid bacteria (LAB) comprise a group of Gram-positive and facultative anaerobic microorganisms able to convert carbohydrates into lactic acid via homo- or heterofermentative metabolism (Stackebrandt and Teuber, 1988; Thompson, 1988). Due to this latter feature, some food-grade LAB species (Lactobacillus sp., Leuconostoc sp., Pediococcus sp., Streptococcus sp., and Lactococcus sp.) are widely used in the production of fermented food (vegetables, milk and meat), where these bacteria are responsible for both preservation and sensory characteristics, such as color, flavor and texture. Moreover, probiotic properties are attributed to some species of lactobacilli (Seegers, 2002).

Lactococcus lactis is a mesophilic- (optimal growth temperature around 30°C) and microaerophilic-fermenting LAB (Duwat et al., 2000) with a relatively small genome (2.5 Mbp; Bolotin et al., 2001). This bacteria is commonly found in nature, on plant and animal surfaces and is widely used in the dairy industry for the production of fermented products, like cheese and buttermilk. Since over 10⁷ tons of cheeses are annually produced worldwide, L. lactis is of great economic importance (Henriksen et al., 1999).

Until the late 1970s, L. lactis ssp. lactis and L. lactis ssp. cremoris were empirically used by the dairy industry (Davidson et al., 1996; Henriksen et al., 1999). Thereafter, impelled by the advent of recombinant DNA technology, our knowledge about many biological aspects (e.g., microbiology, physiology, biochemistry, and mainly genetics) of these bacteria, and of other LAB, has greatly improved. Since then, the use of genetic tools has enabled the construction of many genetically modified strains that exhibit better controlled and/or improved growth and acidification, bacteriophage resistance and improved proteolytic properties (de Vos, 1999). Today, L. lactis is the most extensively characterized LAB (Bolotin et al., 2001), and it has been used to produce heterologous proteins of high biotechnological and medical interest, such as enzymes and antigens; it also has potential as a live vaccine (Langella and Le Loir, 1999).

However, despite all the available data, the loss of some bacterial phenotypes, affecting viability, reproducibility, organoleptic, and/or fermentative capabilities, remains difficult to circumvent. In most cases, these problems are caused by the exposure of LAB to a range of stressful environmental conditions in both natural and/or industrial habitats. Industrial processing exposes L. lactis to multiple stress conditions, such as low and high temperature, low pH, high osmotic pressure, nutrient starvation, and oxidation.

During the last two decades, several research groups have intensively studied stress responses in L. lactis. We describe general and specific aspects of oxidative stress in L. lactis and current insights related to the construction and isolation of both recombinant and non-recombinant L. lactis oxidative stress-resistant strains.

OXIDATIVE STRESS

Bacterial stress can be defined as a physiological perturbation, caused by environmental modifications (physical, chemical and/or nutritional) that can have many consequences for the bacteria, such as retarded growth and cell death (Farr and Kogoma, 1991; Fridovich, 1998; Duwat, 1999). Oxidative stress can cause several types of damage to the bacterial cell, including: i) metabolic pathway disruptions, ii) spontaneous mutations, and iii) bacteriostatic and bactericidal effects (Berlett and Stadtman, 1997; Fridovich, 1998).

Oxygen by itself is unable to cause any damage to the cell; however, during the cellular processes (metabolic pathway), O₂ is partially reduced to water, leading to the formation of reactive O₂ species, which are the superoxide anion radical (O₂^-), the hydroxyl radical (OH^·), and hydrogen peroxide (H₂O₂). These intermediates have a high oxidizing potential and thus are responsible for cellular oxygen toxicity (Farr and Kogoma, 1991; Fridovich, 1998; Storz and Imlay, 1999).

OXIDATIVE STRESS IN LACTOCOCCUS LACTIS

Described as a microaerophilic-fermenting LAB, L. lactis can, under certain conditions, tolerate and even use O₂ (Condon, 1987; Duwat et al., 2001). Nevertheless, O₂ consumption results in an altered redox state, and consequently greater NADH oxidase activity. Sugar fermentation is shifted towards mixed fermentation (Figure 1); hence, O₂ participates in the oxidoreduction steps of NADH to NAD⁺. Through the action of NADH oxidases, H₂O₂ is formed (Table 1; Condon, 1987). The O₂^- can be generated through the following reaction: NADH + 2O₂ ® NAD⁺ + H⁺ + 2O₂^-; which occurs because the flavin group of NADH oxidase carries out single-electron transfer, as well as the transfer of two or four electrons (Thomas and Pera, 1983; Imlay and Fridovich, 1991). The OH^· can be formed during the Fenton reaction (H₂O₂ + Fe²⁺ + H⁺ ® OH^· + H₂O + Fe³⁺) (Duwat et al., 1995) or by spontaneous reactions (O₂^- + H₂O₂ ® OH^- + OH^· + O₂) (Condon, 1987).

