植物病原细菌Ralstonia solanacearum GMI1000中分泌蛋白信号肽分析

利用SignalP 3.0、TMHMM 2.0、TargetP 1.01、LipoP 1.0和PSORTb蛋白分析软件并结合L值计算,对植物病原细菌Ralstonia solanacearum GMI1000菌株基因组中的全部3 440个ORFs进行了分析预测,确定其中186个ORFs所编码蛋白质的N-端有信号肽序列,且它们的氨基酸残基相对保守.其中134条具有分泌型信号肽,22条具有RR-motif型信号肽,30条具有信号肽酶Ⅱ型信号肽.对各类信号肽及其结构域的长度作了系统的分析.未发现Prepilin-like信号肽和细菌素和信息素信号肽.     

繁缕核糖体失活蛋白基因克隆及序列分析

采用同源克隆结合RACE法,克隆了繁缕核糖体失活蛋白的全长cDNA,命名为q3(GenBank accession GQ870262)。序列分析结果表明,q3的开放阅读框(ORF)长780 bp,编码259个氨基酸。序列G+C含量为41.5%,与大部分Ⅰ型RIP基因相近。q3编码的蛋白质命名为Q3,理论分子量为28.16 kD,pI为9.44,均与Ⅰ型核糖体失活蛋白相近;包含由23个氨基酸组成的信号肽。功能结构域分析发现,该蛋白含有3个蛋白激酶磷酸化位点、4个络氨酸蛋白激酶磷酸化位点和7个N-肉豆蔻酰化位点。三级结构预测发现,有35.52%的氨基酸残基参与了α螺旋,24.32%的氨基酸残基组成延伸链,40.15%的氨基酸残基随机缠绕其中。基于繁缕及其近缘种核糖体失活蛋白的氨基酸序列构建的系统发育树显示,其结构与经典分类结果基本一致。     

信号肽结构有种属特异性变异

虽然与蛋白质分泌有关的信号肽在原核和真核生物中具有相同的模式,即由带正电荷的短氨基末端(N区)、具有较强极性并含有信号肽酶切割位点的羧基末端(C区)和处于两者之间的中心疏水区(H区)组成的一段短肽,但在应用统计学方法对原核和真核信号肽的长度和三个区的氨基酸组成进行比较之后,发现信号肽在种属之间存在明显的不同。首先,真核生物的信号肽平均长度较来自原核的短,而革兰氏阳性菌的信号肽又长于革兰氏阴性菌。其次,信号肽N区碱性氨基酸残基的组成存在种属差异。在H区,真核生物信号肽具有更高的疏水性。在不同种类生物中,信号肽C区的差异则与信号肽酶的专一性的细小差别     

立枯丝核菌AG-3全基因组分泌蛋白的预测分析

[目的]预测、分析立枯丝核菌AG-3全基因组范围内的分泌蛋白，并明确其基本特征，筛选其效应蛋白。[方法]依据已经公布的立枯丝核菌AG-3全基因组数据库中的12 726个蛋白序列，利用信号肽预测软件SignalP-4.1,细胞器定位分析软件ProtComp 9.0,跨膜螺旋结构预测软件TMHMM 2.0, GPI-锚定位点预测软件big-PI Fungal Predictor和亚细胞器中蛋白定位分布预测软件TargetP-1.1进行典型分泌蛋白的预测分析，并用LipoP-1.0进行信号肽切割位点的预测分析，最后对预测得到的分泌蛋白通过EffectorP进行效应蛋白的预测。[结果]在立枯丝核菌AG-3中有401个蛋白被预测为分泌蛋白，其编码蛋白长度集中于100～600 aa。信号肽长度介于11～35 aa之间，-3至-1位置上的氨基酸相对保守，切割位点为A-X-A类型，可被SpⅠ型信号肽酶识别并切割。在预测得到的分泌蛋白中通过EffectorP筛选得到140个效应蛋白。[结论]通过全基因组预测得到401个具有典型分泌蛋白特征的蛋白，从预测的分泌蛋白中筛选得到140个效应蛋白。     

