A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang and its mutant with enhanced lactic acid tolerance

Lactobacillus casei has traditionally been recognized as a probiotic and frequently used as an adjunct culture in fermented dairy products, where acid stress is an environmental condition commonly encountered. In the present study, we carried out a comparative physiological and proteomic study to investigate lactic-acid-induced alterations in Lactobacillus casei Zhang (WT) and its acid-resistant mutant. Analysis of the physiological data showed that the mutant exhibited 33.8% higher glucose phosphoenolpyruvate:sugar phosphotransferase system activity and lower glycolytic pH compared with the WT under acidic conditions. In addition, significant differences were detected in both cells during acid stress between intracellular physiological state, including intracellular pH, H⁺-ATPase activity, and intracellular ATP pool. Comparison of the proteomic data based on 2D-DIGE and i-TRAQ indicated that acid stress invoked a global change in both strains. The mutant protected the cells against acid damage by regulating the expression of key proteins involved in cellular metabolism, DNA replication, RNA synthesis, translation, and some chaperones. Proteome results were validated by Lactobacillus casei displaying higher intracellular aspartate and arginine levels, and the survival at pH 3.3 was improved 1.36- and 2.10-fold by the addition of 50-mM aspartate and arginine, respectively. To our knowledge, this is the first demonstration that aspartate may be involved in acid tolerance in Lactobacillus casei. Results presented here may help us understand acid resistance mechanisms and help formulate new strategies to enhance the industrial applications of this species.     

精氨酸代谢途径抗酸关键基因对乳酸乳球菌Lactococcus lactis NZ9000胁迫抗性的影响

【目的】寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。【方法】通过在Lactococcus lactis NZ9000中分别过量表达来源于Lactobacillus casei Zhang的精氨酰琥珀酸合成酶(ASS)和精氨酰琥珀酸裂解酶(ASL)改变精氨酸代谢提高酸胁迫抗性。【结果】与对照菌株对比,重组菌株在环境胁迫下表现了较高的生长性能、存活率和发酵性能。生理学分析发现,酸胁迫环境下,重组菌株细胞有较高的胞内NH4+、ATP含量和H+-ATPase活性,并显著提高了精氨酸脱亚胺酶(ADI)途径中的氨基酸浓度。进一步的转录分析发现,天冬氨酸合成、精氨酸代谢相关的基因转录水平上调。【结论】在L.lactis NZ9000中过量表达ASS或ASL可以引发精氨酸代谢流量的上调,进而提高了细胞的多种胁迫抗性。精氨酸合成途径广泛存在于多种微生物中,为微生物,尤其是工业微生物提高胁迫抗性提供了新思路。     

Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H₂O₂) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H₂O₂/min/10⁸ colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H₂O₂, survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10⁵ CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.     

Acid tolerance inLeuconostoc oenos. Isolation and characterization of an acid-resistant mutant

The acid tolerance ofLeuconostoc oenos was examined in cells surviving at pH 2.6, which is lower than the acid limit of growth (about pH 3.0). Acid-adapted cells survived better than non-adapted cells. Tolerance to acid stress was found to be dependent upon the adaptive pH. Acid resistance was increased by an order of magnitude for cultures adapted to a pH of about 2.9. Inhibiting protein synthesis with chloramphenicol prior to acid shock revealed that acid adaptation may involve two separate systems, one of which appears to be independent of protein synthesis. The acid-resistant mutant LoV8413, isolated during a long-term survival screen at pH 2.6, was found to be able to grow in acidic media and was characterized by a high H⁺-ATPase activity at low pH. The data from electrophoretic analysis of total proteins labeled with [³⁵S] methionine indicate that large amounts of a protein of 42 kDa molecular mass were produced within this acid-resistant mutant.     

Enhancement of succinic acid production by osmotic-tolerant mutant strain of Actinobacillus succinogenes

Fermentation and succinic acid production by Actinobacillus succinogenes YZ0819 was inhibited by high NaCl. To enhance the resistance of this strain to osmotic stress, an NaCl-tolerant mutant strain of A. succinogenes (CH050) was screened and selected through a continuous culture using survival in 0.7 M NaCl as the selection criterion. Using Na₂CO₃ as the pH regulator and glucose as the carbon source in batch fermentation, the isolated osmo-resistant stain, A. succinogenes CH050, produced up to 66 g/l succinic acid with a yield of 73.37% (w/w). The concentration of succinic acid and mass yield were increased by 37.5 and 4.37%, respectively, compared to the parent strain. The dry cell weight reached 10.1 g/l, which is 37% higher than that of the parent strain. The high tolerance of A. succinogenes CH050 to osmotic stress increased improved the succinic acid production from batch fermentation.     

