十字花科黑腐病菌中转录调控因子HpaR1调控一个纤维素酶基因的表达

十字花科黑腐病菌（Xcc8004）中的一个转录调控因子XC₂736（HpaR1）在致病过程中具有重要的作用。前期研究发现该转录调控因子可能调控胞外纤维素酶的合成。为了解HpaR1对纤维素酶的转录调控机理,本研究对HpaR1进行原核表达纯化,并与488bp的包含XC₀639的启动子区DNA片段进行凝胶电泳迁移率试验,发现HpaR1与XC₀639启动子可以发生结合。将488bp的XC₀639的启动子DNA片段与报告基因gus融合,构建XC₀639的报告质粒pGUS0639r,分别导入野生型8004菌株和缺失突变体DM2736中,分析发现在突变体背景下GUS的表达水平比野生型背景明显降低。表明HpaR1正调控XC₀639的表达。构建XC₀639的极性整合突变体PK0639,检测发现PK0639几乎丧失胞外纤维素酶的活力;通过功能反式互补构建的互补菌株CPK0639可以恢复纤维素酶活性。研究结果表明HpaR1通过调控纤维素酶基因XC₀639的表达来调控细胞的纤维素酶活性。本研究为更深入地了解HpaR1如何调控细菌生理生化功能奠定了基础。     

用生物信息学方法寻找肝癌特异性表达基因转录调控模式

为了对肝癌(hepatocellularcarcinoma,HCC)的分子发病机理进行研究,首先对肝癌基因表达谱数据用t-检验算法进行了分析,找到了肝癌中特异性表达基因(characteristicgenes).然后把这些基因结合已知的肝HNF家族转录因子染色质免疫共沉淀结合DNA启动子芯片(ChIP-chip)实验数据用SAEM算法进行分析,得到了肝癌特异性表达基因的转录调控关系,并寻找到了多个HNF家族转录因子调控单基因的转录调控模式.结果表明HNF家族转录因子对大量具有重要功能的肝癌特异性表达基因进行了转录调控,并且多个HNF家族转录因子调控单基因可以形成前馈环和多输入调控等模式.     

十字花科黑腐病菌中转录因子HpaR1与Clp调控一个糖苷水解酶基因表达的分析

十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)是一种维管束致病菌,能够引起寄主的黑腐病,是研究植物病原细菌与植物互作的一种重要模式菌株.在Xcc中,GntR家族的全局性转录调控因子HpaR1参与调控Xcc的运动、胞外多糖和胞外酶的合成等许多细胞过程,并与Xcc的过敏...     

CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

GFP用于单核细胞增生李斯特菌PrfA调控毒力基因actA转录表达的研究

PrfA是单核细胞增生李斯特菌(LM)中迄今为止发现的惟一个调控绝大多数毒力基因转录表达的蛋白因子.为了研究PrfA转录调控毒力基因表达的分子机制,将无启动子的绿色荧光蛋白(GFP)基因与毒力基因actA的启动子融合,连接到穿梭载体pLSV16质粒上,构建成表达融合载体pLSV16-PactA-gfp,然后将其电转化入LM野生株P14、PrfA高表达突变株P14a和prfA基因等位缺失突变株A42中表达.利用荧光显微镜和荧光酶标仪检测上述3株细菌中绿色荧光蛋白的不同表达强度,从而评价actA基因依赖于PrfA的转录活性强弱.结果显示,绿色荧光蛋白在P14a中发出的荧光强度最高,P14次之,A42最弱,两两比较均有显著差异(P＜0.01),表明毒力基因actA的转录水平高低与PrfA的活性成正相关,其转录表达依赖于PrfA的调控;该试验同时也显示GFP能方便、有效地用于研究PrfA调控LM不同毒力基因的转录表达水平.     

细菌DeoR家族转录调控因子的研究进展

细菌基因组中存在大量的转录调控家族,这些转录调控家族在细菌的生长、代谢、外界信号感知与传递等方面发挥着至关重要的作用.DeoR家族是一类广泛分布于原核生物中的转录调控因子,主要参与调控细胞中多个生理过程,包括核苷酸类代谢、糖类代谢、致病菌的毒力以及链霉菌的次级代谢等.DeoR蛋白C末端的配体结合结构域,通常能够以相关代...     

转录因子FboR调控耻垢分枝杆菌抗氧化生长的机制研究

结核病的致病菌结核分枝杆菌(Mycobacterium tuberculosis)在宿主内面临着多种氧化胁迫环境因子的压力,因而进化形成了一系列自己的抗氧化生长机制。转录因子作为细菌快速响应外界环境的重要因子,通过调控其靶基因的表达来帮助细菌适应环境胁迫如抗氧化等。然而,目前分枝杆菌(Mycobacterium)中有关转录因子调控细菌抗氧化生长的分子机制还不是十分清楚。本研究以耻垢分枝杆菌(Mycobacterium smegmatis)作为模式菌株,发现了转录因子FboR调控分枝杆菌的抗氧化能力并检测了相关重组菌株的抗氧化生长情况,证实了FboR负调控细菌的抗氧化能力。随后通过转录组测序分析、凝胶阻滞实验(electrophoretic mobility shift assay, EMSA)、实时定量PCR(real-time quantitative PCR, RT-qPCR)和β-半乳糖苷酶活性检测鉴定了影响细菌抗氧化生长的相关靶基因,成功解析了具体的调控通路与分子机制。     

