Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni

We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C₂ to C₁₈. EstB28 exhibited greatest specificity for C₂ to C₄ pNP-linked substrates.The quality of fermented foods and beverages is affected in part by their composition of aroma compounds. In winemaking, the malolactic fermentation is used to deacidify wine and is typically carried out by Lactobacillus spp., Pediococcus spp., and particularly Oenococcus oeni (10, 7). Numerous reports clearly show that outside of this core function, lactic acid bacteria (LAB) can also bring about significant changes of sensorial importance (2, 5, 12, 32, 40, 49, 55). Such studies typically examined the action of active cultures or whole cells; however, the promise of LAB as a source of purified enzymes for use as additives in winemaking has recently been highlighted (43).Oenococcus oeni is acidophilic and indigenous to wine and similar environments. While the genome of the commercial PSU-1 strain has been sequenced and analyzed (44), there is limited information on the genes or their potential contribution to food and beverage aroma. The only such genes which have been cloned and partially characterized are alsS and alsD (24), which are thought to be responsible for the production of diacetyl, the principle compound conferring “buttery” aroma and flavor in wine (reviewed in reference 4). Analogous characterization of other flavor-related genes and enzymes not only may have practical implications for processes using LAB but also may be of fundamental interest.As a group, esters are a quantitatively significant constituent of beverages such as wine (total of >100 mg·liter⁻¹) (15). Included in this group are the C₄ to C₁₀ ethyl esters of organic acids, ethyl esters of straight-chain fatty acids (and branched-chain fatty acids to a lesser degree), and acetates of higher alcohols which are largely, if not exclusively, responsible for the fruity aroma of wine (13, 14). Some volatile esters are frequently found in fermented beverages in only trace amounts, often below threshold concentrations (3, 21, 25, 29, 50). However, they are extremely important for the flavor profile of these products, with different esters often having a synergistic effect to collectively affect aroma when their individual threshold concentrations are not exceeded. The fact that most esters are present in wine at concentrations around the threshold value implies that minor concentration changes might have a dramatic effect on the wine''s flavor (3, 21, 25, 29, 50). For this reason, an understanding of the hydrolysis and synthesis of esters in winemaking and how these may be manipulated is essential.A large amount of esters is formed during the primary fermentation by yeast; after this, LAB can contribute by increasing and decreasing the ester concentration (2, 5, 12, 40, 49, 55). Ester hydrolysis and synthesis can be catalyzed by esterases (6, 35, 38, 54). These enzymes commonly contain a catalytic triad composed of Ser, His, and Asp/Glu residues and a nucleophilic elbow structural motif (GXSXG), which contains the active-site serine residue (1, 31, 36, 48). They also contain an oxyanion hole, of which two residues donate their backbone amide protons to stabilize the substrate in the transition state. The oxyanion hole residues (in bold) have been divided into two groups termed GX and GGGX, with the glycine and a hydrophobic residue (X) being highly conserved (48).While extensive research has been carried out on the enzymes responsible for ester formation by wine strains of Saccharomyces cerevisiae (22, 23, 45, 51), esterase activity for wine-related LAB is not well documented. Most characterization of esterases in LAB has focused on dairy isolates (9, 16-18, 20). Parallel work in a wine context is limited despite general acceptance of the importance of esters in wine. Until recently, most evidence that wine LAB possess esterase activity came from wine volatile profiling studies which investigated the changes in concentration of individual esters during malolactic fermentation (12, 40, 55). Such changes in ester concentration were strain specific and had the potential to greatly affect the final aroma of wine.Our survey of the esterase activities of whole LAB cells found variations within species and even greater variation between the genera (42), with O. oeni showing greatest activity toward the p-nitrophenyl (pNP)-linked substrates tested. More recently (41), the esterase activities of whole O. oeni, lactobacillus, and pediococcus cells was determined under conditions with some relevance to wine. At least partial resistance to the harsh conditions used was observed, thereby demonstrating a necessary requirement of any enzyme intended for application in analogous environments. To more completely characterize esterases of LAB, the enzymes and their structural genes must be fully investigated. This study represents an effort to dissect the complex array of ester synthesis and hydrolysis activities in whole cells by cloning, heterologous expression, partial purification, and biochemical characterization of a single esterase from O. oeni. With a view to applying such an esterase under conditions found in wine and perhaps other industrial settings, enzyme function under the harsh physicochemical conditions frequently encountered in wine was examined.     

The ftsH Gene of the Wine Bacterium Oenococcus oeni Is Involved in Protection against Environmental Stress

The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.     

Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.     

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis. Received: 8 February 1996 / Accepted: 11 March 1996     

Growth and Arginine Metabolism of the Wine Lactic Acid Bacteria Lactobacillus buchneri and Oenococcus oeni at Different pH Values and Arginine Concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

The groESL Operon of the Halophilic Lactic Acid Bacterium Tetragenococcus halophila

The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45°C), and 4-fold by high NaCl (3-4 M). The Bacillus subtilis σ^A-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.     

一种新型反义RNA技术:RNA错折叠

反义RNA技术的发现对现代医疗技术的发展有着重要的作用,常规的反义RNA技术是设计出与靶RNA互补的寡核苷酸,通过它与靶RNA结合来干扰转录或翻译过程,使蛋白质不能被表达出来。目前,一项新的技术——寡核苷酸导向的RNA错折叠将反义RNA技术进一步简化,从另一个角度丰富了反义RNA技术。     

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5-5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 x 10(9) cfu sponge(-)(1) in at least four subsequent batch fermentations.     

