全基因组预测Phenylobacterium zucineum HLK1~T的外排蛋白
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摘要
Phenylobacterium zucineum是一种我们在人红白血病细胞株K562中发现的兼性胞内寄生菌,我们把首次发现的这一菌株命名为HLK1~T。由于该菌寄生在肿瘤细胞内,而目前大多数胞内寄生菌都是病原体,因此该菌很可能和致病性有关;由于该菌是兼性胞内寄生菌,所以在胞内和胞外的细菌生存形态不同,研究其侵入人宿主细胞的机制有着重要意义;由于本组的生物学实验研究表明,该菌的侵入方式为“拉链”方式,即通过表达一些表面蛋白,包括膜蛋白和分泌蛋白(即外排蛋白),与真核细胞表面受体相互作用,该受体-配体的结合引起宿主细胞骨架发生重排、细胞膜延伸并包裹该菌,形成吞噬泡并侵入细胞。所以寻找其表达的蛋白及受体-配体相互作用机制是研究中的一个重点。通过全基因组测序、基因注释、蛋白翻译以及搜索蛋白的N-端信号肽,跨膜α-螺旋等外排信号来预测其外排蛋白是一个很好的根本方法,它可以促进潜在生物学意义的发掘。
细菌基因组的全基因组测序目前已经是相当成熟的技术了,截至2006年5月,NCBI上公布的完成全基因组测序的细菌已达343种。
细菌基因组测序带来的意义和应用相当广阔,从最基本的来说它可以帮助我们鉴定相关的基因,为下一步的研究工作奠定基础。
本文中所用到的是“全基因组散弹法”(Whole-genome-shotgun)对HLK1~T全基因组进行测序和分析。在注释阶段,我们采用了国际通用的Glimmer软件预测开放阅读框(ORFs),用RBSfinder对蛋白的起始密码进行校正后用BLAST对ORFs进行NCBI的非冗余数据库的同源性检索,以预测每个编码序列(CDS)的功能。并且对P.zucineum基因组编码的所有蛋白进行COG分类。对注释得到的基因应用Translate工具软件进行翻译获得其氨基酸序列为下一步预测外排蛋白做准备,最后应用SingalP v3.0,LipoP v1.0,Phobius和TMHMM 2.0对HLK1~T基因组所编码的外排蛋白进行综合预测。对Ⅳ型蛋白和Tat通路蛋白这种具有保守序列的外排蛋白采用手工方法进行搜索。
最后结果显示,P.zucineum基因组大小为4,379,231bp,由一个大小为3,996,255bp的环状染色体和一个大小为382,976bp的质粒组成。染色体和质粒的GC含量分别为71.4%和68.5%。我们在染色体上鉴定到42个tRNA基因和1个16S-23S-5S rRNA操纵子。
HLK1~T基因组的一个显著特征是高GC含量,位于已全基因组测序的343种细菌基因组中的第四位。而另外几种高GC的细菌大多属于放线菌纲。由于放线菌同时具有细菌和真菌的特征,从生物进化的角度看,它还是属于介于细菌与真菌之间的过渡类型。P.zucineum基因组的高GC含量不仅预示着复杂的二级结构,而且可以进一步探求该菌与放线菌之间在高GC含量上的潜在关系。
在已全基因组测序的细菌基因组中,与P.zucineum同源性最高的是新月柄杆菌(C.crescentus)。C.crescentus是一种用于研究细胞分化、不对称分裂、细胞周期调控的单细胞模式生物,单个菌细胞可分裂成两个不同类型的细胞,与病原体有许多共同的基因。其中的ctrA基因是C.crescentus中控制细胞周期的主调控基因,具有细菌生存必须的并具有转录的自我调控能力,编码一种二元信号转导系统(Two-component signal-transduction system)关键应答调控子。二元信号转导系统能够感应外界环境的变化,继而把信息传递给内部系统,在病原菌对宿主的识别和侵袭、进一步的致病或共生等过程中起着非常重要的作用。我们在P.zucineum中找到了ctrA与同源性高达93%的序列,命名为ctrZ,对于进一步探讨P.zucineum可能的致病作用具有重要的意义。
根据注释的结果我们发现,P.zucineum共编码3681条蛋白(染色体3534条,质粒327条)。在这其中,3,715个可以和非冗余蛋白库比对上。而在能够比对上的这些蛋白中,866个是保守的假定蛋白,2,849个蛋白有已知或者预测的功能。大量功能未知的序列提示着新功能和新基因存在的可能。
P.zucineum的基因组中含有丰富的二元信号系统,转录调控子,和热休克反应蛋白,使得菌株能够在转录和翻译后水平对外界的刺激做出相应的反应。在这102个二元信号转导系统蛋白中(11个编码基因位于质粒上),有36个组氨酸激酶,48个反应调控子,18个和组氨酸激酶、反应调控子融合的杂交蛋白。16对组氨酸激酶和反应调控子紧密连锁并可能形成功能性操纵子。这些紧密连锁的元件使得二元信号系统对外界环境的变化能够有效地做出反应。HLK1~T的基因组中包含18个ESFσ-因子(4个位于质粒上),它们能够协调胞外刺激和细菌的基因表达。
我们还找到了170个转录调控子(154个有染色体编码,16个由质粒编码)(表3),这使得HLK1~T拥有对任何已知的环境刺激做出有效应答的潜能,包括青霉素抗性,低温抗性,以及金属抗性。
HLK1~T基因组有3个热休克σ-因子基因rpoH(2个位于染色体上)和35个热休克分子伴侣基因(15个位于染色体上,20个位于质粒上)。根据文献报道rpoH可以协助E.coli对抗温度的剧烈变化,而热休克分子伴侣可以在多种环境压力的诱导下产生,包括细胞能量消耗,极高浓度的重金属,和各种有毒物质。