十字花科黑腐病菌的XC1193基因与致病相关

十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)是一种重要植物病原细菌,在全球范围内侵染十字花科植物引起黑腐病,其σ因子在基因表达中起到重要调节作用。XC1193基因在Xcc 8004菌株编码一个σ~70因子,为进一步研究该σ因子在Xcc中的调控作用,利用自杀质粒p K18mobsac B构建了XC1193基因的缺失突变体DM1193。与野生型菌株的比较发现,XC1193基因突变不影响菌体在丰富培养基和基本培养基上的生长;突变体的胞外蛋白酶、纤维素酶、淀粉酶等生化表型也与野生型一致;植株实验表明XC1193突变不影响过敏反应。采用剪叶接种法进行致病性检测,突变体致病力与野生型一致。而采用喷雾接种法,突变体致病力显著降低,互补菌株致病力恢复至野生型水平。结果表明,该σ因子在十字花科黑腐病菌致病过程中发挥作用,并与Xcc侵染寄主的早期事件有关。     

茶树GRF基因家族的全基因组鉴定及表达分析

生长调控因子(growth regulating factor,GRF)是植物特有的转录因子家族,在植物生长发育过程中起重要调控作用。该研究利用生物信息学的方法,从茶树基因组中鉴定获得11个CsGRF转录因子家族成员,且均具有完整的特征结构域QLQ和WRC。CsGRF家族成员含有3~6个外显子,基于系统进化关系将CsGRF家族分为6组,且与葡萄及猕猴桃GRF家族的亲缘关系更为接近。不同组织转录组数据分析结果表明,该家族在生长活跃的茶树嫩梢部位高表达。上游启动子区域分析发现大量与植物发育、激素及胁迫响应密切相关的顺式作用元件。荧光定量检测发现,分别有10个和2个CsGRF成员在低温和干旱胁迫处理后呈现显著上调表达,其中CsGRF8和CsGRF11对2种非生物胁迫均有响应;并发现受ABA、MeJA和GA激素处理诱导分别有9个、3个和6个CsGRF基因的表达水平差异显著。研究表明,CsGRF基因家族在茶树生长发育过程及应激反应中起作用,推测CsGRF基因可能通过各种激素信号转导途径参与胁迫响应过程。     

鼠疫菌毒力调控研究

鼠疫菌通过一系列转录调控子(如CRP、PhoP、RovA和Fur)控制着一些关键毒力因子(如Pla、强毒力岛、III型分泌系统等)的基因表达。鼠疫菌可感应宿主体内信号刺激,紧密调控毒力因子的表达。在这个紧密调控过程中,调控子、毒力相关基因构成了一个动态网络。鼠疫菌在从假结核菌祖先演化的进程中,基因表达调控网络的重塑在鼠疫菌毒力进化过程中发挥着不可取代的作用。     

十字花科黑腐病菌中影响致病相关基因XC3814表达的基因鉴定

十字花科黑腐病菌8004菌株的XC3814基因与致病性和胞外多糖合成有关。文章将XC3814的启动子与报告基因sacB融合, 构建了XC3814的表达报告质粒pL3814sac。将该质粒导入野生型菌株8004, 获得了报告菌株8004/pL3814sac。利用转座子EZ::Tn5对报告菌株的基因组进行随机诱变, 分离到3株耐蔗糖的突变体。分析发现其中的1株突变体是由EZ::Tn5插入到编号为XC3882的未知功能的基因所产生的。将由XC3814启动子与报告基因gusA融合得到的报告质粒pGUS3814分别导入8004菌株和XC3882的转座子Tn5gusA5插入突变体, 测定比较pGUS3814的GUS表达水平, 结果显示在XC3882突变体背景下GUS的表达水平比在野生型背景下降低81.3%, 表明XC3814基因的表达水平受XC3882基因的影响。     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.     

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.     

Genome-scale mutagenesis and phenotypic characterization of two-component signal transduction systems in Xanthomonas campestris pv. campestris ATCC 33913

The gram-negative bacterium Xanthomonas campestris pv. campestris is the causal agent of black rot disease of cruciferous plants. Its genome encodes a large repertoire of two-component signal transduction systems (TCSTSs), which consist of histidine kinases and response regulators (RR) to monitor and respond to environmental stimuli. To investigate the biological functions of these TCSTS genes, we aimed to inactivate all 54 RR genes in X. campestris pv. campestris ATCC 33913, and successfully generated 51 viable mutants using the insertion inactivation method. Plant inoculation identified two novel response regulator genes (XCC1958 and XCC3107) that are involved in virulence of this strain. Genetic complementation demonstrated that XCC3107, designated as vgrR (virulence and growth regulator), also affects bacterial growth and activity of extracellular proteases. In addition, we assessed the survival of these mutants under various stresses, including osmotic stress, high sodium concentration, heat shock, and sodium dodecyl sulfate exposure, and identified a number of genes that may be involved in the general stress response of X. campestris pv. campestris. Mutagenesis and phenotypic characterization of RR genes in this study will facilitate future studies on signaling networks in this important phytopathogenic bacterium.     

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.