Validation of reference genes for real-time quantitative PCR studies in gene expression levels of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.     

Immunogenicity of Orally Administrated Recombinant <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang Expressing <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> Surface Adhesion Protein P23 in Mice

Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals.     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.     

一株耐盐碱乳酪短杆菌G20响应盐碱胁迫的差异代谢物分析

【目的】探究耐盐碱乳酪短杆菌G20响应盐碱胁迫的代谢物组成以及代谢物合成潜力,为潜在功能分子和盐碱诱导的快速稳定响应逻辑门基因线路的挖掘提供参考。【方法】利用液相色谱-质谱联用技术(LC-MS)检测乳酪短杆菌G20盐碱环境与正常环境下4个生长时期的代谢产物。着重对富含高差异变化倍数代谢物的适应期与指数期进行分析。【结果】乳酪短杆菌G20可以在pH 10.0、9%NaCl环境中正常生长,同时环境pH值会随菌株生长逐步下降。综合正负离子2种模式,乳酪短杆菌G20在盐碱环境下各生长时期间差异代谢物数量分别为正常环境的0.69、0.75和0.81倍。盐碱胁迫诱导下适应期与指数期差异代谢物主要为苯环型化合物、有机酸及其衍生物与有机杂环类化合物。其中上调的有机酸化合物吲哚-3-乙酸、犬尿酸和葡萄糖酸指数期质谱信号强度低于适应期。菌株中可能存在的渗透保护剂有L-瓜氨酸、L-脯氨酸、N-乙酰鸟氨酸和左旋肉碱等。适应期变化倍数较大或质谱信号强度较高的差异化合物有毛果芸香碱、植物鞘氨醇和柠檬酸等,指数期有组胺、L-脯氨酸和硫胺素等。菌株差异代谢通路集中在氨基酸代谢与碳水化合物代谢。菌株代谢物中存在甜菜碱和...     

p LA-PEDV-S1质粒拷贝数与发酵重组乳酸菌S1表达量之间的相关性研究

探究质粒拷贝数以及目标蛋白S1表达量与发酵时间的关系,从而确定放大生产p LA-PEDV-S1/Lactobacillus casei的最佳发酵时间。通过发酵重组干酪乳杆菌,绘制重组干酪乳杆菌的生长曲线,确定其生长的最佳时期。将p LA-PEDV-S1/L. casei分别接种至添加抗生素和不添加抗生素的MRS培养基中传代培养,进行稳定性实验。使用荧光定量PCR方法检测重组干酪乳杆菌中质粒的拷贝数,使用流式细胞术检测乳酸菌表达的目标蛋白。重组菌在7 h达到生长顶点,传至120代时外源质粒并无丢失情况出现。质粒拷贝数在9 h达到峰值29. 34,表达目标蛋白的重组菌在7 h达到峰值97. 98%。结果显示在细菌生长的对数生长期末期,质粒拷贝数最高且S1表达量最多;在平台期随着发酵时间的增加,质粒拷贝数逐渐降低,S1表达量也相应减少。发酵的最佳时间为7～10 h,质粒拷贝数与S1表达量之间存在着正相关性。     

Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3′ end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, β-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.     

Genomic DNA standards for gene expression profiling in Mycobacterium tuberculosis

A fundamental problem in DNA microarray analysis is the lack of a common standard to compare the expression levels of different samples. Several normalization protocols have been proposed to overcome variables inherent in this technology. As yet, there are no satisfactory methods to exchange gene expression data among different research groups or to compare gene expression values under different stimulus–response profiles. We have tested a normalization procedure based on comparing gene expression levels to the signals generated from hybridizing genomic DNA (genomic normalization). This procedure was applied to DNA microarrays of Mycobacterium tuberculosis using RNA extracted from cultures growing to the logarithmic and stationary phases. The applied normalization procedure generated reproducible measurements of expression level for 98% of the putative mycobacterial ORFs, among which 5.2% were significantly changed comparing the logarithmic to stationary growth phase. Additionally, analysis of expression levels of a subset of genes by real time PCR technology revealed an agreement in expression of 90% of the examined genes when genomic DNA normalization was applied instead of 29–68% agreement when RNA normalization was used to measure the expression levels in the same set of RNA samples. Further examination of microarray expression levels displayed clusters of genes differentially expressed between the logarithmic, early stationary and late stationary growth phases. We conclude that genomic DNA standards offer advantages over conventional RNA normalization procedures and can be adapted for the investigation of microbial genomes.     

