Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.     

CCK39/UreB双基因融合表达载体的构建及其在大肠杆菌中的表达

本研究报道了猪源性胆囊收缩素39肽(Cholecystokinin 39, CCK39)和猪肠道大肠菌群脲酶B亚基(UreaseB, UreB)融合的原核表达载体的构建, 以及兼有CCK39和UreB功能活性域的融合蛋白的表达。利用RT-PCR从猪十二指肠总RNA中克隆CCK39, 再通过PCR从猪肠道产脲酶大肠菌群质粒中克隆UreB, 之后将扩增所得的两基因先后定向插入原核表达载体pET43a(+)中, 构建双基因融合表达载体pET43a(+)/CCK39/UreB, 并转化至大肠杆菌(Escherichia coli) BL-21(DE3)中。重组质粒pET43a(+)/CCK39/UreB经PCR、双酶切鉴定和序列分析, 证实已成功构建双基因融合表达载体。重组表达菌经isopropylthio-b-D-galactoside (IPTG)诱导表达了分子量约为80 kD的融合蛋白, 与预期分子量相同, 主要以可溶蛋白的形式存在于细胞质中; 用Ni2+-NTA对可溶蛋白进行亲和纯化, 纯度大于95%; Western blotting分析结果显示重组融合蛋白可分别为CCK8和UreB抗血清识别, 具有良好的反应原性。本研究所表达的融合蛋白为研制抗CCK/Urease双效疫苗奠定了基础。     

Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows' milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73 degrees C for 15 s or 25 s with and without prior homogenization (2,500 lb/in(2) in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73 degrees C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73 degrees C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73 degrees C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

A survey of the cellular responses in Pseudomonas putida KT2440 growing in sterilized soil by microarray analysis

Genome-wide scanning of gene expression by microarray techniques was successfully performed on RNA extracted from sterilized soil inoculated with Pseudomonas putida KT2440/pSL1, which contains a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoic acid (3CB). The genes showing significant changes in their expression in both the triplicate-microarray analysis using amplified RNA and the single-microarray analysis using unamplified RNA were investigated. Pathway analysis revealed that the benzoate degradation pathway underwent the most significant changes following treatment with 3CB. Analysis based on categorization of differentially expressed genes against 3CB revealed new findings about the cellular responses of the bacteria to 3CB. Genes specifically involved in the transport of 3CB were upregulated, including a K(+)/H(+) antiporter complex, a universal stress protein, two cytochrome P450 proteins and an efflux transporter. The downregulated expression of several genes involved in carbon metabolism and the genes belonging to a prophage in the presence of 3CB was observed. This study demonstrated the applicability of the method of soil RNA extraction for microarray analysis of gene expression in bacteria growing in sterilized soil.     

东亚砂藓取材部位对提取其总RNA及DDRT-PCR的影响

分别采用新鲜东亚砂藓植物体先端、基部及中部作为材料,采用改良的SDS法,提取及纯化三部分的总RNA。比较RNA产率、纯度及分析电泳图谱来确定适于东亚砂藓RNA分离的最佳部位。并运用mRNA差异显示方法比较了东亚砂藓不同部位的差异。实验结果显示,东亚砂藓植物体的先端适于其总RNA提取及mRNA差异显示的研究。     

Optimization of methods for detecting Mycobacterium avium subsp. paratuberculosis in environmental samples using quantitative, real-time PCR

Detection of Johne's disease, an enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), has been impeded by the lack of rapid, reliable detection methods. The goal of this study was to optimize methodologies for detecting M. paratuberculosis in manure from an infected dairy cow or in contaminated soil samples using a quantitative, real-time PCR (QRT-PCR) based analysis. Three different nucleic acid extraction techniques, the efficiency of direct versus indirect sample extraction, and sample pooling were assessed. The limit of detection was investigated by adding dilutions of M. paratuberculosis to soil. Results show that the highest yield (19.4+/-2.3 microg(-1) DNA extract) and the highest copy number of the targeted M. paratuberculosis IS900 sequence (1.3+/-0.2x10(8) copies g(-1) manure) were obtained with DNA extracted from manure using Qbiogene's Fast DNA Spin kit for soil. Pooling ten samples of M. paratuberculosis-contaminated soil improved the limit of detection ten fold (between 20 and 115 M. paratuberculosis cells g(-1) soil). Detection was between 65% and 95% higher when samples were extracted directly using bead-beating than when using pre-treatment with cell extraction buffers. The final soil-sampling and extraction regime was applied for detection of M. paratuberculosis in pasture soil after the removal of a M. paratuberculosis culture positive dairy cow. M. paratuberculosis remained in the pasture soil for more than 200 days. Results from these studies suggest that DNA extraction method, sampling protocol and PCR conditions each critically influence the outcome and validity of the QRT-PCR analysis of M. paratuberculosis concentrations in environmental samples.     

Paratuberculosis in saiga antelope (Saiga tatarica) and experimental transmission to domestic sheep.

Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted.     

Mycobacterium avium sub. paratuberculosis in tissue samples of Crohn's disease patients

Crohn's disease is a non-specific chronic transmural inflammatory disease. The disease was associated with a frameshit mutation in the NOD2 gene. Nevertheless, other researchers associated the presence of M. paratuberculosis within the intestinal tissues of patients with the disease. An adapted "in situ hybridization" technique was used to detect IS900 M. paratuberculosis DNA in paraffin embedded tissue from Crohns tissue disease samples. We were able to identify M. paratuberculosis DNA in around 69% of the paraffine embedded intestinal samples of Crohn's disease patients analysed. The presence of M. paratuberculosis DNA in the intestinal samples analysed does not necessarily mean that M. paratuberculosis is responsible for Crohn's disease. Our results support the hypothesis that infection may be caused by cell wall defective M. paratuberculosis since no bacteria were detected by Ziehl Neelsen stain.     

Possible association of GroES and antigen 85 proteins with heat resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65 degrees C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65 degrees C (D(65 degrees C) values; times required to reduce the concentration of bacteria by a factor of 10 at 65 degrees C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D(65 degrees C) value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 °C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.Communicated by G. Antranikian