Genetic characterization of non-spoilage variant isolated from beer-spoilage Lactobacillus brevis ABBC45

AIMS: To characterize the non-spoilage variant obtained from beer-spoilage Lactobacillus brevis ABBC45C and to identify a potential genetic marker capable of discriminating beer-spoilage L. brevis strains from non-spoilers. METHODS AND RESULTS: A non-spoilage variant was obtained from beer-spoilage L. brevis ABBC45C by repeatedly subculturing the strain at 37 degrees C. Genetic characterization of the variant revealed that 12,605 bp portion of one plasmid, designated pRH45II, was lost in the variant. The sequence analysis indicates the presence of 12 ORFs in the deleted region of pRH45II. The PCR and Southern hybridization study revealed that the homologues of ORF5 found in the deleted region were present in all of the beer-spoilage L. brevis strains examined in this study. In contrast, the homlogues appeared to be absent in non-spoilage L. brevis strains. CONCLUSIONS: The presence or absence of ORF5 homologues was found to be highly correlated with the beer-spoilage ability of L. brevis strains, indicating this ORF is potentially a useful genetic marker capable of differentiating beer-spoilage strains among L. brevis. SIGNIFICANCE AND IMPACT OF THE STUDY: A non-spoilage variant was successfully isolated from beer-spoilage L. brevis ABBC45C. This study could facilitate the understanding of mechanisms underlying beer-spoilage ability of L. brevis.     

Genetic marker for differentiating beer-spoilage ability of Lactobacillus paracollinoides strains

AIMS: To determine whether the beer-spoilage ability is an intrinsic character of Lactobacillus paracollinoides and identify a genetic marker for differentiating the beer-spoilage ability of strains belonging to this species. METHODS AND RESULTS: The ribotype of a nonspoilage strain, Lact. brevis ATCC8291, was found to be identical with that of Lact. paracollinoides LA7. The 16S rDNA sequence analysis and DNA-DNA hybridization study indicates that nonspoilage ATCC8291 should belong to Lact. paracollinoides. We further isolated nonspoilage variants from Lact. paracollinoides LA2(T) and LA9 by incubating these strains at 30 degrees C. To identify a genetic marker for differentiating the beer-spoilage ability of Lact. paracollinoides, open reading frames 5 (ORF5), the previously reported genetic marker for Lact. brevis, was evaluated. As a result, ORF5 homologues were detected in all of the 12 beer-spoilage strains of Lact. paracollinoides, while this ORF was not found in ATCC8291 or the two nonspoilage variants obtained from LA2(T) and LA9. CONCLUSIONS: Lactobacillus paracollinoides is not an intrinsic beer-spoiler and the nonspoilage strain Lact. brevis ATCC8291 should be reclassified as Lact. paracollinoides. ORF5 was found to be useful for differentiating beer-spoilage ability of this species. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that Lact. paracollinoides includes nonspoilage strains necessitates brewers to use a genetic marker that is associated with the beer-spoilage ability of this species.     

Comparative analysis of conserved genetic markers and adjacent DNA regions identified in beer-spoilage lactic acid bacteria

AIMS: To conduct an inter-species comparative study on the nucleotide sequences of the conserved DNA regions surrounding ORF5, a genetic marker for differentiating beer-spoilage lactic acid bacteria. METHODS AND RESULTS: The conserved DNA regions surrounding ORF5 were examined by PCR analysis, using three beer-spoilage strains, Lactobacillus brevis ABBC45C, L. paracollinoides LA2T and Pediococcus damnosus ABBC478. As a result, the DNA regions containing ORF1-7, originally found in ABBC45C, appeared to be conserved among the three strains, while the downstream region was not found in L. paracollinoides LA2T and P. damnosus ABBC478. The sequencing analysis of the conserved DNA regions of LA2T and ABBC478 revealed ca 99% nucleotide sequence identities with that of ABBC45C. CONCLUSIONS: The nucleotide sequences of the ca 8.2 kb DNA regions containing ORF1-7 were virtually identical among the three strains belonging to different species. The internal organizations of the ORFs were found to be remarkably similar. SIGNIFICANCE AND IMPACT OF THE STUDY: The level of nucleotide sequence identities suggests the DNA regions surrounding ORF5 were horizontally acquired by these beer-spoilage strains belonging to the three different species of lactic acid bacteria.     

Genetic characterization and specific detection of beer-spoilage Lactobacillus sp. LA2 and related strains

AIMS: Lactobacillus sp. LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability. The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L. collinoides, generally considered to be nonbeer-spoilage bacteria. The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp. LA2 group and L. collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp. LA2 group. METHODS AND RESULTS: The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp. LA2 and L. collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups. As a result, 300 and 500 bp ITS regions of Lactobacillus sp. LA2 were found to be almost identical with those of L. collinoides JCM1123T. Sequence comparison analysis between Lactobacillus sp. LA2 and L. collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp. LA2. Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp. LA2 group and nonbeer-spoilage L. collinoides. CONCLUSIONS: The PCR-based method has been developed to identify Lactobacillus sp. LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity. The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp. LA2 group strains from its close relatives that exhibit no beer-spoilage ability.     

