Genes Required for Plasmid R64 Thin-Pilus Biogenesis: Identification and Localization of Products of the pilK, pilM, pilO, pilP, pilR, and pilT Genes

We have previously shown that the pilL, pilN, pilQ, pilS, pilU, and pilV genes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation. In this work, we characterized the products of the remaining essential genes, pilK, pilM, pilO, pilP, pilR, and pilT, with regard to their localization and processing. Overexpression systems containing pilM, pilO, and pilP genes fused with N-terminal glutathione S-transferase (GST) or a His tag were constructed. Overproduced proteins were purified and used to raise specific antibodies. Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively. The pilK, pilR, and pilT products were produced with a C-terminal His tag and then detected by anti-His tag antibody. Subcellular fractionation experiments with Escherichia coli cells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins. PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins. Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein. We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.     

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.     

The plasmid R64 thin pilus identified as a type IV pilus.

The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.     

铜绿假单胞菌蹭行运动相关基因的研究

应用Mu转座重组技术研究铜绿假单胞菌 (Pseudomonasaeruginosa)蹭行运动 (Twitchingmotility)的相关基因。通过转座突变、表型筛选 ,得到 8个Twitchingmotility缺陷或减弱的突变子。经过基因克隆、核苷酸测序研究 ,鉴定转座子插入到基因组中的位置。结果表明 ,在其中 4个突变子中 ,转座子分别插入到与IV型菌毛生物合成和功能相关的 3个已知基因中 (其中有两个突变子转座子插入到同一基因的不同位置 ) ,它们是pilV ,pilQ ,algR。另外 4个突变子中 ,有 3个是转座子分别插入到基因pilL基因的前端 ,中部和后端 ,均引起Twitchingmotility功能缺失。另一个突变子中 ,转座子插入到基因PA1 82 1中 ,引起Twitchingmotility功能减弱。PilL和PA1 82 1的编码产物均属于 3 类蛋白质 ,它们的功能是根据其保守的氨基酸基序或基因序列与已知功能基因的相似性推测得出的。但缺乏详细的试验证据。研究结果为pilL控制Twichingmotility提供了有力的证据。并证实基因PA1 82 1与Twitchingmotility有关。将Mu转座重组技术应用到假单胞菌的研究中 ,国内外均未见报道。由于该技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点。保证了表型的改变与转座子插入位点的基因突变的一一对应关系。为进一步研究铜绿假     

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.     

Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803

The relevance of pilus-related genes to motility, pilus structure on the cell surface and competency of natural transformation was studied by gene disruption analysis in the unicellular motile cyanobacterium SYNECHOCYSTIS: sp. PCC 6803. The genes disrupted in this study were chosen as related to the pil genes for biogenesis of the type IV pili in a Gram-negative bacterium PSEUDOMONAS: aeruginosa. It was found that motility of SYNECHOCYSTIS: cells was lost in the mutants of slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 together with a simultaneous loss of the thick pili on the cell surface. Competency of the natural transformation was lost in the mutants listed above and slr0197-disruptant. The gene slr0197 was previously predicted as a competence gene by a search with sequence-independent DNA-binding structure [Yura et al. (1999) DNA Res. 6: 75]. It was suggested that both DNA uptake for natural transformation and motility are mediated by a specific type IV-like pilus structure, while a putative DNA-binding protein encoded by slr0197 is additionally required for the DNA uptake. Based on the homology with the pil genes in P: aeruginosa, slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 were designated pilB1, pilM, pilN, pilO, pilQ and pilA1, respectively. The gene slr0197 was designated comA.     

Synthesis and metabolism of arachidonyl- and eicosapentaenoyl-CoA in rat aorta

In studies on the metabolism of polyunsaturated fatty acids, acyl-CoA synthetase for 5,8,11,14-20:4 (arachidonic acid) and 5,8,11,14,17-20:5 (eicosapentaenoic acid) and the incorporation of these fatty acids into complex lipids and their conversion to CO2 were investigated in rat aorta. The activity of acyl-CoA synthetase was 35.9 for arachidonic acid and 63.0 for eicosapentaenoic acid (nmol/mg protein per 10 min) and the apparent Km values were 45 microM for arachidonic acid and 56 microM for eicosapentaenoic acid. Inhibition of eicosapentaenoyl-CoA synthesis by arachidonic acid was stronger than that of arachidonyl-CoA synthesis by eicosapentaenoic acid. Arachidonic acid and eicosapentaenoic acid were mostly incorporated into phospholipids. The incorporation of these fatty acids into cholesterol ester and their conversion to CO2 were less than those of palmitic acid, but their incorporation into triacyglycerol was greater. The incorporation of these fatty acids into phosphatidylserine + phosphatidylinositol and phosphatidylethanolamine was also greater than that of palmitic acid. The patterns of incorporation of arachidonic acid and eicosapentaenoic acid were similar. The physiological roles of these polyunsaturated fatty acids and the interference of eicosapentaenoic acid in arachidonic acid metabolism are discussed on the basis of these results.     