Oxidative stress damages at the molecular level

At the molecular level, O₂^-, OH^· and H₂O₂ can react with cellular targets, such as proteins and nucleic acids. O₂^- has a moderate oxidizing potential and can attack compounds such as polyphenols, ascorbate and catecholamines (Farr and Kogoma, 1991; Fridovich, 1998). H₂O₂ can directly oxidize protein cysteinyl residues, thus inactivating enzymes (Storz and Imlay, 1999). It can also react with cations, such as Fe²⁺ and Cu⁺, and give rise to OH^·, through the Fenton reaction (Farr and Kogoma, 1991; Duwat et al., 1995; Fridovich, 1998). OH^· is a strong oxidant agent that can attack most organic compounds and can cause strand breaks and a wide spectrum of base modifications in DNA (Czapski, 1984; Farr and Kogoma, 1991; Fridovich, 1998). Another type of damage is the peroxidation of membrane lipids and membrane protein alterations, affecting cell permeability and osmoregulation (Harley et al., 1978; Kong and Davison, 1980).

Oxidative stress damages at the metabolic level

Many effects of O₂ are also observed at the metabolic level. Under anaerobic conditions, L. lactis has a fermentative metabolism that enables the transformation of various types of carbohydrates into lactic acid (Figure 1) (Holt et al., 1994; Lopez de Felipe et al., 1997, 1998). In this case, 2 NADH molecules, generated from the oxidation of glyceraldehyde-3-phosphate, are reoxidized to favor the reduction of pyruvate to lactic acid by the action of lactate dehydrogenase (LDH). However, the NADH/NAD⁺ ratio plays a determinant role in the shift control from homolactic acid to mixed-acid fermentation in L. lactis (Garrigues et al., 1997). Under aerobic conditions, increased expression and activities of NADH oxidase and NADH peroxidase (Table 1) compete with LDH for NADH molecules (Murphy and Condon, 1984). Consequently, lactic acid production is reduced and glycolytic flux is shifted towards the production of acetate, ethanol, acetoin, diacetyl and CO₂ (mixed-fermentation) by the action of pyruvate dehydrogenase (PDH), pyruvate-formate lyase and a-acetolactate synthase (Figure 1). These changes also lead to the formation of H₂O₂, which causes a reduction of the growth rate of L. lactis, and even its death. Concentrations of around 0.2 mM H₂O₂ inhibit the growth of this bacterium by 50% and concentrations >1.15 mM H₂O₂ can compromise cell viability (Anders et al., 1970; Duwat et al., 1999). Although NADH peroxidase (Table 1) contributes to cellular H₂O₂ detoxification, its activity is generally low (10 to 30 times lower than that of NADH oxidase) and endogenous or exogenous H₂O₂ overcomes the cell’s capability to deal with this oxygen species (Anders et al., 1970; Condon, 1987).

OXIDATIVE STRESS RESISTANCE MECHANISMS IN LACTOCOCCUS LACTIS

How does L. lactis overcome damage caused by active oxygen species? Experimental data and genomic analyses indicate that L. lactis, similar to Escherichia coli and Bacillus subtilis, is equipped with general and specific stress response mechanisms (Duwat et al., 1999). Several genes (see below) that participate in these mechanisms have been identified, and the respective encoded proteins have been shown to contribute to oxidative stress resistance. Moreover, induction of some of these genes is growth phase-dependent (exponential or stationary), and their products confer multi-stress resistance (Farr and Kogoma, 1991; Kjelleberg et al., 1993; Walker, 1996; Duwat et al., 2000).