Mechanical Stress Is a Pro-Inflammatory Stimulus in the Gut: In Vitro,In Vivo and Ex Vivo Evidence

Aims

Inflammatory infiltrates and pro-inflammatory mediators are found increased in obstructive and functional bowel disorders, in which lumen distention is present. However, what caused the low level inflammation is not well known. We tested the hypothesis that lumen distention- associated mechanical stress may induce expression of specific inflammatory mediators in gut smooth muscle.

Methods

Static mechanical stretch (18% elongation) was applied in vitro in primary culture of rat colonic circular smooth muscle cells (RCCSMCs) with a Flexercell FX-4000 Tension Plus System. Mechanical distention in vivo was induced in rats with an obstruction band placed in the distal colon.

Results

In the primary culture of RCCSMCs, we found that static stretch significantly induced mRNA expression of iNOS, IL-6, and MCP-1 in 3 hours by 6.0(±1.4), 2.5(±0.5), and 2.2(±0.5) fold (n = 6∼8, p<0.05), respectively. However, gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In the in vivo model of colon obstruction, we found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, but not in the sham controls or distal segments. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of NF-κB p65. This treatment further increased mRNA expression of inflammatory mediators in the naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against rat IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators.

Conclusions

Our studies demonstrate that mechanical stress induces gene expression of inflammatory mediators i.e. iNOS, IL-6, and MCP-1 in colonic SMC. Further ex vivo study showed that mechanical stress functions as a pro-inflammatory stimulus in the gut.     

Characterization of a Novel Integrative Element, ICESt1, in the Lactic Acid Bacterium Streptococcus thermophilus

The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3′ end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element.     

Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity

Mutants of Oenococcus oeni were isolated as spontaneous neomycin-resistant mutants. Three of these mutants harbored a significantly reduced ATPase activity that represented 50% of that of the wild-type strain. Their growth rates were also impaired at pH 5.3 (46-86% of the wild-type level). However, the profiles of sugar consumption appeared identical to those of the parental strain. At pH 3.2, all the mutant strains failed to grow and a drastic decrease in viability was observed after an acid shock. Surprisingly, all the isolated mutants were devoid of malolactic activity. These results suggest that the ATPase and malolactic activities of O. oeni are linked to each other and play a crucial role in the mechanism of resistance to an acid stress.     

DNA Relatedness,Divergence, and Sibling Species of the Lactic Acid Bacterium Streptococcus thermophilus

Previously, five distinct groups with 80–90% intragroup DNA homology values were revealed among 19 lactic acid–producing bacterial strains. The study of 39 new strains of thermophilic streptococci in the present work allowed us to reveal the sixth DNA homology group. The nine strains of this group are close, at 55–70% DNA homology levels, to the type strain Streptococcus thermophilus ATCC 19258. Group VI showed a low level of DNA–DNA reassociation (20–30%) with the DNA homology groups I, II, III, and V. The intergroup DNA–DNA reassociation values determined from DNA renaturation rates varied from 20 to 50%. These data were interpreted as indicative of the existence of at least four sibling species among the thermophilic streptococci studied.     

Catabolism of N-Acetylneuraminic Acid,a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei,Involves Two Newly Characterized Proteins

In silico analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB) Lactobacillus sakei 23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use N-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other L. sakei strains that were shown to be unable to grow on NANA. Moreover, L. sakei 23K nanA, nanT, nanK, and nanE genes were able to complement Escherichia coli mutants. Construction of different mutants in L. sakei 23K ΔnanR, ΔnanT, and ΔnanK and the double mutant L. sakei 23K Δ(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from nanK, lsa1644 and lsa1645, are involved in the catabolism of sialic acid in L. sakei 23K, as a L. sakei 23K Δlsa1645 mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster nanTEAR-nanK-lsa1644-lsa1645 is indeed involved in the use of NANA as an energy source by L. sakei.     

Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

We describe the dynamics of changes in the intracellular pH (pH_i) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH_ex). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH_i within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH_ex from 7.0 to 5.0. Under these conditions, the pH_i of L. innocua remained neutral (between 7 and 8). In contrast, the pH_i values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH_ex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH_i at pH_ex 7.0, while different kinetics of pH_i regulation were observed in different species and also in different strains of S. thermophilus.     

Investigation of Lactic Acid Bacteria Isolated from Giant Panda Feces for Potential Probiotics In Vitro

Probiotics and Antimicrobial Proteins - The present study aimed to isolate an optimal lactic acid bacterial strain from the feces of healthy giant pandas. The strain exhibited good stability at low...     

Genomic Features of Lactococcus lactis IO-1, a Lactic Acid Bacterium That Utilizes Xylose and Produces High Levels of L-Lactic Acid

Lactococcus lactis IO-1 (JCM7638) produces L-lactic acid predominantly when grown at high xylose concentrations, and its utilization is highly desired in the green plastics industry. Therefore it is worthwhile studying its genomic traits. In this study, we focused on (i) genes of possible horizontal transfer derivation (prophages, the nisin-sucrose transposon, and several restriction-modification systems), and (ii) genes for the synthetic pathways of amino acids and vitamins in the IO-1 genome. In view of the results of this analysis, we consider their meanings in strain IO-1.     

Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis

In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.