全基因组预测禾谷炭疽菌的分泌蛋白

目的:禾谷炭疽菌侵染玉米、小麦等农作物引起的炭疽病,给农业生产造成了巨大经济损失。分泌蛋白在植物病原菌对植物侵染、定殖、扩展等致病过程中,发挥着重要作用。对禾谷炭疽菌分泌蛋白进行预测,并对其特征进行明确。方法:利用SignalP、ProtComp、TMHMM、big-PI Fungal Predictor和TargetP预测程序对禾谷炭疽菌中120 006条蛋白质序列进行分泌蛋白找寻,同时,对上述分泌蛋白的氨基酸分布、信号肽长度大小、信号肽切割位点以及理化性质等进行分析。结果:禾谷炭疽菌含有分泌蛋白为630个,其氨基酸长度多集中于100~600 aa之间;信号肽长度以17~20个aa的序列最为集中;信号肽切割位点属于A-X-A类型;分泌蛋白在分子量、等电点、稳定系数方面均存在差异,但大多数分泌蛋白属于亲水性蛋白。此外,上述分泌蛋白中,具有预测功能的蛋白为330个,其功能较多的集中于酶类,包括α-半乳糖苷酶、β-木聚糖酶、β-木糖苷酶等。结论:通过上述生物信息学分析方法有效地实现了禾谷炭疽菌分泌蛋白的预测,分泌蛋白的信号肽切割位点类型与已经报道的致病疫霉、粗糙脉孢霉等分泌蛋白信号肽切割位点一致,分泌蛋白功能涉及较多的酶类,也与其他已经报道的病菌分泌蛋白功能相类似。     

黄鳝cGnRH-Ⅱ的cDNA克隆与序列分析

促性腺激素释放激素（Conadotropin-releasing hormone,GnRH）是一个保守的十肽神经家族激素,在脊椎动物的性腺发育和繁殖功能的维持方面起着重要的调控作用。本文通过运用RACE和RT-PCR方法,从黄鳝脑组织中克隆得到cGnRH-ⅡcDNA全序列,其核苷酸序列长度为617bp。该cDNA编码的cGnRH-Ⅱ的前体氨基酸序列结构组成与其他物种的cGnRH-Ⅱ前体结构一致,其推导的蛋白前体长度为83个氨基酸,包括一个信号肽、cGnRH-Ⅱ十肽和一个由蛋白水解位点（Gly—Lys—Arg）连接的GnRH联接肽,其中信号肽和联接肽的长度分别为21和49个氨基酸。cGnRH-Ⅱ的氨基酸序列和其他相关物种cGnRH-Ⅱ氨基酸序列比较结果显示,cGnRH-Ⅱ cDNA的蛋白编码区高度保守,而非编码区的保守性程度很低。     

基于Z曲线方法重新注释脑膜炎奈瑟菌MC58株基因组

已测序的微生物基因组中包含的注释开放阅读框(open reading frames,ORFs)可以分为两大类:第一类对应于功能已知的蛋白质编码基因;第二类则为功能未知的假设ORFs,其中通常有一部分实际上不编码蛋白质。采用基于Z曲线的方法从属于第一类的功能已知基因出发训练参数,进而确定第二类ORFs中非编码的部分。通过支持向量机的学习及分类,结果显示十重交叉检验平均正确率为98.45%,说明Z曲线联合支持向量机是一种高度准确的基因识别方法。最终,确定216个假设ORFs实际上不编码蛋白质。通过采用Blastp进行序列比对,保留的假设ORFs中有341个在高可靠性的条件下获得了功能信息。根据蛋白质直系同源簇方法进行功能分类,分别有30、53、59和159个新注释的假设ORFs属于信息储存和加工类、细胞加工和信号传递类、新陈代谢类和特征不明显类。另外还有70个不属于其中的任何一类。注释结果比RefSeq及GenBank提供的原注释更加准确,更加完整。     

