Advanced Progress on Adaptive Stress Response of Oenococcus oeni

Zhao Wen-ying, and Kang Zhen-kui

1 College of Chemical Engineering and Environment, North University of China, Taiyuan 030051, Shanxi, China;

2 Tianjiaohong Biotechnology Company, Jiaocheng 030500, Shanxi, China

Introduction

Malolactic fermentation (MLF)follows alcoholic fermentation during the process of winemaking.For many years, MLF has been recognized as an indispensable step in the elaboration of most wines(Zhao et al., 2008). Such bacterium genera as Leuconostoc, Pediococcus, Lactobacillus have been reported to carry out MLF in wine (Alexandre et al., 2004).They have to cope with very harsh environmental conditions in wine, such as low pH, high alcohol content, high concentration of SO2, low temperature and low nutrient concentration. Oenococcus oeni is recognized as the most advantageous and tolerant bacterium during MLF (Nielsen et al., 1996). The bacterium was identified in the mid-1960s and reclassified as O. oeni on the basis of 16S rRNA sequence analysis (Dicks et al., 1995). The genomic analysis of O. oeni strains showed that this bacterium had a compact genome of approximately 1.8 Mb with a 38% GC content. And 1701 ORFs could be predicted from the sequence and 75% of them were functionally classif i ed (Mills et al., 2005).

Due to its particular ability to grow in a hostile medium, O. oeni has been regarded as a good model organism for the study of stress response in lactic acid bacteria (LAB). Till now, three main mechanisms for stress tolerance response of O. oeni during culturing process has been reported: metabolic energy and homeostasis mechanism, adaptive stress response of cell membrane and synthesis of stress proteins.

Metabolic Energy and Homeostasis Mechanism

O. oeni responds to changes in their immediate surroundings via metabolic reprogramming, which leads to a cellular state of the enhanced resistance.

Activation of membrane-bound H+-F0F1-ATPases

H+-F0F1-ATPases, coupled to the ATP hydrolysis,extrude protons out of the cells to the media which can generate proton gradient, and this proton gradient exerts an inwardly directed force on the protons as proton motive force (pmf). The influx of protons driven by pmf results in the release of energy can be used to drive metabolic energy requiring membranebound processes, this process is therefore called as'secondary metabolic energy generation' (Konings et al., 2006). Moreover, H+-ATPase also plays a key role in the acid tolerance of bacteria. A spontaneous O. oeni mutant lacking H+-F0F1-ATPases is very sensitive to acid environment. H+-ATPase activity is induced at low pH, not directly affected by malic acid metabolism, and regulation of H+-ATPase activity seems to occur at the level of transcription. However,stress compounds in wine such as SO2, fatty acids and copper can reduce ATPase activity, thereby they play an inhibit effect on cell growth and the MLF duration(Fortier et al., 2003). Therefore, H+-F0F1-ATPases is a good indicator of the physiological state of the O. oeni cells. Nevertheless, other cation transports,such as K+-translocating ATPase, can also participate in the pH homeostasis mechanism (Mills et al., 2005).

Malolactic fermentation

As cellular functions are inhibited at very acidic pH,the ATP synthesis by substrate-level phosphorylation would not proceed as eff i ciently (Drici-Cachon et al.,1996), the MLF becomes important part of regulatory mechanism of the ATP synthesis and pH homeostasis in O. oeni. The pathway of MLF includes the uptake of L-malate, its decarboxylation to L-lactic acid and CO2,and excretion of the end products (including a proton)(Drici-Cachon et al., 1996). As a proton is consumed in the decarboxylation reaction, the internal pH increase results in creation of a proton potential gradient across the membrane (ΔpH)and forms pmf across the cytoplasmic membrane (chemiosmotic mechanism),together with the Δψ, an electrical potential gradient,generated by the transport of monovalent malate, and such pmf is sufficient to drive the ATP synthesis via the membrane-bound F1F0-ATPase (Galland et al.,2003). Thereby, the MLF pathway results in not only the proton consumption during the decarboxylation of L-malate which participates in the regulation of pHin, but also the generation of a pmf sufficient to drive ATP synthesis via the membrane-bound F1F0-ATPase. Such a pmf generated by secondary transport processes is called as secondary metabolic energy generation. The secondary transport systems convert the (electro)chemical energy of a solute gradient into electrochemical energy of proton (Konings, 2006).

Other metabolic processes

Citrate metabolism in O. oeni can lead to the generation of pmf and participation in response to stress conditions (Olguín et al., 2009). It is proposed that decarboxylation of amino acid associate with ammonia production by O. oeni might fulf i ll a protective function against intracellular and extracellular acidification (Lonvaud-Funel et al., 2001). And it is reported that arginine and fructose trigger the expression of a subset of stress responsive genes that is independent of pH (Bourdineaud et al., 2006). This area merits further studies.