亚硝酸盐影响Lactobacillus brevis 4903发酵的研究

通过研究可知,亚硝酸盐对Lactobacillusbrevis4903发酵有抑制作用,环境中亚硝酸盐一旦分解掉,这种抑制作用就会被解除。分析其原因:①亚硝酸盐抑制了乳酸菌生长,从而抑制了乳酸发酵;②在发酵初期可能因亚硝酸盐还原酶的作用,使亚硝酸盐酶解生成NH3,NH3中和了乳酸菌生成的酸(H ),从而使环境pH值的下降和酸的积累变得缓慢。     

一株短乳杆菌所产细菌素的部分特性

为了研究分离自内蒙古传统发酵乳制品——焦克的短乳杆菌KLDS1.0373所产细菌素的部分生物学特性(抑菌谱,对酶、pH和温度的敏感性,作用方式)。短乳杆菌KLDS1.0373发酵液经硫酸铵沉淀和葡聚糖凝胶纯化后,测定其部分生物学特性,并采用Tricine-SDS-PAGE方法确定细菌素的分子量范围。结果表明:短乳杆菌KLDS1.0373所产细菌素的抑菌活性对热和pH不敏感,在100°C或121°C处理30 min后抑菌活力略有增强,可被多种蛋白酶失活,但对α-淀粉酶不敏感。该细菌素分子量约为3.8 kD,对多种革兰氏阳性和阴性菌有抑制作用,作用方式为杀菌。     

短乳杆菌谷氨酸脱羧酶的生物信息学分析

以短乳杆菌(Lactobacillus brevis)Lb-2菌株cDNA为模板克隆了谷氨酸脱羧酶(Glutamate decarboxylase,GAD)基因。采用在线分析工具及相应软件分析预测了GAD基因核苷酸和氨基酸序列的组成、理化性质、信号肽以及高级结构等,并构建系统发育树。该基因序列全长1 407 bp,为一个完整的阅读框,编码468个氨基酸。GAD相对分子量理论预测值和等电点分别是53 517.8 u和5.42,没有跨膜区,没有其他亚细胞定位序列,为亲水性蛋白,与植物乳杆菌(Lactobacillus plantarum)和德氏乳酸杆菌(Lactobacillus delbrueckii)的GAD进化关系最近。     

短乳杆菌DM9218核苷酸代谢过程中的蛋白组学分析

目的 探讨短乳杆菌DM9218在核苷酸代谢过程中的蛋白表达差异。方法 分别提取DM9218菌株与底物（肌苷+鸟苷）反应前后的菌体蛋白,利用蛋白双向凝胶电泳（2-DE）技术,找出该菌株与底物反应前后的差异蛋白质点,选取其中差异变化较大的蛋白点进一步做蛋白质谱分析。结果 2-DE分析显示两样品蛋白点主要分布在等电点4~9和分子量11~90 kD范围内,将所得的蛋白点结合其蛋白得率、浓度、储存蛋白含量进行比较,得到匹配的蛋白点数为732个。从中选取14个差异显著的蛋白点进行质谱分析,质谱结果显示所选取蛋白质点主要与物质代谢、能量转换及基因水平转录和翻译等生物学功能密切相关。结论 本研究为后期分析研究短乳杆菌DM9218在核苷酸代谢过程中蛋白的表达奠定了基础。     

Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.     

Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809 isolated from wine and L. brevis ATCC 367

Tyramine, a frequent amine in wines, is produced from tyrosine by the tyrosine decarboxylase (TDC) activity of bacteria. The tyramine-producing strain Lactobacillus brevis IOEB 9809 isolated from wine and the reference strain L. brevis ATCC 367 were studied. At the optimum pH, 5.0, K(m) values of IOEB 9809 and ATCC 367 crude extracts for L-tyrosine were 0.58 mM and 0.67 mM, and V(max) was higher for the wine strain (115 U) than the ATCC 367 (66 U). TDC exhibited a preference for L-tyrosine over L-DOPA as substrate. Enzyme activity was pyridoxal-5'-phosphate (PLP)-dependent and it was stabilized by the substrate and coenzyme. In contrast, glycerol and beta-mercaptoethanol strongly inhibited TDC. Tyramine competitively inhibited TDC for both strains. Citric acid, lactic acid and ethanol had an inhibitory effect on cells and crude extracts, but none could inhibit TDC at the usual concentrations in wines.     

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation

ABSTRACT

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.     