通过对蛋白的注释我们也发现在HLK1~T基因组编码的蛋白中有Sec依赖的、Sec-非依赖的、Ⅱ型以及Ⅳ型外排系统的组成成分。注释找到了蛋白转运的Sec依赖的通路的组成蛋白的编码基因例如SecA(PHZ_c0758),secB(PHZ_c0006),secD(PHZ_c1954),secE(PHZ_c1211),SecF(PHZ_c1953),secG(PHZ_c1752),和secY(PHZ_c1250)。也有蛋白外排所需要的信号肽酶(PHZ_c1871),脂蛋白信号肽酶(PHZ_c3162)和信号识别小体srp54(PHZ_c0117)和受体(ftsY,PHZ_c0131)。Tat通路是用来转运已经折叠的蛋白通过质膜的Sec非依赖的外排通路,在5个己知的Tat蛋白中,TatABC存在于P.zucineum中。而13个编码Ⅱ型分泌系统的蛋白互相连锁形成一个操纵子。与此同时,P.zucineum中还存在3套Ⅳ型分泌系统,其中1套由质粒编码。因此无论是利用哪种通路进行外排的蛋白都能顺利实现其从胞内向胞外的转运。
HLK1~T基因组中还含有对抗氧化压力的各种成分。包括超氧化物歧化酶,过氧化氢还原酶等。此外,还含有组成对抗胞内氧化压力的GSH-氧化还原循环的各种组成成分。
为了适应胞内的生存环境,P.zucineum还编码能够利用宿主的离子资源为自己所用的蛋白,1个ABC型的铁载体转运体系,60个能够摄取离子复合物的TonB-依赖的受体(2个由质粒编码)。
预测分析得到基因组编码的蛋白中有35.4%为外排蛋白,其中以跨膜蛋白为最多,共预测到735个,占总蛋白的19.0%,外排蛋白的53.3%。此外,它还编码Ⅰ型分泌蛋白(499个,占总蛋白的12.9%,外排蛋白的36.2%)和脂蛋白(101个,占总蛋白的2.6%,外排蛋白的7.3%),以及4个Ⅳ型蛋白和12个Tat通路蛋白。
直系同源簇方法,即从同一簇中的已知基因注释未知基因的功能的这种COG(Cluster of orthologous group)中,目前已有25种,P.zucineum基因组含有其中的20种,并以未知功能的COG居多。鉴于外排蛋白的预测结果以及P.zucineum的兼性胞内寄生菌的特征和“拉链”式侵入细胞的特性,在这20类COG中尤以与细胞壁/膜/荚膜合成相关、无机离子转运和代谢有关的、氨基酸转运和代谢有关的、脂质转运和代谢有关的、信号传导机制相关、胞内传输/分泌/小跑传输相关的蛋白这几类最有意义。
HLK1~T编码的蛋白中有大量外排蛋白,这些蛋白可能在细菌与宿主的相互作用以及胞内寄生的过程中发挥重要作用。
P.zucineum is a novel facultative intracellular bacterium which was recently discovered in the tumor cell line K562 by our group and the type strain was named HLK1~T.Since this microbe is a facultative intracellular microbe which can survive in human cells,it may have a pathogenic relevance with human and mammals, Previously,we found that this microbe invaded cells via a "Zipper" entry mechanism, whereby the microbe expresses some surface proteins,including transmembrane proteins and secreted proteins(exported proteins),which react with surface receptors of eukaryotic cells and trigger alteration and redistribution of the cell skeleton structure,extension of cell membrane,which permits the entrance of the.Thus,it is a pivot to identify the exported proteins.In this study,on the basis of the whole genom of P.zucineum,the gene identification and annotation,protein translation and exported proteins prediction by exported signal sequences identification has been carried out.