NADH oxidase/NADH peroxidase

The most common oxidative stress resistance mechanism found in L. lactis, as well as in other LAB, is performed by a coupled NADH oxidase/NADH peroxidase system (Table 1; Condon, 1987). Initially, intracellular O₂ can be eliminated by NADH oxidase activity, which utilizes O₂ to oxidize NADH into NAD⁺, thereby giving rise to H₂O₂. Furthermore, NADH peroxidase reduces H₂O₂, generating H₂O. However, NADH peroxidase activity is low, and considering that L. lactis does not possess any catalase activity, cellular H₂O₂ detoxification is inefficient. It was recently proposed that at high glycolytic fluxes, L. lactis ATCC19435 (L. lactis subsp. lactis, lacking both NADH peroxidase and superoxide dismutase (Sod) activities) metabolically produced pyruvate reacts nonenzymatically with H₂O₂ to form H₂O and acetate (van Niel et al., 2002), thus providing the cell with an alternative mechanism of protection against H₂O₂.

Superoxide dismutase

A second common resistance mechanism is provided by the action of a Sod that removes O₂^- radicals (Table 1; Condon, 1987). In prokaryotes, the classification of these enzymes depends on the metal cofactor content (Cu-Zn, Fe or Mn). Most LAB have Sod activity. A unique manganese-containing Sod (MnSod) has been identified in L. lactis, during an analysis of acid stress-induced protein expression (Sanders et al., 1995). The growth rate of sodA mutants, under anaerobic conditions, is similar to that of the wild type strain, whereas under aerobic conditions the growth rate of this mutant strain is reduced (Sanders et al., 1995). Moreover, under anaerobic conditions, L. lactis sodA has a low initial expression, which is shifted to a high gene expression level under aerated conditions. An alternative mechanism to eliminate O₂^- radicals, which could compensate the low Sod activity, can be provided by high levels of intracellular glutathione (Fahey et al., 1978; Wiederholk and Steele, 1993).

RecA

Another oxidative stress resistance mechanism is supported by RecA activity. This protein is commonly found in bacteria, such as E. coli, where it plays a key role in the SOS response and homologous recombination (Miller and Kokjohn, 1990; Walker, 1996). Duwat et al. (1995) constructed a L. lactis recA mutant strain, and showed that RecA is involved in resistance to oxidative and thermal stresses. They observed that during exponential and stationary growth phases, recA mutants are highly sensitive to aeration, resulting in a reduction in growth rate and viability. They also postulated that in the absence of RecA activity, the OH^· radical, generated during the Fenton reaction, is responsible for damage to DNA. To test this hypothesis, an iron chelator was added to aerated cultures of L. lactis recA mutants; bacterial doubling time was restored close to that of anaerobic conditions, leading to the observation that i) O₂ radicals are responsible for oxygen toxicity and that ii) RecA plays an essential role in the repair of DNA damage caused by these compounds. They also studied the function of RecA under thermal stress. Under both oxidative and thermal stress, recA mutants have an elevated expression of HflB protein, which transcriptionally down-regulates heat-shock proteins, such as DnaK, GroEL and GrpE, and thus keeping these heat-shock proteins from being induced under elevated temperature conditions (above 37°C). Moreover, recA of L. lactis is co-transcribed with another DNA repair gene, formamidopyrimidine DNA glycosylase (fpg) (Duwat et al., 1992, 1995). In conclusion, activation of recA by oxidative stress can confer cross-protection against thermal stress. A single stimulus can activate various stress resistance mechanisms, conferring protection against various types of stress in L. lactis.

Cytochrome d oxidase (cydA)

Another way to cope with oxidative stress would be efficient O₂ uptake and utilization, as in the case of bacteria able to respire. Despite its fermentative metabolism classification, there is evidence that L. lactis can undergo respiration in aerated culture media in the presence of heme (an iron-containing porphyrin molecule that forms the oxygen-binding portion of molecules such as hemoglobin) (Sijpesteijn, 1970). More recently, based on L. lactis IL1403 genome data, it was found that this bacterium possesses some of the genes needed for respiration, such as cydA, which encodes for cytochrome d oxidase (Bolotin et al., 1999). During respiration, CydA directly participates in the electron transfer chain, where O₂ is the terminal electron acceptor, thus generating ATP. To test these possibilities and the respiration capacity of L. lactis, a cydA mutant strain was constructed (Duwat et al., 2001). Under aerated conditions, in the presence of heme, it was observed that i) L. lactis cydA expression is induced during the stationary growth phase; ii) this bacterium has an extended growth period and improved long-term survival, and iii) its metabolism is shifted from fermentation to respiration, as demonstrated by reduced lactic acid production and elevated acetate, acetoin and diacetyl formation. Based on this evidence, it is clear that L. lactis is able to respire, and probably this ability enables it to overcome oxidative stress injuries.