On the other hand, as the uptake of amino acids requires a major fraction of the available metabolic energy of the LAB, its energy cost can be reduced drastically by accumulating oligopeptides instead of the individual amino acids. The utilization of complex nitrogen sources like peptides or proteins has been demonstrated in O. oeni by the production of proteases and peptidases (Alexandre et al., 2004). Moreover,bacterial growth yield of O. oeni is higher in the presence of nitrogen from peptides than that from free amino acids (Remize et al., 2006). This mechanism might be related to adaptation to wine conditions,but little is still known about nitrogen metabolic signif i cance of O. oeni up to now.

Adaptive Stress Response of Cell Membrane

Membrane physical properties and fluidity need constant regulation, since they determined both the role of barrier and the enzymatic activities of permeases. Those permeases are closely related with generation of metabolic energy for growth of O. oeni in wine. Membrane fl uidity is an important characteristic of living microorganisms that are influenced by environment factors and active cellular control.

Adjustment of cell membrane composition

Since the plasma membrane is considered as the primary target for the deleterious effects of the environment, one of the most important adaptive responses to stress exposure is related to changes in membrane fatty acid (FA)composition which can lead to alterations in saturation degree, carbon chain length, branching position, cis/trans isomerisation.Conversion of unsaturated fatty acids (UFAs)into cyclopropanes plays an important role in O. oeni to modulate membrane chemico-physical properties (Li et al., 2009; Montanari et al., 2010). Another strategy is about of change of the ratio of phospholipids to proteins of membrane (Garbay et al., 1996).

Ethanol stress and membrane

It is agreed that ethanol could perturb membrane structure and function. Studies showed that membrane fluidity and permeability increases with increasing concentrations of ethanol (Graca Silveira et al., 2004).The increase of permeability of the membrane for protons or other ions might lead to a dissipation of the pmf, resulting in a less effective energy transduction.At the same time, an increased permeability of the cells might also affect the internal pH control of the cells or could result in the loss of enzyme activity.Therefore, the failure of MLF after direct inoculation of O. oeni in wine might be explained by the deleterious effects of ethanol in combination with low pH of the wine (Carrete et al., 2002).

However, those adapted cells grown in the presence of 8% (vol/vol)ethanol reduced membrane fluidity and increased its integrity compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of unsaturation of fatty acid and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol)ethanol (Chu-Ky et al.,2005). Additionally, ethanol has an effect on the cell growth of O. oeni. But ethanol-adapted cells are more eff i cient in degrading malic acid and generating ATP than control cells in wine (Graca Silveira et al.,2004), which should be related with the enhancement of membrane integrity. Therefore, appropriate ethanol addition to culture medium should play a part in enhancing O. oeni survival rate and malolactic activities in wine (Zhao et al., 2011).

Acid stress and membrane

In contrast, little work has been carried out to elucidate the physical effect of acidity on cytoplasmic membrane. In LAB, acid tolerance (AT)increases in at least two distinct physiological states. (i)During logarithmic growth, an adaptive response referred to as L-ATR can be induced by incubation at a non-lethal acidic pH; (ii)after entry in the stationary phase, AT increases as a result of the induction of a general stress response (Guchte et al., 2002).

It is proposed that the medium pH considerably modif i es the fatty acid distribution in Lo107 (an acidophilic strain)and Lo8413 (a moderately acidophilic strain), at pH2.9, and Lo107 has a remarkably high level of C19-cyclopropane fatty acids which might participate in stress adaptive processes (Drici-Cachon et al., 1996). Phenolic acids are also deemed to increase the cell membrane permeability in wine lactic acid bacteria. And different types of phenolic acids have different effects on membrane permeability that could be related to differences in their structure and lipophilic character (Campos et al., 2009).

Synthesis of Stress Proteins

Stress gene response plays a key role in the cell adaptation to various environmental conditions. To survive in hostile environment, cells could synthesize proteins including both chaperones and proteases to prevent accumulation of abnormal proteins. The purpose of study on stress response mechanisms of O. oeni is to fi nd the stress proteins which play a key role in protecting cells under stress conditions.