Using a "whole-genome-shotgun" method,we sequenced P.zucineum's genome.At the step of annotation,"Glimmer" was used to predict ORFs(open reading frame), "RBSfinder" was used to readjust the results of Glimmer and "BLAST" was used to search homologous sequences in non-redundant database of NCBI,in order to predict functions of every CDS(coding sequence).All the proteins were classified by COG and the gene sequences were then translated into amino acid sequences.We predicted the exported proteins using consensus between multiple predictive tools,such as SignalP v3.0,LipoP v1.0,Phobius and TMHMM 2.0 by searching the export signals such as N-terminal signal peptides,α-transmembrane helices,etc.TypeⅣsignal peptides and proteins exported via Tat machinery were searched manually based on their conservative motifs.
We sequenced its 4,379,231bp genome,including a 3,996,255 bp ring chromosome and a 382,976 bp plasmid.The GC content of chromosome and plasmid is 71.4%and 68.5%,respectively.We identified 42 tRNA genes and 1 16S-23S-5S rRNA operon in the chromosome.
One of the interesting features of the P zucineum is its high GC content,which is the fourth highest in 343 genome-sequenced bacteria.The feature prefigures the complex secondary structure in the genome and possible relationship with actinobacteria which also have high GC content.
Sequences of P.zucineum have the highest homology to genome of C.crescentus,a single-cell model system to study cellular differentiation,asymmetric division,and their coordination with cell cycle progression.One cell of C.crescentus can divide into two types of cells.Its master regulator of cell cycle is ctrA gene,which controls one quarter of cell cycle regulators and participates in many progresses in cell cycle. ctrA encodes a key response regulator of two-component signal-transuction system, an important system in recognition,invasion,pathogenesis and symbiosis of pathogens to host.A sequence named ctrZ in P zucineurn has 93%homology to ctrA. It may also be an essential component of two-component-transduction system and may play an important role in the pathogenesis to the human host cells.
According to the annotation,the genome of P.zucineum encodes 3861 proteins (3534 proteins encoded by chromosome and 327 proteins encoded by plasmid),of which,3,715 have significant matches to the non-redundant protein database.Of the matches,866 are conserved hypothetical proteins,and 2,849 with known or predicted funtions.The existence of a lot of function unknown sequences indicates that the novel genes and functions of P.zucineum.