Adaptation

Another resistance mechanism in L. lactis is adaptation. It is known that in response to environmental changes, bacteria can trigger adaptive mechanisms that enable cells to tolerate harsh conditions (Condon, 1987; Woojin et al., 1999, 2002). Previously, it was observed that lactococci exposed to sublethal concentrations of H₂O₂ became able to grow in the presence of lethal concentrations (³1.15 mM) of H₂O₂ (Anders et al., 1970; Condon, 1987). More recently, it has been shown that adaptation to a specific stress, such as UV irradiation, starvation or high temperature, can lead to multi-stress resistance acquisition, probably due to the induction of genes that have functions in common in the resistance mechanisms against various types of stress (Duwat et al., 1995; Hartke et al., 1996, 1997; Duwat, 1999).

CONTROL OF OXIDATIVE STRESS RESPONSES IN LACTOCOCCUS LACTIS

Oxidative stress resistance mechanisms in L. lactis seem to be overlap, and thus cells under one specific stress condition can trigger different stress responses. Some studies have provided us with interesting clues concerning the control of oxidative stress responses in L. lactis (Duwat et al., 1999, 2000; Rallu et al., 2000; Scott et al., 2000; O’Connell-Motherway et al., 2000).

Sigma factors

Control at the transcriptional level, by sigma (s) factors would be an efficient way to modulate stress responses. However, whereas E. coli and B. subtilis have stress specific s-factors (Lange and Hengge-Aronis, 1994; Herman et al., 1995; Hecker et al., 1996), L. lactis only has three s-factors: RpoD, ComX and SigX (Araya et al., 1993; Bolotin et al., 1999, 2001). The vegetative s-factor, RpoD (Araya et al., 1993), allows the binding of RNA polymerase and the initiation of transcription at the so-called “vegetative promoters”, whereas ComX function is likely to induce competence genes (Bolotin et al., 1999, 2001). Although SigX, an extracytoplasmic function (Helmann, 2002) RNA polymerase s-factor in B. subtilis has been associated with heat stress resistance (Huang et al., 1997), no function for the lactococcal SigX has been proposed.

Metabolite flux sensors

The cAMP receptor protein, the fumarate and nitrate reduction regulator (FNR) and the FNR-like proteins (Flp) are members of a superfamily of structurally related transcriptional factors that could be involved in the control of stress responses (Green et al., 2001). Commonly found in a wide range of bacteria, these proteins possess structural properties that provide cells with versatile sensory (transducing environmental and metabolic signals) and DNA/RNA-regulatory mechanisms (DNA recognition motifs and RNA polymerase interaction) that trigger general and specific responses to particular physiological conditions, such as stress. In both E. coli (Jordan et al., 1997) and Lactobacillus casei (Gostick et al., 1998), FNR and Flp play a key role in oxidative stress by interaction with an oxygen-labile [4Fe-4S] cluster. L. lactis possesses two Flp encoding genes, flpA and flpB, located in two paralogous operons (orfX-orfY-flp), where the proximal genes appear to encode potential metal ion transport systems (Gostick et al., 1999). To further characterize the L. lactis Flp proteins, single and double mutants of flpA/B were constructed (Scott et al., 2000). No phenotypic effects were observed between the single mutant and the wild-type strain when submitted to oxidative stress conditions (H₂O₂). However, under the same conditions the double mutant was more affected, as evidenced by i) an increased lag phase, ii) an altered polypeptide profile, and iii) a lower intracellular zinc [Zn(II)] content. This indicates that at least one of the L. lactis Flp must be functional in order to be able to cope with oxidative stress. Based on these observations, it was suggested that FlpA/B may be involved in the control of Zn(II) uptake and stress resistance mechanisms. It was also postulated that L. lactis uses Zn(II) to protect protein thiol groups from oxidation and that intracellular levels of this compound may be functioning as a “metabolite flux sensor” (MFS), sensing and controlling general stress responses.

