Adaptation of Lactobacillus plantarum IMDO 130201, a wheat sourdough isolate, to growth in wheat sourdough simulation medium at different pH values through differential gene expression

Sourdough is a very competitive and challenging environment for microorganisms. Usually, a stable microbiota composed of lactic acid bacteria (LAB) and yeasts dominates this ecosystem. Although sourdough is rich in carbohydrates, thus providing an ideal environment for microorganisms to grow, its low pH presents a particular challenge. The nature of the adaptation to this low pH was investigated for Lactobacillus plantarum IMDO 130201, an isolate from a laboratory wheat sourdough fermentation. Batch fermentations were carried out in wheat sourdough simulation medium, and total RNA was isolated from mid-exponential-growth-phase cultures, followed by differential gene expression analysis using a LAB functional gene microarray. At low pH values, an increased expression of genes involved in peptide and amino acid metabolism was found as well as that of genes involved in plantaricin production and lipoteichoic acid biosynthesis. The results highlight cellular mechanisms that allow L. plantarum to function at a low environmental pH.     

A two component system is involved in acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus

The Gram-positive bacterium Lactobacillus delbrueckii subsp. bulgaricus is of vital importance to the food industry, especially to the dairy industry. Two component systems (TCSs) are one of the most important mechanisms for environmental sensing and signal transduction in the majority of Gram-positive and Gram-negative bacteria. A typical TCS consists of a histidine protein kinase (HPK) and a cytoplasmic response regulator (RR). To investigate the functions of TCSs during acid adaptation in L. bulgaricus, we used quantitative PCR to reveal how TCSs expression changes during acid adaptation. Two TCSs (JN675228/JN675229 and JN675230/JN675231) and two HPKs (JN675236 and JN675240) were induced during acid adaptation. These TCSs were speculated to be related with the acid adaptation ability of L. bulgaricus. The mutants of JN675228/JN675229 were constructed in order to investigate the functions of JN675228/JN675229. The mutants showed reduced acid adaptation compared to that of wild type, and the complemented strains were similar to the wild-type strain. These observations suggested that JN675228 and JN675229 were involved in acid adaptation in L. bulgaricus. The interaction between JN675228 and JN675229 was identified by means of yeast two-hybrid system. The results indicated there is interaction between JN675228 and JN675229.     

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.     

基于RNA-Seq的向日葵脂肪酸合成基因筛选与分析

向日葵(Helianthus annuus L.)是世界四大油料作物之一。为解析向日葵主要脂肪酸组分的生物合成分子机制,选用高油酸自交系J9、低油酸自交系P50为研究材料;采用错期播种同期采样试验,以液相色谱测定授粉后不同样品籽粒的脂肪酸组分与含量;利用RNA-Seq技术,对不同样品测序,获得各样品与脂肪酸合成相关的基因表达数据,以气象学数据、液相色谱数据、向日葵基因组数据和转录组数据为基础,采用生物信息学方法分析相关数据。结果表明：(1)高油酸向日葵材料脂肪酸组分和含量受温度影响小,而低油酸材料易受温度影响;(2)J9和P50的12个样本的高表达基因数在7500个左右,而中、低表达基因数相当,是高表达基因的两倍,约15000个;(3)品种特异性表达基因和品种间特异表达基因去除重复后,在J9和P50在-CK和S数据集得到15885个,GO功能富集获得的与脂质代谢直接相关的403个DEGs 映射到与的脂质代谢相关的KEGG代谢通路,累计29个DEGs直接参与了19条相关KEGG通路。本研究将为深入解析向日葵脂肪酸代谢分子机制提供新信息。     

Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

DNA microarray analyses of the long-term adaptive response of Escherichia coli to acetate and propionate

In its natural environment, Escherichia coli is exposed to short-chain fatty acids, such as acetic acid or propionic acid, which can be utilized as carbon sources but which inhibit growth at higher concentrations. DNA microarray experiments revealed expression changes during exponential growth on complex medium due to the presence of sodium acetate or sodium propionate at a neutral external pH. The adaptive responses to acetate and propionate were similar and involved genes in three categories. First, the RNA levels for chemotaxis and flagellum genes increased. Accordingly, the expression of chromosomal fliC'-'lacZ and flhDC'-'lacZ fusions and swimming motility increased after adaptation to acetate or propionate. Second, the expression of many genes that are involved in the uptake and utilization of carbon sources decreased, indicating some kind of catabolite repression by acetate and propionate. Third, the expression of some genes of the general stress response increased, but the increases were more pronounced after short-term exposure for this response than for the adaptive response. Adaptation to propionate but not to acetate involved increased expression of threonine and isoleucine biosynthetic genes. The gene expression changes after adaptation to acetate or propionate were not caused solely by uncoupling or osmotic effects but represented specific characteristics of the long-term response of E. coli to either compound.     

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.     

Changes in gene expression of Actinobacillus pleuropneumoniae in response to anaerobic stress reveal induction of central metabolism and biofilm formation

Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry worldwide. Oxygen deprivation is a stress that A. pleuropneumoniae will encounter during both early infection and the later, persistent stage. To understand modulation of A. pleuropneumoniae gene expression in response to the stress caused by anaerobic conditions, gene expression profiles under anaerobic and aerobic conditions were compared in this study. The microarray results showed that 631 genes (27.7% of the total ORFs) were differentially expressed in anaerobic conditions. Many genes encoding proteins involved in glycolysis, carbon source uptake systems, pyruvate metabolism, fermentation and the electron respiration transport chain were up-regulated. These changes led to an increased amount of pyruvate, lactate, ethanol and acetate in the bacterial cells as confirmed by metabolite detection. Genes encoding proteins involved in cell surface structures, especially biofilm formation, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis were up-regulated as well. Biofilm formation was significantly enhanced under anaerobic conditions. These results indicate that induction of central metabolism is important for basic survival of A. pleuropneumoniae after a shift to an anaerobic environment. Enhanced biofilm formation may contribute to the persistence of this pathogen in the damaged anaerobic host tissue and also in the early colonization stage. These discoveries give new insights into adaptation mechanisms of A. pleuropneumoniae in response to environmental stress.