According to our analysis,the genome of P.zucineurn contains abundant two component systems,transcriptional regulator,and heat shock response proteins, enabling the strain to respond to a plethora of stimuli coordinately at transcriptional and post-translational levels.In the total 102 two component systems transduction proteins(11 in plasmid),there are 36 histidine kinase,48 response regulator,and 18 hybrid proteins fused with histidine kinase and response regulator.16 pairs of histidine kinase and response regulator are adjacently aligned and may act as functional operons.These tightly linked modules make two component systems respond to environmental changes efficiently.We identified 170 transcriptional regulators(154 in the chromosome and 16 in the plasmid),which have the potential to respond to almost all known environmental stimulus,including penicillin resistance, low-temperature resistance,and metal resistance.There are 3 heat shock sigma factors rpoH(2 in the plasmid)and 35 heat shock molecular chaperons in the genome(15 in the chromosome and 20 in the plasmid).It was reported that rpoH helped E.coli to respond to the drastic change of temperature,and that heat shock molecular chaperons could be induced by a variety of stresses,including cellular energy depletion,extreme concentration of heavy metal,and various toxic substances.
According to the annotation,the P.zucineum genomes contains sec-dependent and sec-independent,typical typeⅡand typeⅣsecretion systems,which are known to play an important role in adapting to diverse conditions.The see-dependent pathway for protein translocation such as SecA(PHZ_c0758),secB(PHZ_c0006),secD (PHZ_c1954),secE(PHZ_c1211),SecF(PHZ_c1953),secG(PHZ_c 1752),and secY (PHZ_c1250)were identified.Signal peptidases(PHZ_c1781),lipoprotein signal peptidase(PHZ_c3162),and the signal recognition particle srp54(PHZ_c0117),and the receptor(ftsY,PHZ_c0131)are present.The twin arginine motif translocation(Tat) pathway accomplishes the secretion of folded cofactor-containing proteins across the membrane.Of the 5 known Tat proteins,TatABC are present.13 proteins coding a set of typeⅡsecretion system are aligned tightly with each other to form an operon. There are 3 sets of typeⅣsecretion system,one of which is in the plasmid.Therefore, it is possible for any exported proteins to translocate across the membrane no matter which pathway they utilize.
The genome of P.zucineum contains machineries to adhering and subsequently invading host cells.We identified 1 pili biosynthesis gene(pilA)and two operons related to pili biosynthesis located in chromosome.
The genome also has fundamental compositions against oxidative stress, including superoxide dismutases,and hydroperoxide reductase,etc.In addition,the compositions of GSH-redox cycling,a major defense system against intracellular oxidative stress is also present.
To survive in the intracellular environment,P.zucineum encodes various proteins to utilize the host iron source.The genome has one ABC type siderophore transporter system and 60 TonB-dependent(2 in the plasmid)which could uptake iron-siderophore complexes.
Among all these proteins,35.4%was predicted to encode exported proteins, most of which(totally 735,19.0%of the proteome and 53.3%of all the exported proteins)utilize uncleavable transmembrane helices.In addition,typeⅠsignal peptides(499,12.9%of the proteome and 36.2%of the exported proteins), lipoproteins(101,2.6%of the proteome and 7.3%of the exported proteins)were also identified.Four proteins contain typeⅣsignal peptides and 12 proteins which exported by Tat pathway were detected as well.
There are 25 COGs in NCBI and P.zucineum contains 20 of them.Among them, the most abundant group is "function unknown".Based on our exported protein prediction and the characteristics of facultative intracellular bacterium and its "Zipper" invasion mode,some categories of COGs are more important than the others. These categories are cell wall/membrane/envelope biogenesis;inorganic ion transport and metabolism;amino acid transport and metabolism;lipid transport and metabolism; signal transduction mechanisms;intracellular trafficking,secretion,and vesicular transport.
The genome of HLK1~T encodes various types of exported proteins which may play an important role in the interaction with the host cells and facilitate the strain to invade the latter.