An example of how MFS can control stress responses was given by the construction of L. lactis recA-derived mutant strains. The L. lactis recA strain is sensitive to both oxidative and thermal stresses. Therefore, in order to identify other mechanisms related to this sensitivity, recA-derived strains were isolated by random insertional mutagenesis (Duwat et al., 1999). Most mutated genes were found to encode proteins involved in metabolic pathways for the biosynthesis of guanosine phosphate (GP) (deoB, guaA and tktA) or in phosphate uptake (pstS and pstB). These mutations conferred resistance to multiple stresses and it was postulated that low intracellular levels of GP and/or phosphate are functioning as MFS, controlling general stress responses. This hypothesis was later confirmed by Duwat et al., 2000, who showed that depletion of GP in growth media of wild-type strains was able to induce general stress resistance; otherwise, the addition of guanine (over 15 µg/ml) to the growth medium from a guaA/recA-derivative mutant strain resulted in complete loss of the stress-resistant phenotype. The involvement of phosphate pools was also demonstrated with the pstB/recA-derivative mutant strain (Duwat et al., 2000). Similar results were obtained during an independent selection of acid-resistant mutants of L. lactis, which were also found to be resistant to oxidative stress caused by H₂O₂ (Rallu et al., 2000).

Two-component regulatory systems

Control of stress responses could also be achieved by cellular systems that sense and transmit environmental signals into the cell, thereby modulating gene expression and physiological changes. These systems have been described in both prokaryotic and eukaryotic organisms, and are known as “two-component regulatory systems” (Chang and Stewart, 1998). Basically, these systems are composed of a membrane-anchored sensor protein, usually a histidine protein kinase, and an intracellular response regulator. In L. lactis, six of these systems have been identified (O’Connell-Motherway et al., 1997) and three of these (systems B, D and F) are involved in specific susceptibility to acid, osmotic and oxidative stress (O’Connell-Motherway et al., 2000). In the latter (system F), the L. lactis mutant strain, obtained by insertional mutagenesis, has greater H₂O₂ sensitivity than the wild-type strain (O’Connell-Motherway et al., 2000). At least in this case, it seems that the control of stress responses are stress specific.

In summary, many questions remain and it is possible that control of stress resistance mechanisms in L. lactis operates through pleiotropic effects or even through multiple regulatory mechanisms, some of which may be unique to this bacterium.

PRESENT AND FUTURE DIRECTIONS

Despite all the efforts that have been made to identify and improve our knowledge about oxidative stress resistance and its control mechanisms in L. lactis, a number of questions remain unanswered. Many aspects of general and specific stress in this bacterium differ from model microorganisms, such as E. coli and B. subtilis. Thus, it becomes an important challenge to isolate and construct new stress resistant strains that can help to answer these questions.

Most studies concerning stress in L. lactis concern the production of stress-resistant mutant strains; depending on the method employed to obtain these strains, they can be classified as: i) “non-genetically modified mutant strains” (non-GMM) or ii) “genetically modified mutant strains” (GMM). In the former, stress resistant strains are acquired through screening in selective media, or are isolated from industrial sources or from their natural habitat. Nevertheless, these types of mutants are not very informative, due to difficulties in identifying possible genes and/or metabolic pathways involved in stress resistance. Alternatively, such genes or pathways can be characterized by the identification and analysis of stress-induced proteins in two-dimensional polyacrylamide gel electrophoresis (Giard et al., 2002). GMM strains can be constructed by site-directed mutagenesis (homologous recombination utilizing non-replicative plasmids; Leloup et al., 1997; O’Connell-Motherway et al., 2000) or by random insertional mutagenesis (transposition elements; Schafer et al., 1991; Maguin et al., 1992). One of the most powerful tools that has been developed is the thermo-sensitive plasmid pG+host:ISS1 (Maguin et al., 1996), which permits insertional gene inactivation and subsequent determination of its involvement in bacterial stress.

In addition to basic knowledge of the L. lactis stress response, obtained through the construction of stress resistant strains, new information about oxidative and other types of stress has opened new possibilities for their practical application. Today, similar findings have been applied to the metabolic engineering of sugars and other molecules (Lopez de Felipe et al., 1997, 1998; Hugenholtz et al., 2002; Koebmann et al., 2002; Cocaign-Bousquet et al., 2002), in the development of new expression systems (Sanders et al., 1998; Madsen et al., 1999), and to develop new anti-oxidant strains of L. lactis that could become useful in medicine and industry.

ACKNOWLEDGMENTS

We are grateful to Elisabeth Azevedo, Herbert P. Schweizer, and Santuza Teixeira for discussion and text corrections. We are also grateful to Alexandra Gruss for scientific discussion and to our team members and colleagues (Brazilian and French) for their friendly support. We have a special thought for Patrick Duwat (deceased January 5, 2000) who initiated the work on L. lactis oxidative stress in the URLGA-INRA. Research supported by grants from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil) and COFECUB (Comité Français d’Evaluation de la Coopération Universitaire avec le Brésil).

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