A small heat shock protein (smHSP)Lo18

The small HSPs (smHSP)are membrane-associated proteins localized within specif i c membrane domains and thereby used for correcting in part the lipid order and phase-state of the membranes (Horváth et al., 2012). 8-ku polypeptide named Lo18 is greatly induced in O. oeni after heat (42℃), acid (pH 3)or ethanolic (12% (v/v))shocks and also induces in stationary growth phase. Its corresponding gene(hsp18)encodes a polypeptide consisting of 148 amino acid residues with a calculated molecular mass of 16 938 ku, and Lo18 protein has a signif i cant identity with small heat shock proteins of the α-crystallin family (Jobin et al., 1997). In O. oeni, Lo18 can be induced by heat shock and membrane association of Lo18 depends on the temperature upshift. After heat shock at 46℃, Lo18 is associated with the membrane fraction to a greater extent. Additionally, ethanol or benzyl alcohol also can induce Lo18 expression. It is indicated that Lo18 could maintain cell integrity by direct action on membrane phospholipids under various stress conditions (Coucheney et al., 2005a).Based on the stress specif i c high expression of Lo18,this smHSP is identified as a good marker for stress tolerance of O. oeni. (Coucheney et al., 2005b; Olguín et al., 2010). The tested strain presents the highest malolactic activity on intact cells at pH 3.0 and a high level of Lo18 induction and shows a high growth rate and a high specific kinetic of malate consumption.And so the expression amounts of Lo18 can be used for the selection of effective strains intended for direct inoculation into wine. Studies showed that different amino acid residues in Lo18 are involved in the thermostabilization of proteins and in membrane fluidity regulation and some different residus are localized in the α-crystallin domain (Weidmann et al.,2010).

Syntheses of other stress proteins and adaptive stress gene response

In addition to a small heat shock protein Lo18, many other stress proteins are also found to be involved in the Oenococcal stress responses. These include ClpX(Olguín et al., 2010)which is preferentially expressed at the beginning of the exponential phase, and is heat inducible; TrxA (Jobin et al., 1999b), similar to known thioredoxins, whose expression increases signif i cantly in the presence of hydrogen peroxide in the medium or after heat shock; FtsH (Bourdineaud et al., 2003)whose expression increases at high temperature and under osmotic shock; ClpLP (Beltramo et al., 2004)whose higher levels of expression are detected in stressed cells compared to unstressed cells; OmrA(Bourdineaud et al., 2004)whose expression increases at high temperature or under osmotic shock; GroESL and DnaK which are universal chaperone complexes(Mills et al., 2005).

Different expression patterns are found during the growth of O. oeni. While clpX is more strongly expressed at the beginning of growth, hsp18 and clpL1 expression are highly induced upon entry into the stationary phase. The clpP and trxA transcript level is detected with a slight increase at the beginning of growth and at the stationary phase. In order to characterize O. oeni performances in wine, the transcriptional response of the genes related to MLF,citrate metabolism and stress response are compared among strains (Beltramo et al., 2006; Olguín et al.,2010). The results suggested that an improved basal level of some genes expression can confer upon the O. oeni cells a capacity for adaptation to stress.

Regulation of stress gene expression

Although many stress response genes have been characterized in O. oeni, it is necessary to understand the regulatory mechanisms controlling stress gene expression to enhance O. oeni viability under harsh wine conditions. Apart from the clpX gene, all of the tested molecular chaperone genes in O. oeni (e.g. ctsR,clpP, clpL2, hsp18, groES, and grpE)have a CtsR box target, but those stress genes present different induction patterns (Grandvalet et al., 2005). Genomic sequence analysis of O. oeni IOB 8413 and PSU-1 reveals that apart from CtsR, no alternative sigma factors and known genes encoded regulators of stress response, such as HrcA, could be identified in both genomes (Grandvalet et al., 2005; Mills et al., 2005).O. oeni is the fi rst example where dnaK and groESL are controlled by CtsR but not by HrcA. Under optimal growth conditions, CtsR protein would prevent the synthesis of unnecessary stress proteins until environmental changes (ethanol, acid, nutritional stresses, etc.)strongly induce transcription of stress genes, thus enhancing the adaptability of O. oeni to adverse environmental conditions. Further work should be focused on identification of members of the CtsR regulon and analysis of the effects of mutations in individual CtsR-dependent genes on stress adaptation in order to address whether and to what extent CtsR-dependent genes contribute to Oenicoccus survival in wine and resistant to freeze-drying.

Concluding Remarks

A better understanding of the mechanisms of stress resistance of O. oeni should obtain targets: (i)To develop more effective and reliable biochemical or gene tools to screen for highly tolerant strains and master the characterization of malolactic starters.Distinct strains of the same pieces can differ in their behavior when direct inoculation in wine and during drying and storage in the dried state, so it is necessary to make a choice of the inherently stress-tolerant strains as starter cultures. (ii)To rationalize the specif i c preparation procedure used during the production of commercial active dried wine starter cultures.The induction of adaptive responses of O. oeni can undoubtedly enhance its growth performance and robustness, so as to make MLF eff i cient and reliable.Moreover, the growth conditions are likely to play a role in survival subsequent to freeze-drying. Freezedrying techniques have commonly been used to guarantee long-term delivery of stable cultures in terms of viability and functional activity, but these techniques can produce undesirable side effects, such as denaturation of sensitive proteins and decreased viability. So further research is still required to understand the mechanisms of O. oeni stress response to freezing and freeze-drying, which may eventually lead to the development of starter cultures technology.(iii)Heterologous expression of Oenococcus useful genes could be used to confer noval behaviour on strains of biotechnological interest. In addition, O. oeni genome and transcriptome analyses will undoubtedly complement the proteome and genetic information of LAB available today.

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