Sterol and bile acid metabolism during development. 1. Studies on the gallbladder and intestinal bile acids of newborn and fetal rabbit

Bile acid composition and content in the intestine and gallbladder of newborn and fetal rabbits were investigated. Unlike the circumstances in adult rabbits, the bile acids were conjugated with both taurine and glycine. The major bile acids of the fetus and newborn rabbit were cholic acid, chenodeoxycholic acid, and deoxycholic acid. This is different from the known bile acid composition of adult rabbits, in which deoxycholic acid is the major bile acid (> 80%). The proportion of chenodeoxycholic acid was higher in the fetal than in the newborn tissues. The total bile acid pool in the newborn was higher than in the fetus. In the fetus, large proportions of bile acids (60.9%) were associated with the gallbladder fraction, whereas in the newborn the bulk of the bile acids were found with the intestinal fraction (64.4%),     

Aromatic and fatty acids of triterpene esters and rubber content of Hoya latices and their taxonomic significance

The acid composition and the rubber content of the latices of 20 Hoya species were determined. All acids isolated from the latices were esterified with triterpenols and particle-bound. Cinnamic acid was the main acid in most latices. Acetic acid occurred in all latices, and predominated in three of the investigated species. Isovaleric acid and benzoic acid were present in some species; in one latex (H. bella) isovaleric acid was the predominant acid. Almost all species contained small amounts of phenylpropionic acid, and in two species this acid was a major compound. Longchain fatty acids were present as minor compounds. Rubber occurred in all particle-containing latices; its contribution to the total lipid fraction ranged from 1.7–24.0%. The acid profiles proved to be species-specific in Hoya and may be useful in species identification. Patterns of latex particle synthesis are proposed.     

金刺梨果实和叶中酚类、Vc含量及其抗氧化能力分析

为了解贵州金刺梨（Rosa sterilis D.Shi）果实和叶片中的活性成分及其抗氧化活性,以贵州普定县金刺梨种植基地的果实和叶片为试材,测定其活性成分含量及其抗氧化活性,并对各项指标进行相关性分析。结果显示：没食子酸、芦丁、槲皮素、儿茶素、鞣花酸、绿原酸、阿魏酸是供试金刺梨果实和叶片的主要酚类成分,金刺梨果实和叶片中活性组分差异显著（P<0.05）,果实中p-香豆酸、总黄酮和抗坏血酸的含量相对较高,而叶片中没食子酸、儿茶素、绿原酸、表儿茶素、阿魏酸、鞣花酸、芦丁、槲皮素和总酚含量均高于果实;金刺梨果实抗氧化活性值均显著高于叶片（P<0.05）;相关性分析发现：总黄酮对总还原力（TRPA）值的贡献极强,抗坏血酸对Fe³⁺还原抗氧化能力（FRAP）值贡献最强,槲皮素对ABTS值的贡献最强,说明金刺梨果实和叶片是一种具有较高开发价值的药食同源资源。     

浓香型白酒窖泥中可培养细菌的分离鉴定及产酸研究

为系统了解浓香型白酒窖泥中可培养细菌的多样性,采用平板稀释涂布法分离筛选窖泥中可培养菌株。扩增纯培养细菌的16SrRNA基因,测序并与EzBioCloud数据库比对,所有序列已在GenBank中注册。结果共从窖泥中筛选出42株差异性较大的菌株,其中5株与模式菌株相似性低于97％,共包括14个属,以Bacillus、Lysinibacillus、Sporosarcina、Staphylococcus四个属为主;高效液相色谱检测各个菌发酵液结果表明,有机酸包括乙酸、乳酸、酒石酸、苹果酸、柠檬酸、琥珀酸、α-酮戊二酸、草酸;其中尤以乙酸、乳酸的产量较高。     

Isolation and characterisation of bis-phosphatidic acid and its partially deacylated derivatives from cultured BHK-cells