酸胁迫下短乳杆菌NCL912蛋白质的差异表达及其作用

【目的】本文从蛋白质组水平,对本实验室分离的一株高产γ-氨基丁酸的短乳杆菌NCL912(Lactobacillus brevis)在酸胁迫下蛋白质的差异表达及其应激机理进行探讨。【方法】利用双向凝胶电泳技术对pH 5.0和pH 4.0条件下,不含L-谷氨酸钠的培养物的蛋白质组电泳图谱进行了分析,并对酸胁迫下差异表达的蛋白进行了比较。利用质谱检测技术和生物信息学技术对这些差异表达的蛋白进行了鉴定、功能分类和代谢途径分析等。【结果】通过双向凝胶电泳技术,可以得到均匀、背景清晰、分辨率高、重复性好的Lb.brevis NCL912的双向凝胶电泳图谱。对pH 5.0和pH 4.0条件下培养的该菌总蛋白质电泳图谱进行比较,发现有25个差异表达的蛋白点。对这25个差异表达的蛋白进行了质谱鉴定。由于缺乏短乳杆菌NCL912的全基因组,所以其中只有8个蛋白点被质谱鉴定和分析得到。它们分别参与了蛋白质的合成、核苷酸的合成、糖酵解代谢、细胞能量水平的调节等。【结论】酸应激下这些表达蛋白质可通过其相应的功能来保护细胞耐受酸胁迫,从而使菌能够在酸性环境下生存增值。这可能就是Lb.brevis NCL912的酸胁迫应激机理之一。     

Detection of resistance of lactic acid bacteria to a mixture of the hop analogue compounds tetrahydroiso-alpha-acids by noninvasive measurement of intracellular pH

AIMS: To examine the resistance of beer isolates of lactic acid bacteria (LAB) towards a mixture of tetrahydroiso-alpha-acids (Tetra) by growth experiments as well as by measurement of intracellular pH. METHODS AND RESULTS: Beer LAB isolates were identified to species level by SDS-PAGE of whole-cell proteins. Beer isolates of Lactobacillus brevis showed better ability for growth in the presence of Tetra than nonbeer isolates of the L. brevis or other species of LAB including beer and nonbeer isolates. The antimicrobial effect of Tetra was also examined by noninvasive measurement of intracellular pH by fluorescence ratio imaging microscopy for selected beer isolates of L. brevis and Pediococcus inopinatus. Strains of L. brevis showing limited decrease of intracellular pH during exposure to Tetra also showed better ability for growth in the presence of these compounds as well as in commercial beer products. CONCLUSIONS: It was possible to apply a method for noninvasive measurement of intracellular pH to predict the resistance of beer spoilage LAB towards the Tetra hop analogue compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the usability of a new rapid method for detecting hop-resistant variants of known beer spoilage LAB species.     

短乳杆菌DM9218肌苷水解酶基因的异源表达及活性检测

目的 探讨短乳杆菌DM9218肌苷水解酶基因A0008的异源表达及其对肌苷的分解活性检测。方法 克隆来源于短乳杆菌DM9218基因组的肌苷水解酶基因A0008,构建原核表达载体,转入大肠埃希菌BL21诱导重组蛋白表达并纯化,进行体外酶活检测。结果 成功构建了肌苷水解酶A0008-pET28a原核表达载体,表达并纯化出重组蛋白,酶活结果显示该重组蛋白具有水解肌苷的能力。结论 短乳杆菌DM9218基因A0008可能编码肌苷水解酶并参与DM9218对肌苷的分解。     

Kinetics of sterilization of Lactobacillus brevis cells by the application of high voltage pulses

The technique of irreversible electroporation has been successfully applied to cause a lethal effect on Lactobacillus brevis cells suspended in phosphate buffer solution, Na(2)HPO(4)/NaH(2)PO(4) . H(2)O (0.845/0.186 mM) between parallel plane electrodes. Tests were carried out at different temperatures (24,45,60, and 80 degrees C) to determine if there was a synergistic effect of temperature and electric pulse treatment on the destruction of L. brevis. Experimental results indicate that the viability (log N/N(0); where N(0) and N are the number of cells survived per milliliter before and after pulse voltage application, respectively) of L. brevis decreased with electric field strength E and temperature T and treatment time t(t). The relations between log(N/N(0)) and t(t) and log(N/N(0)) and E indicate that higher field strengths are more effective than higher treatment times in causing destruction of L. brevis cells. It was also found that as the temperature of the liquid medium containing L. brevis cells increased from 24 to 60 degrees C, the death rate of L. brevis cells increased with a decrease in the total treatment time t(t) (pulse width x number of pulses applied). The application of an electric field strength E = 25 kV/cm at 60 degrees C and treatment time t(t) = 10 ms resulted in very high destruction levels of L. brevis cells (N/N(0) = 10(-9)). In comparison with existing steam sterilization technology, this new method of sterilization using relatively low temperature and short treatment time could prove to be an excellent method to minimize thermal denaturation of important nutrient components in liquid media. (c) John Wiley & Sons, Inc.     

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.