转录调节因子σ~(38)介导铜绿假单胞菌绿脓菌素合成代谢调控

【目的】为了进一步鉴定铜绿假单胞菌转录调控因子σ~(38)对2个拷贝吩嗪合成基因簇(phz A1-G1和phz A2-G2)的具体调控方式并推定介导绿脓菌素合成代谢的可能调控机制。【方法】根据铜绿假单胞菌基因组信息,利用同源重组原理构建rpo S基因缺失突变株Δrpo S以及克隆全长rpo S基因作互补分析;再以单一吩嗪基因簇缺失突变株Δphz1和Δphz2为出发菌株,分别构建rpo S缺失突变株Δrpo Sphz1和rpo S插入突变株Δrpo Sphz2,测定并比较野生株及相关突变株的绿脓菌素合成量,初步推定σ~(38)因子对2个不同吩嗪基因簇表达的调控方式。【结果】在GA培养基中,突变株Δrpo S的绿脓菌素合成量比野生株显著增加;互补分析证实,σ~(38)可使突变株Δrpo S的绿脓菌素降低并接近野生株PAO1水平;与对照株Δphz1相比,突变株Δrpo Sphz1的绿脓菌素合成量因σ~(38)因子缺失而显著减少;而与对照株Δphz2相比,突变株Δrpo Sphz2的绿脓菌素合成量因σ~(38)因子缺失显著增加。【结论】转录调控因子σ~(38)对铜绿假单胞菌绿脓菌素的合成代谢的确具一定的负调控作用;结合已报道的研究结果,初步推定:σ~(38)因子通过负调控吩嗪基因簇phz1,正调控吩嗪基因簇phz2的表达实现对绿脓菌素合成代谢的调控。     

铜绿假单胞菌全局调控因子RsmA的缺失影响2个吩嗪基因簇的表达

[目的]为了研究铜绿假单胞菌全局调控因子RsmA对两个吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制.[方法]采用基因缺失和抗性基因(gentamycin resistance cassette,aacC1)插入相结合的策略构建了rsmA基因缺失突变株PA-RG ;通过构建互补表达载体和过表达载体,进一步确认RsmA对绿脓菌素的调控作用 ;采用电转化方法将构建的翻译融合表达载体pMEZ1(phz1'-'lacZ)和pMEZ2(phz2'-'lacZ)分别导入铜绿假单胞菌突变株PA-RG和野生株PAO1,采用Miller法测定融合β-半乳糖苷酶活性.[结果]在GA培养基中,互补分析和过表达分析表明,RsmA抑制绿脓菌素的合成.此外,pMEZ1在突变株PA-RG中的表达增强,为野生株的2-3倍 ;而pMEZ2在突变株PA-RG中的表达降低,野生株是突变株的2倍.[结论]由此初步判定,铜绿假单胞菌全局调控因子RsmA对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上RsmA负调控phz1,正调控phz2.     

藤黄绿脓菌素的自诱导及假单胞菌M18抗生物质代谢相关性初步分析

假单胞菌M18的生防功能归功于其分泌吩嗪-1-羧酸和藤黄绿脓菌素。为了研究抗生物质合成代谢相关性及调控机制,分别构建了两种抗生物质合成基因簇插入突变株M18T和M18Z1。用翻译融合表达载体pMEAZ(pltA′-′lacZ)分别转化野生株和突变株M18T、发酵培养并测定β-半乳糖苷酶活性,结果显示,添加藤黄绿脓菌素使突变株M18T(pMEAZ)的β-半乳糖苷酶活性比野生株M18(pMEAZ)增加约6倍,表明藤黄绿脓菌素对自身基因簇具正向自诱导作用。抗生物质的测定结果显示,突变株M18T无藤黄绿脓菌素合成,而吩嗪-1-羧酸的合成量与野生株相同;突变株M18Z1与野生株相比,吩嗪-1-羧酸明显减少,藤黄绿脓菌素却显著提高。过量的吩嗪-1-羧酸又抑制藤黄绿脓菌素的合成。表明,假单胞菌M18中独有的代谢相关方式为:藤黄绿脓菌素不影响吩嗪-1-羧酸,但吩嗪-1-羧酸负调控藤黄绿脓菌素。     

rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控

【目的】为了研究铜绿假单胞菌rpoS基因对吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用抗庆大霉素基因(gentamycin resistance cassette,aacC1)插入失活的策略构建了rpoS基因突变株PA-SG;同时利用lacZ的翻译融合表达载体pME6015,构建了phz1′-′lacZ和phz2′-′lacZ翻译融合表达载体pMEZ1和pMEZ2。采用电转化法分别将pMEZ1、pMEZ2和pME6015导入铜绿假单胞菌突变株PA-SG和野生株PAO1,用Miller法检测融合β-半乳糖苷酶活性。【结果】在KMB或PPM培养基中,pMEZ1在突变株PA-SG中的表达均增强,为野生株的4-5倍;而pMEZ2在突变株PA-SG中的表达均降低,野生株是突变株的2-3倍。【结论】由此推测,铜绿假单胞菌rpoS基因对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上,rpoS负调控phz1,正调控phz2。     