Bis-phosphatidic acid, semi-lyso-bis-phosphatidic acid and lyso-bis-phosphatidic acid were isolated from cultured baby hamster kidney fibroblasts (BHK-cells). They constituted about 0.02%, 0.1% and 1.6% respectively of total lipid phosphorus of fresh BHK-cell monolayers. These three phospholipids were also prepared by partial synthesis. The natural lipids were identical with the synthetic samples as determined by thin-layer chromatography and by partial degradation by controlled alkaline methanolysis, acetic acid hydrolysis and acetolysis. The three derivatives of bis-phosphatidic acid contained 60–80% of octadecenoic acid; in this respect they were different from other BHK-cell phospholipids.     

Linoleic acid and linolenic acid elongation products in muscle tissue of Syncerus caffer and other ruminant species

The metabolic elongation products of both linoleic acid and linolenic acid were found in muscle tissues of Syncerus caffer and other ruminants. The acids with four double bonds were predominantly in the linoleic acid series, whereas the higher degrees of unsaturation, mainly five double bonds, were in the linolenic acid series. The total linoleic acid and linolenic acid groups were present in the relative proportions of about 4:1, in contrast with the fish oils, where the acids are mainly in the linolenic acid series. The consistent occurrence of members of both groups of acids in the animals studied here suggests to us that both may be important for structural purposes.     

Bile acid profiles in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis

Bile acid profiles of bile, urine, and feces obtained from a patient with cerebrotendinous xanthomatosis on the same day have been analyzed by gas-liquid chromatography-mass spectrometry after fractionation into groups by mode of conjugation by an ion-exchange chromatography. The predominant biliary bile acid was cholic acid conjugated with glycine and taurine. Lesser amounts of the amino acid conjugates of chenodeoxycholic acid, ursodeoxycholic acid, 7-ketodeoxycholic acid, allocholic acid, and deoxycholic acid, and of unconjugated norcholic acid and allonorcholic acid were also present in the bile. The major fecal bile acid was 7-epicholic acid. Relatively large amounts of bile acids were excreted in the urine. Unconjugated 7-epicholic acid, norcholic acid, allonorcholic acid, and cholic acid predominated. The bile acid profiles of the patient were different from those of normal subjects and should be useful for the diagnosis.     

源于解淀粉芽胞杆菌酚酸脱羧酶的克隆与表达

【背景】酚酸脱羧酶催化分解酚酸产生的4-乙烯基酚类物质可用于食品添加剂及香精香料行业,而酚酸脱羧酶的表达水平相对较低,因此,高水平的酚酸脱羧酶是工业规模生产4-乙烯基酚类物质的先决条件。【目的】克隆解淀粉芽胞杆菌的酚酸脱羧酶基因,实现在大肠杆菌中的高效异源表达,分析酚酸脱羧酶的底物特异性,并对其表达条件进行优化。【方法】通过PCR技术获得酚酸脱羧酶的基因,构建重组基因工程菌,将测序结果与其他酚酸脱羧酶序列进行比对,利用IPTG诱导方法高效表达蛋白。将重组酚酸脱羧酶与4种不同的底物进行反应,设计响应面试验对诱导条件进行优化。【结果】酚酸脱羧酶对对香豆酸、阿魏酸、咖啡酸、芥子酸的比酶活比率为:100:23.33:15.39:10.51。结合与其他酚酸脱羧酶比对结果发现酚酸脱羧酶家族的C末端区域氨基酸序列的变异率最高,这与酚酸脱羧酶的底物特异性和催化机制有关。通过单因素和响应面试验得到酚酸脱羧酶诱导表达的最佳条件为:2×YT培养基,诱导温度30°C,接种量1.78%,诱导时机3.8 h,IPTG1.25mmol/L,诱导时间18h,此时预测酶活和实际酶活分别为47.61IU/mL和47.55...     