细菌基因组的全基因组测序目前已经是相当成熟的技术了,截至2006年5月,NCBI上公布的完成全基因组测序的细菌已达343种。
细菌基因组测序带来的意义和应用相当广阔,从最基本的来说它可以帮助我们鉴定相关的基因,为下一步的研究工作奠定基础。
本文中所用到的是“全基因组散弹法”(Whole-genome-shotgun)对HLK1~T全基因组进行测序和分析。在注释阶段,我们采用了国际通用的Glimmer软件预测开放阅读框(ORFs),用RBSfinder对蛋白的起始密码进行校正后用BLAST对ORFs进行NCBI的非冗余数据库的同源性检索,以预测每个编码序列(CDS)的功能。并且对P.zucineum基因组编码的所有蛋白进行COG分类。对注释得到的基因应用Translate工具软件进行翻译获得其氨基酸序列为下一步预测外排蛋白做准备,最后应用SingalP v3.0,LipoP v1.0,Phobius和TMHMM 2.0对HLK1~T基因组所编码的外排蛋白进行综合预测。对Ⅳ型蛋白和Tat通路蛋白这种具有保守序列的外排蛋白采用手工方法进行搜索。
最后结果显示,P.zucineum基因组大小为4,379,231bp,由一个大小为3,996,255bp的环状染色体和一个大小为382,976bp的质粒组成。染色体和质粒的GC含量分别为71.4%和68.5%。我们在染色体上鉴定到42个tRNA基因和1个16S-23S-5S rRNA操纵子。
HLK1~T基因组的一个显著特征是高GC含量,位于已全基因组测序的343种细菌基因组中的第四位。而另外几种高GC的细菌大多属于放线菌纲。由于放线菌同时具有细菌和真菌的特征,从生物进化的角度看,它还是属于介于细菌与真菌之间的过渡类型。P.zucineum基因组的高GC含量不仅预示着复杂的二级结构,而且可以进一步探求该菌与放线菌之间在高GC含量上的潜在关系。
在已全基因组测序的细菌基因组中,与P.zucineum同源性最高的是新月柄杆菌(C.crescentus)。C.crescentus是一种用于研究细胞分化、不对称分裂、细胞周期调控的单细胞模式生物,单个菌细胞可分裂成两个不同类型的细胞,与病原体有许多共同的基因。其中的ctrA基因是C.crescentus中控制细胞周期的主调控基因,具有细菌生存必须的并具有转录的自我调控能力,编码一种二元信号转导系统(Two-component signal-transduction system)关键应答调控子。二元信号转导系统能够感应外界环境的变化,继而把信息传递给内部系统,在病原菌对宿主的识别和侵袭、进一步的致病或共生等过程中起着非常重要的作用。我们在P.zucineum中找到了ctrA与同源性高达93%的序列,命名为ctrZ,对于进一步探讨P.zucineum可能的致病作用具有重要的意义。
根据注释的结果我们发现,P.zucineum共编码3681条蛋白(染色体3534条,质粒327条)。在这其中,3,715个可以和非冗余蛋白库比对上。而在能够比对上的这些蛋白中,866个是保守的假定蛋白,2,849个蛋白有已知或者预测的功能。大量功能未知的序列提示着新功能和新基因存在的可能。
P.zucineum的基因组中含有丰富的二元信号系统,转录调控子,和热休克反应蛋白,使得菌株能够在转录和翻译后水平对外界的刺激做出相应的反应。在这102个二元信号转导系统蛋白中(11个编码基因位于质粒上),有36个组氨酸激酶,48个反应调控子,18个和组氨酸激酶、反应调控子融合的杂交蛋白。16对组氨酸激酶和反应调控子紧密连锁并可能形成功能性操纵子。这些紧密连锁的元件使得二元信号系统对外界环境的变化能够有效地做出反应。HLK1~T的基因组中包含18个ESFσ-因子(4个位于质粒上),它们能够协调胞外刺激和细菌的基因表达。
我们还找到了170个转录调控子(154个有染色体编码,16个由质粒编码)(表3),这使得HLK1~T拥有对任何已知的环境刺激做出有效应答的潜能,包括青霉素抗性,低温抗性,以及金属抗性。
HLK1~T基因组有3个热休克σ-因子基因rpoH(2个位于染色体上)和35个热休克分子伴侣基因(15个位于染色体上,20个位于质粒上)。根据文献报道rpoH可以协助E.coli对抗温度的剧烈变化,而热休克分子伴侣可以在多种环境压力的诱导下产生,包括细胞能量消耗,极高浓度的重金属,和各种有毒物质。