<Emphasis Type="Italic">rpoS</Emphasis> Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H₂O₂ and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

Enhancement of the activity and alkaline pH stability of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> xylanase A by directed evolution

Directed evolution has been used to enhance the catalytic activity and alkaline pH stability of Thermobifida fusca xylanase A, which is one of the most thermostable xylanases. Under triple screened traits of activity, alkaline pH stability and thermostability, through two rounds of random mutagenesis using DNA shuffling, a mutant 2TfxA98 with approximately 12-fold increased k _cat/K _m and 4.5-fold decreased K _m compared with its parent was obtained. Moreover, the alkaline pH stability of 2TfxA98 is increased significantly, with a thermostability slightly lower than that of its parent. Five amino acid substitutions (T21A, G25P, V87P, I91T, and G217L), three of them are near the catalytic active site, were identified by sequencing the genes encoding this evolved enzyme. The activity and stabilizing effects of each amino acid mutation in the evolved enzyme were evaluated by site-directed mutagenesis. This study shows a useful approach to improve the catalytic activity and alkaline pH stability of T. fusca xylanase A toward the hydrolysis of xylan.     

Mycothiol peroxidase MPx protects Corynebacterium glutamicum against acid stress by scavenging ROS

Objectives

To investigate mycothiol peroxidase (MPx) of Corynebacterium glutamicum that is a novel CysGPx family peroxidase using both the mycoredoxin and thioredoxin reducing systems as proton donors for peroxide detoxification and may be involved in the relief of acid stress.

Results

A Δmpx mutant exhibited significantly decreased resistance to acid stress and markedly increased accumulation of reactive oxygen species (ROS) and protein carbonylation levels in vivo. Over-expression of mpx increased the resistance of C. glutamicum to acid stress by reducing ROS accumulation. The stress-responsive extracytoplasmic function-sigma (ECF-σ) factor, SigH, mediated acid-induced expression of mpx in the wild-type under acid conditions, which in turn directly contributed to tolerance to acid stress.

Conclusion

MPx is essential for combating acid stress by reducing intracellular ROS levels induced by acid stress in C. glutamicum, which adds a new dimension to the general physiological functions of CysGPx.

     

XRE家族转录因子XrpA调控粘质沙雷氏菌的耐酸能力

【背景】工业菌株的耐酸能力是发酵过程中的一大挑战。粘质沙雷氏菌(Serratia marcescens)作为肠杆菌科的一种细菌,可生成2,3-丁二醇、乙偶姻和灵菌红素等高附加值产品。然而目前对于粘质沙雷氏菌酸耐受能力的分子机制尚不清楚。【目的】通过对转录调控因子XrpA的挖掘以及对其功能的研究,探究粘质沙雷氏菌酸耐受能力的分子机制,为改善工业菌株耐酸能力提供新的策略。【方法】通过对粘质沙雷氏菌进行转座子插入突变,构建了一个Tn5G转座子插入突变文库,利用文库筛选了一株酸敏感型突变株,并对其进行测序鉴定;同时还对突变菌株中与耐酸相关关键基因的转录水平以及细胞膜通透性、细胞膜完整性和H+-ATPase的活性变化进行检测。【结果】发现了一个响应酸胁迫的转录调控因子BVG90_23400,其属于XRE超级家族转录调控因子,命名为XrpA。在酸性条件下,与野生型菌株(JNB5-1)相比,xrpA被阻断后导致了粘质沙雷氏菌多种表型的变化,其中包括生物量显著下降、H+-ATPase活性降低、细胞膜的通透性以及完整性受到破坏。【结论】 XrpA影响粘质沙雷氏菌耐酸能力的分子机制是通过对细胞膜通透性、细胞膜完整性以及H+-ATPase活性的正向调节来维持细胞在酸性条件下的内环境稳态。同时,XrpA可以通过调节酸性应激反应基因的转录水平来影响细胞内环境稳态,从而调控粘质沙雷氏菌对低pH的耐受能力。     

A physiological comparative study of acid tolerance of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZDY 2013 and <Emphasis Type="Italic">L. plantarum</Emphasis> ATCC 8014 at membrane and cytoplasm levels

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.     

Isolation and basic characterization of a β‐glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

Aims: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular β‐glucosidase from this strain. Methods and Results: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for β‐glycosidase activities. A strain of Lact. brevis showing high intracellular β‐d ‐glucosidase, β‐d ‐xylosidase and α‐l ‐arabinosidase activities was selected for purification and characterization of its β‐glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3·5. The K_m for p‐nitrophenyl‐β‐d ‐glucopyranoside and p‐nitrophenyl‐β‐d ‐xylopyranoside is 0·22 and 1·14 mmol l^?1, respectively. The β‐glucosidase activity was strongly inhibited by gluconic acid δ‐lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7·0 (t_1/2 = 50 day) but not at pH 4·0 (t_1/2 = 4 days). Conclusions: The β‐glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei ( Coulon et al. 1998 ) and Lactobacillus plantarum ( Sestelo et al. 2004 ). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long‐term stability could be of great interest for the release of aroma compounds during winemaking. Significance and Impact of the study: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular β‐glucosidase isolated from a wine‐related strain of Lact. brevis.     

Acid- and multistress-resistant mutants of Lactococcus lactis : identification of intracellular stress signals

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.