全基因组预测希金斯炭疽菌的候选效应分子

[目的]希金斯炭疽菌可以侵染十字花科诸多植物引起炭疽病,给各国农业生产造成了巨大经济损失。对该菌候选效应分子进行预测,并对其特征进行明确。[方法]利用Signal P、Prot Comp等程序对该菌中分泌蛋白进行找寻,并对分泌蛋白所具有的氨基酸大小、信号肽长度以及理化性质等进行分析。结合已经报道的真菌效应分子具有的典型特征,明确该菌存在的候选效应分子数量及特征。[结果]希金斯炭疽菌含有658个分泌蛋白,氨基酸长度集中于50~300 aa之间;信号肽长度以17~21个aa的序列最为集中;信号肽切割位点属于A-X-A类型;分泌蛋白在分子量、等电点、不稳定系数等方面均存在差异,但大多数分泌蛋白属于亲水性蛋白。其中,预测功能的蛋白为290个,其功能较多的集中于酶类,包括脂肪酶、α-L-鼠李糖苷酶、果胶酯酶等。依据候选效应分子典型特征,明确该菌中含有388个候选效应分子。[结论]通过上述生物信息学分析方法,结合真菌效应分子典型特征,有效地实现了希金斯炭疽菌候选效应分子的预测,其信号肽切割位点类型与其他已经报道的致病疫霉、粗糙脉孢霉等分泌蛋白信号肽切割位点一致,分泌蛋白功能涉及较多的酶类,也与其他已经报道的病菌分泌蛋白功能相类似。     

全基因组预测糙皮侧耳经典胞外分泌蛋白

糙皮侧耳(平菇)作为目前木质纤维素降解模式真菌,其产生的胞外分泌蛋白在植物纤维素、半纤维素及木质素降解过程中发挥着重要作用。为了给蛋白质组学鉴定糙皮侧耳胞外分泌蛋白实验提供参考,利用常见的生物信息学工具SignalP、ProtComp、TMHMM、big-PI Fungal Predictor和TargetP对糙皮侧耳全基因组中的12 186条蛋白质序列进行经典分泌蛋白预测,同时,对上述分泌蛋白的氨基酸分布、信号肽长度和切割位点以及其理化性质等进行分析。结果表明,糙皮侧耳全基因组中含有359个经典分泌蛋白,其氨基酸长度主要集中于101~600之间;信号肽长度集中在18~21之间;信号肽切割位点属于A-X-A类型,除此之外还含有5条具有RR-motif的信号肽。对这些分泌蛋白进行功能注释,结果表明225个蛋白质为未知功能蛋白质;114个为已知功能蛋白质,主要功能注释集中于木质纤维素降解酶类,包括纤维素酶、半纤维素酶、木质素降解相关酶等。以上结果表明通过上述生物信息学分析实现了对糙皮侧耳全基因组经典分泌蛋白的有效预测。     

牛催乳素基因组及其cDNA全长序列的分子克隆和分析

通过LongPCR等技术首次克隆得到全长9388bp的牛催乳素（bPRL)基因组序列（GenBank登录号AF426315）,其中包括bPRL基因全部5个外显子和4个内含子,5′端854bp的上游调控区以及3′端69bp的UTR,AF426315基因编码的蛋白质在GenBank中的序号为AAL28075,由229个氨基酸残基组成,1-30位氨基酸残基为信号肽序列,成熟的多肽含有199个氨基酸残基,将bPRL基因组DNA真核表达载体转染COS-7细胞后通过RT-PCR得到长度为804bp的bPRLcDNA序列,该序列涵盖了bPRL基因的全部ORF区,证明本研究所获得的bPRL基因组DNA具有转录的生物学功能,Blast搜索结果显示,GenBank数据库中收集有多条bPRL基因的mRNA和EST序列,各序列间存在多个SNP位点,主要分布于下游编码区和3′端的UTR,这些位点均未改变相应的氨基酸残基的性质,此外,5′端编码信号肽序列的区域呈现高度保守性。     

Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato

Avirulence gene D, cloned from Pseudomonas syringae pv. tomato, caused P. s. pv. glycinea to elicit a hypersensitive defense response on certain cultivars of soybean. Nucleotide sequence data for a 5.6-kb HindIII fragment containing avrD disclosed five long open-reading frames (ORFs) occurring in tandem. The phenotype conferred by avrD was expressed in P. s. pv. glycinea solely by the first of these ORFs (933 bases) that encoded a protein of 34,115 daltons. Neither a signal peptide sequence nor significant regions of hydrophobicity were present that would indicate secretion of the protein or its membrane association. Hybridization analyses revealed that some but not all P. syringae pathovars contained DNA homologous to avrD. This included weak hybridization to all tested races of P. s. pv. glycinea, although none of them express the phenotype conferred by avrD. The avrD gene occurred on an indigenous 75-kb plasmid in several P. s. pv. tomato isolates.     

Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility of Pseudomonas syringae pv. tomato DC3000

Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to "mine" the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.     

Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.     

Production and comparison of peptide siderophores from strains of distantly related pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352

The production of peptide siderophores and the variation in siderophore production among strains of Pseudomonas syringae and Pseudomonas viridiflava were investigated. An antibiose test was used to select a free amino acid-containing agar medium favorable for production of fluorescent siderophores by two P. syringae strains. A culture technique in which both liquid and solid asparagine-containing culture media were used proved to be reproducible and highly effective for inducing production of siderophores in a liquid medium by the fluorescent Pseudomonas strains investigated. Using asparagine as a carbon source appeared to favor siderophore production, and relatively high levels of siderophores were produced when certain amino acids were used as the sole carbon and energy sources. Purified chelated siderophores of strains of P. syringae pv. syringae, P. syringae pv. aptata, P. syringae pv. morsprunorum, P. syringae pv. tomato, and P. viridiflava had the same amino acid composition and spectral characteristics and were indiscriminately used by these strains. In addition, nonfluorescent strains of P. syringae pv. aptata and P. syringae pv. morsprunorum were able to use the siderophores in biological tests. Our results confirmed the proximity of P. syringae and P. viridiflava; siderotyping between pathovars of P. syringae was not possible. We found that the spectral characteristics of the chelated peptide siderophores were different from the spectral characteristics of typical pyoverdins. Our results are discussed in relation to the ecology of the organisms and the conditions encountered on plant surfaces.     

Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast

The plant apoplast is the intercellular space that surrounds plant cells, in which metabolic and physiological processes relating to cell wall biosynthesis, nutrient transport, and stress responses occur. The apoplast is also the primary site of infection for hemibiotrophic pathogens such as P. syringae, which obtain nutrients directly from apoplastic fluid. We have used apoplastic fluid extracted from healthy tomato leaves as a growth medium for Pseudomonas spp. in order to investigate the role of apoplastic nutrients in plant colonization by Pseudomonas syringae. We have confirmed that apoplast extracts mimic some of the environmental and nutritional conditions that bacteria encounter during apoplast colonization by demonstrating that expression of the plant-induced type III protein secretion pathway is upregulated during bacterial growth in apoplast extracts. We used a modified phenoarray technique to show that apoplast-adapted P. syringae pv. tomato DC3000 expresses nutrient utilization pathways that allow it to use sugars, organic acids, and amino acids that are highly abundant in the tomato apoplast. Comparative analyses of the nutrient utilization profiles of the genome-sequenced strains P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, P. syringae pv. phaseolicola 1448A, and the unsequenced strain P. syringae pv. tabaci 11528 with nine other genome-sequenced strains of Pseudomonas provide further evidence that P. syringae strains are adapted to use nutrients that are abundant in the leaf apoplast. Interestingly, P. syringae pv. phaseolicola 1448A lacks many of the nutrient utilization abilities that are present in three other P. syringae strains tested, which can be directly linked to differences in the P. syringae pv. phaseolicola 1448A genome.     