Synthesis and antiviral activity of ureides and carbamates of betulinic acid and its derivatives

Ureides and carbamates of betulinic acid and its derivatives were prepared in good yields by interaction of betulinic acid, betulonic acid, and betulonic acid 3-oxime with amines, amino acids, and alcohols. Ureides of betulonic acid containing L-Val and L-Met residues were found to be effective against herpes simplex type 1 virus. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

猫儿屎和三叶木通种子油中脂肪酸成分的GC-MS分析

采用索氏提取法提取木通科植物猫儿屎和三叶木通种子的脂溶性成分,甲酯化处理后用气相色谱-质谱联用技术(GC-MS)分离和鉴定其组成和含量。从猫儿屎种子油中鉴定出9种脂肪酸,占检出物总质量分数的94.67%(其中饱和脂肪酸占12.63%,不饱和脂肪酸占82.04%),主要成分为9-十六烯酸(47.22%)、9-油酸(27.13%)、棕榈酸(10.75%)、亚油酸(7.47%)和硬脂酸(1.61%)。从三叶木通种子油中鉴定出10种脂肪酸,占检出物总质量分数的99.75%(其中饱和脂肪酸占23.39%,不饱和脂肪酸占76.36%),主要成分为11-油酸(47.63%)、亚油酸(27.05%)、棕榈酸(20.14%)、16-甲基-十七烷酸(3.03%)和8-油酸(1.07%)。结果表明,猫儿屎和三叶木通种子油中脂肪酸含量丰富,在食用、医疗保健等方面具有较高的应用潜力和综合开发前景。     

Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells

Improved methods for lipid analysis that have been developed recently were employed to reevaluate the phospholipid composition, the fatty acid and fatty aldehyde composition of the total phospholipid, and the fatty acid composition of the individual phospholipids of normal human red cells. Thirty-three fatty acids and five fatty aldehydes were estimated and tentatively identified in the total phospholipid of normal human red cells. Additional minor components were evident. The major individual phospholipids were isolated by silicic acid thin-layer chromatography and quantified. The fatty acid compositions of phosphatidyl ethanolamine, phosphatidyl serine, lecithin, and sphingomyelin were determined. Each of these phospholipids showed a distinctive and characteristic fatty acid pattern.     

商品鹿鞭中氨基酸含量比较

采用氨基酸自动分析仪测定不同品种与产地的9份鹿鞭药材中氨基酸成分的种类及含量。这9份样品中均含17种氨基酸,其中人体必需氨基酸6种。17种氨基酸中以甘氨酸含量最高,必需氨基酸中亮氨酸含量最高;比较不同样品的含量,以辽宁西丰的梅花鹿鞭的总氨基酸含量最高,黑龙江梅花鹿鞭的必需氨基酸含量最高。结论为不同产地鹿鞭中氨基酸含量有一定差异,该方法可用于鹿鞭药材的质量评价。     

Influence of sex and gonadal hormones on rat-liver and carcass lipids during the development of an essential fatty acid deficiency

1. Groups of intact male and female rats and castrated rats injected with oestradiol or testosterone were given a diet containing hydrogenated coconut oil for 9 weeks, and at intervals the amounts and fatty acid compositions of the carcass and liver lipids were determined. 2. Male rats grew faster and larger, and exhibited typical external essential fatty acid deficiency symptoms sooner than did females. Testosterone-treated castrated male rats were similar to males, and oestradiol-injected castrated male rats resembled females. 3. Intact females maintained a higher linoleic acid concentration in their carcass than did males. Total amounts of carcass linoleic acid remained similar for all groups, only 200mg. being removed in 9 weeks regardless of body size. 4. The amounts of total cholesteryl esters were independent of liver size. They were higher in males and testosterone-treated castrated male rats than in females and oestrogen-treated castrated male rats. 5. Phospholipids represented about 80% of the liver lipids. The total amounts of the phospholipid linoleic acid and arachidonic acid were similar for all groups regardless of liver size, and were not affected appreciably by the deficiency. Females and oestrogen-treated castrated male rats maintained a higher proportion of phospholipid arachidonic acid for longer periods than did their male counterparts. Both the total amounts and the proportions of eicosatrienoic acid and palmitic acid were higher in males than in females. 6. Supplementation of the essential fatty acid-deficient diet with linoleic acid caused a rapid loss of eicosatrienoic acid and palmitic acid with a concomitant increase in stearic acid and arachidonic acid. 7. There were no obvious differences in the way that the essential fatty acids were metabolized or mobilized from adipose tissue of male or female rats during essential fatty acid deficiency. 8. The results indicated that the greater growth rate of the male rats caused them to require and synthesize more phospholipids than did the females. In the absence of adequate amounts of arachidonic acid, eicosatrienoic acid was substituted into the additional phospholipid. The earlier symptoms of essential fatty acid deficiency in the male rat could therefore be ascribed to the higher tissue concentrations of this unnatural phospholipid and its inability to perform the normal metabolic functions of phospholipids.