MvaT调控铜绿假单胞菌吩嗪的合成机制

【背景】属于H-NS家族的MvaT转录因子参与了铜绿假单胞菌的许多重要代谢过程,如吩嗪合成代谢,但其调控方式仍不十分明确。【目的】确定转录调控因子MvaT是否直接调控铜绿假单胞菌的吩嗪合成过程,即该蛋白是否可以直接结合2个吩嗪-1-羧酸合成基因簇(phzA1G1和phzA2G2)与3个分支转化基因(phzH、phzS和phzM)的上游启动子区域。【方法】以铜绿假单胞菌SJTD-1和其mvaT基因敲除突变株SJTD-1(ΔmvaT)为研究对象,检测其在不同培养基条件下吩嗪化合物的合成量差异。通过体外异源表达与亲和纯化,获得重组蛋白MvaT。利用凝胶阻滞实验,确定MvaT重组蛋白对5个吩嗪代谢基因簇/基因上游启动子的结合情况。【结果】mvaT基因敲除突变株SJTD-1(ΔmvaT)的吩嗪产量较野生型显著提升。MvaT重组蛋白被有效表达与纯化,体外凝胶阻滞实验结果显示,该重组蛋白可与phzA1G1、phzA2G2、phzM、phzS和phzH的上游启动子区域均发生特异性结合。其中,重组蛋白MvaT与phzA1G1和phzA2G2的结合区域位于其上游启动子的200 bp以内,而该蛋白与phzM、phzS和phzH的结合区域则位于其上游启动子的100 bp以内。【结论】MvaT蛋白通过直接结合吩嗪合成代谢基因的上游启动子区域来直接调控假单胞菌的吩嗪类化合物合成。     

RNA聚合酶σ^38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。     

RNA聚合酶σ38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3.1kb EcoRⅠ_XhoⅠ片段。通过抗性基因（抗庆大霉素基因）的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪_1_羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10.2μg/mL分别减少到20.4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0.5μg/mL和20.5μg/mL分别提高到75.4μg/mL和185.6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β_半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果: σ38亚基正调控吩嗪_1_羧酸的表达,而负调控藤黄绿脓菌素的表达。     

假单胞菌GP72 rpeB突变株的构建及其对吩嗪类抗生素合成的调控

[目的]绿针假单胞菌GP72是一株可生产吩嗪类抗生素吩嗪-1-羧酸(PCA)及2-羟基吩嗪(2-OH-PHZ)的生防假单胞菌.RpeA/RpeB双元调控系统是其双元调控系统中的一组,本文旨在研究这一系统中的应答调控子(RR)RpeB对于PCA及2-OH-PHZ的生物合成影响.[方法]通过生物信息学分析获得了rpeA/rpeB双元调控系统的序列,并从GP72中扩增出rpeB基因,通过同源重组技术构建卡那霉素抗性片段插入突变rpeB的突变菌株GP72BN.利用发酵实验、rpeB基因回补实验及荧光定量PCR实验,验证rpeB对于吩嗪类抗生素合成及相关基因表达的调控作用.[结果]在KMB培养基中,rpeB突变株的PCA产量下降为野生型的49.5％,2-OH-PHZ产量下降为野生型的67.3％.rpeB基因的回补可以在一定程度上回复PCA及2-OH-PHZ的产量.荧光定量PCR实验结果表明,rpeB突变株中群体感应系统基因phzI/phzR及吩嗪合成基因簇基因phzE转录水平均显著下调,而PCA转化为2-OH-PHZ的修饰基因phzO转录水平变化不显著.[结论]RpeB正调控PCA与2-OH-PHZ合成途径的表达.RpeB很可能是通过调控群体感应基因phzI/phzR和phz基因簇的表达,从而影响PCA的合成,并间接调控其衍生物2-OH-PHZ的合成.     

铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

【目的】在假单胞菌中,小RNA(sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控,通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用,深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术,构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY,通过基因过表达、lacZ报告基因融合分析实验,进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量,突变菌株M18RY中PCA的产量显著增加,为野生型菌株的5倍左右,而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明,rsmY基因区别性调控PCA和Plt的生物合成。     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Changes in lactate dehydrogenase activity in chicken tissues in hyperthyreosis in ontogenesis

The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.     

Scurvy in capybaras bred in captivity in Argentine

In order to determine if the absence of vitamin C in the diet of capybaras (Hydrochoerus hydrochaeris) causes scurvy, a group of seven young individuals were fed food pellets without ascorbic acid, while another group of eight individuals received the same food with 1 g of ascorbic acid per animal per day. Animals in the first group developed signs of scurvy-like gingivitis, breaking of the incisors and death of one animal. Clinical signs appeared between 25 and 104 days from the beginning of the trial in all individuals. Growth rates of individuals deprived of vitamin C was considerably less than those observed in the control group. Deficiency of ascorbic acid had a severe effect on reproduction of another population of captive capybaras. We found that the decrease in ascorbic acid content in the diet affected pregnancy, especially during the first stages. The results obtained suggest that it is necessary to supply a suitable quantity of vitamin C in the diet of this species in captivity.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.