大肠埃希氏杆菌UTI89分泌蛋白组的预测分析

目的：从大肠埃希氏杆菌UTI89基因组中筛选出全部潜在的分泌蛋白并进行初步研究。方法：使用SignalP3.0、TatP1.0、 SecretomeP2.0等蛋白分析软件对5211个ORF进行预测;对筛选出的信号肽及分泌蛋白的基本特征进行统计学分析;使用Blast 2 Sequences进行同源性分析。结果：共筛选出432个sec途径分泌蛋白,19个Tat途径分泌蛋白,386个非经典分泌蛋白;信号肽、分泌蛋白平均长度分别为25.5aa、282.8aa;信号肽中出现频率最高的3种氨基酸依次为L、A、S;仅有两个信号肽的氨基酸序列完全相同,相应的分泌蛋白高度同源。结论：大肠埃希氏杆菌UTI89基因组中有837个ORF可能编码分泌蛋白;分泌蛋白集中在500aa以下;组成信号肽的氨基酸相对保守,多数为疏水氨基酸;信号肽变异性较大,含相同信号肽的蛋白可能由同源基因编码。     

A gene from Pseudomonas syringae pv. glycinea with homology to avirulence gene D from P. s. pv. tomato but devoid of the avirulence phenotype

A gene was cloned from Pseudomonas syringae pv. glycinea that hybridized to avirulence gene D (avrD), previously cloned from P. s. pv. tomato. Unlike avrD, the hypersensitive response (HR) was not elicited when the P. s. pv. glycinea gene was reintroduced into P. s. pv. glycinea race 4 on a broad host range plasmid and the bacteria were inoculated into soybean leaves. DNA sequence data disclosed that the P. s. pv. glycinea homologue of avrD encoded a protein containing 86% identical amino acids to avrD, with substitutions distributed throughout the protein. Two ORFs immediately downstream from the avrD homologue were more similar in P. s. pv. tomato and P. s. pv. glycinea, with 98 and 99% identical amino acids. Expression of the wildtype P. s. pv. glycinea gene and recombinant genes constructed between the P. s. pv. tomato avrD gene and its P. s. pv. glycinea homologue in both Escherichia coli and P. s. pv. glycinea indicated that the P. s. pv. glycinea gene product was formed less efficiently or was less stable than was the P. s. pv. tomato protein encoded by avrD. The data indicated that the P. s. pv. glycinea homologue represents a recessive allele of the P. s. pv. tomato avrD gene which has been modified by mutation such that it does not lead to an avirulence phenotype on the normal host plant, soybean.     

Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase.

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant.     

Characterization and expression of two avirulence genes cloned from Pseudomonas syringae pv. glycinea.

Two avirulence genes, avrB and avrC, from race 0 of Pseudomonas syringae pv. glycinea, were sequenced and found to encode single protein products of 36 and 39 kilodaltons, respectively. The proteins had neither recognizable signal peptide sequences nor significant stretches of hydrophobic amino acids that might indicate membrane association. Both avrB and avrC had relatively low position 3 and overall G+C contents, which suggests that they may have been recently introduced into P. syringae pv. glycinea. The deduced amino acid sequences of the proteins encoded by avrB and avrC shared 42% identical amino acids. However, when introduced into race 4 of P. syringae pv. glycinea, each gene directed a unique pattern of hypersensitive reactions on several differential soybean cultivars. The avrC protein was overproduced in Escherichia coli cells and deposited as insoluble inclusion bodies in the cell cytoplasm. The avrC protein could be solubilized with urea-octyl glucoside treatment, but neither the solubilized protein nor the intact inclusion bodies elicited a hypersensitive reaction in soybean leaves.