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

Complete Genome Sequence of Lactobacillus casei Zhang,a New Probiotic Strain Isolated from Traditional Homemade Koumiss in Inner Mongolia,China

Lactobacillus casei Zhang is a new probiotic bacterium isolated from koumiss collected in Inner Mongolia, China. Here, we report the main genome features of L. casei Zhang and the identification of several predicted proteins implicated in interactions with the host.Koumiss, a traditional drink made from mare''s milk by nomadic peoples in China and Mongolia, is believed to be beneficial in the cure of digestive diseases and a wide range of chronic diseases, including tuberculosis, bronchitis, and anemia (3). Lactobacillus casei Zhang is a novel probiotic strain identified by screening of lactic acid bacteria isolated from koumiss samples collected in Inner Mongolia, China, and exhibits high-level resistance to acid and bile stresses, as well as antibacterial, antioxidative, and immunomodulatory properties (6, 7, 11).A whole-genome shotgun strategy was used for sequencing of the genome of L. casei Zhang. pUC18 plasmid libraries with insertions of 1.5 to 2.5 kb and 4 to 6 kb were constructed (8). Gaps were closed by sequencing of PCR products. Base calling and sequence assembly were carried out using the Phred/Phrap/Consed software package (http://www.phrap.org/), and reads giving a total of 6.2-fold coverage were assembled with an error rate of <0.0001. Gene prediction and annotation were performed as described previously (10).The complete genome of L. casei Zhang consists of a 2,861,848-bp circular chromosome and a 36-kb plasmid. The average G+C content of the chromosome is 46.5%, while the plasmid has a lower G+C content (10). The L. casei Zhang genome contains 2,804 predicted coding sequences (CDSs), five rRNA operons, and 59 tRNAs. No functional prophages were identified, except for the previously described prophage remnant (9). Genes for 41 transposases were found in the genome, and this number was much lower than (only about 30%) those of transposase genes in L. casei ATCC 334 and BL23 (1, 4), suggesting that insertion element (IS)-mediated genome diversification was less frequent in L. casei Zhang.Comparative genome analysis revealed that the number of phosphotransferase system (PTS)-related proteins varied significantly in L. casei strains. Almost twice as many PTS components were found in L. casei Zhang and BL23 as in L. casei ATCC 334. In contrast to L. casei ATCC 334, L. casei Zhang was found to have 33 PTS components consisting of 11 complete substrate-specific enzyme II (EII) complexes encoded by six genomic islands. The G+C contents of the six islands ranged from 41 to 47%, similar to the average G+C content of the L. casei Zhang genome. In addition, most of the EII components in L. casei Zhang (81 of 96) were conserved in L. casei BL23, suggesting that a large-scale loss of PTSs occurred in L. casei ATCC 334 during its evolution. Conspicuous redundancy of chromosome-encoded PTSs in L. casei Zhang may offer benefits in the transport and use of a large panel of carbon sources.Genes encoding five putative mucus-binding proteins (LCAZH_0407, LCAZH_2292, LCAZH_2478, LCAZH_2398, and LCAZH_1427) and a cluster of genes encoding bacteriocin biosynthetic proteins (LCAZH_2341 to LCAZH_2348) nearly identical to those in L. casei ATCC 334 and BL23 were identified in L. casei Zhang and may provide this bacterium with some competitive advantages in the gastrointestinal environment (2, 5).In conclusion, the comparative analysis revealed the flexibility of L. casei Zhang in sugar utilization. In addition, some possible hints for its interactions with the host were identified. This genome sequence will be the basis for systematic studies into the mechanism for the probiotic properties of L. casei Zhang.     

Correlation between cell lipid content, gene expression and fermentative behaviour of two Saccharomyces cerevisiae wine strains

Aim: To verify a possible correlation between cell lipid composition, expression of key genes in lipid metabolism and fermentative behaviour of Saccharomyces cerevisiae wine strains. Methods and Results: The fermentative abilities of two commercial wine strains of S. cerevisiae were tested under stressful conditions. Cell number, glucose and fructose concentrations, expression of ACS1, ACS2, ACC1, OLE1, ERG9, ERG10, ARE1 and ARE2 and lipid content were evaluated. The strain that failed to complete the fermentation had lower amounts of C16:1 and C16:0 fatty acids at the beginning of fermentation (0 h) and late logarithmic phase (72 h). While the amount of C18:1 in this strain was lower than that in the strain that completed the fermentation at 0 h, same levels were observed for both strains at 72 h. The sterol levels were generally higher in the strain that failed to complete the fermentation. Gene expression generally increased from the beginning of the fermentation to the late logarithmic phase in both strains. Conclusion: A positive correlation between good fermentative ability, elevated fatty acid content and ACC1 gene expression has been identified. Significance and Impact of the Study: The cell lipid content at the time of inoculum and expression of ACC1 gene of starter strains should be carefully considered in order to identify the possible stuck/sluggish fermentations.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA-controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat-shock or stationary culture conditions lead to the induction of additional PrfA-dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient-stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.