通过对蛋白的注释我们也发现在HLK1~T基因组编码的蛋白中有Sec依赖的、Sec-非依赖的、Ⅱ型以及Ⅳ型外排系统的组成成分。注释找到了蛋白转运的Sec依赖的通路的组成蛋白的编码基因例如SecA(PHZ_c0758),secB(PHZ_c0006),secD(PHZ_c1954),secE(PHZ_c1211),SecF(PHZ_c1953),secG(PHZ_c1752),和secY(PHZ_c1250)。也有蛋白外排所需要的信号肽酶(PHZ_c1871),脂蛋白信号肽酶(PHZ_c3162)和信号识别小体srp54(PHZ_c0117)和受体(ftsY,PHZ_c0131)。Tat通路是用来转运已经折叠的蛋白通过质膜的Sec非依赖的外排通路,在5个己知的Tat蛋白中,TatABC存在于P.zucineum中。而13个编码Ⅱ型分泌系统的蛋白互相连锁形成一个操纵子。与此同时,P.zucineum中还存在3套Ⅳ型分泌系统,其中1套由质粒编码。因此无论是利用哪种通路进行外排的蛋白都能顺利实现其从胞内向胞外的转运。
HLK1~T基因组中还含有对抗氧化压力的各种成分。包括超氧化物歧化酶,过氧化氢还原酶等。此外,还含有组成对抗胞内氧化压力的GSH-氧化还原循环的各种组成成分。
为了适应胞内的生存环境,P.zucineum还编码能够利用宿主的离子资源为自己所用的蛋白,1个ABC型的铁载体转运体系,60个能够摄取离子复合物的TonB-依赖的受体(2个由质粒编码)。
预测分析得到基因组编码的蛋白中有35.4%为外排蛋白,其中以跨膜蛋白为最多,共预测到735个,占总蛋白的19.0%,外排蛋白的53.3%。此外,它还编码Ⅰ型分泌蛋白(499个,占总蛋白的12.9%,外排蛋白的36.2%)和脂蛋白(101个,占总蛋白的2.6%,外排蛋白的7.3%),以及4个Ⅳ型蛋白和12个Tat通路蛋白。
直系同源簇方法,即从同一簇中的已知基因注释未知基因的功能的这种COG(Cluster of orthologous group)中,目前已有25种,P.zucineum基因组含有其中的20种,并以未知功能的COG居多。鉴于外排蛋白的预测结果以及P.zucineum的兼性胞内寄生菌的特征和“拉链”式侵入细胞的特性,在这20类COG中尤以与细胞壁/膜/荚膜合成相关、无机离子转运和代谢有关的、氨基酸转运和代谢有关的、脂质转运和代谢有关的、信号传导机制相关、胞内传输/分泌/小跑传输相关的蛋白这几类最有意义。
HLK1~T编码的蛋白中有大量外排蛋白,这些蛋白可能在细菌与宿主的相互作用以及胞内寄生的过程中发挥重要作用。
P.zucineum is a novel facultative intracellular bacterium which was recently discovered in the tumor cell line K562 by our group and the type strain was named HLK1~T.Since this microbe is a facultative intracellular microbe which can survive in human cells,it may have a pathogenic relevance with human and mammals, Previously,we found that this microbe invaded cells via a "Zipper" entry mechanism, whereby the microbe expresses some surface proteins,including transmembrane proteins and secreted proteins(exported proteins),which react with surface receptors of eukaryotic cells and trigger alteration and redistribution of the cell skeleton structure,extension of cell membrane,which permits the entrance of the.Thus,it is a pivot to identify the exported proteins.In this study,on the basis of the whole genom of P.zucineum,the gene identification and annotation,protein translation and exported proteins prediction by exported signal sequences identification has been carried out.
Using a "whole-genome-shotgun" method,we sequenced P.zucineum's genome.At the step of annotation,"Glimmer" was used to predict ORFs(open reading frame), "RBSfinder" was used to readjust the results of Glimmer and "BLAST" was used to search homologous sequences in non-redundant database of NCBI,in order to predict functions of every CDS(coding sequence).All the proteins were classified by COG and the gene sequences were then translated into amino acid sequences.We predicted the exported proteins using consensus between multiple predictive tools,such as SignalP v3.0,LipoP v1.0,Phobius and TMHMM 2.0 by searching the export signals such as N-terminal signal peptides,α-transmembrane helices,etc.TypeⅣsignal peptides and proteins exported via Tat machinery were searched manually based on their conservative motifs.
We sequenced its 4,379,231bp genome,including a 3,996,255 bp ring chromosome and a 382,976 bp plasmid.The GC content of chromosome and plasmid is 71.4%and 68.5%,respectively.We identified 42 tRNA genes and 1 16S-23S-5S rRNA operon in the chromosome.
One of the interesting features of the P zucineum is its high GC content,which is the fourth highest in 343 genome-sequenced bacteria.The feature prefigures the complex secondary structure in the genome and possible relationship with actinobacteria which also have high GC content.
Sequences of P.zucineum have the highest homology to genome of C.crescentus,a single-cell model system to study cellular differentiation,asymmetric division,and their coordination with cell cycle progression.One cell of C.crescentus can divide into two types of cells.Its master regulator of cell cycle is ctrA gene,which controls one quarter of cell cycle regulators and participates in many progresses in cell cycle. ctrA encodes a key response regulator of two-component signal-transuction system, an important system in recognition,invasion,pathogenesis and symbiosis of pathogens to host.A sequence named ctrZ in P zucineurn has 93%homology to ctrA. It may also be an essential component of two-component-transduction system and may play an important role in the pathogenesis to the human host cells.
According to the annotation,the genome of P.zucineum encodes 3861 proteins (3534 proteins encoded by chromosome and 327 proteins encoded by plasmid),of which,3,715 have significant matches to the non-redundant protein database.Of the matches,866 are conserved hypothetical proteins,and 2,849 with known or predicted funtions.The existence of a lot of function unknown sequences indicates that the novel genes and functions of P.zucineum.
According to our analysis,the genome of P.zucineurn contains abundant two component systems,transcriptional regulator,and heat shock response proteins, enabling the strain to respond to a plethora of stimuli coordinately at transcriptional and post-translational levels.In the total 102 two component systems transduction proteins(11 in plasmid),there are 36 histidine kinase,48 response regulator,and 18 hybrid proteins fused with histidine kinase and response regulator.16 pairs of histidine kinase and response regulator are adjacently aligned and may act as functional operons.These tightly linked modules make two component systems respond to environmental changes efficiently.We identified 170 transcriptional regulators(154 in the chromosome and 16 in the plasmid),which have the potential to respond to almost all known environmental stimulus,including penicillin resistance, low-temperature resistance,and metal resistance.There are 3 heat shock sigma factors rpoH(2 in the plasmid)and 35 heat shock molecular chaperons in the genome(15 in the chromosome and 20 in the plasmid).It was reported that rpoH helped E.coli to respond to the drastic change of temperature,and that heat shock molecular chaperons could be induced by a variety of stresses,including cellular energy depletion,extreme concentration of heavy metal,and various toxic substances.
According to the annotation,the P.zucineum genomes contains sec-dependent and sec-independent,typical typeⅡand typeⅣsecretion systems,which are known to play an important role in adapting to diverse conditions.The see-dependent pathway for protein translocation such as SecA(PHZ_c0758),secB(PHZ_c0006),secD (PHZ_c1954),secE(PHZ_c1211),SecF(PHZ_c1953),secG(PHZ_c 1752),and secY (PHZ_c1250)were identified.Signal peptidases(PHZ_c1781),lipoprotein signal peptidase(PHZ_c3162),and the signal recognition particle srp54(PHZ_c0117),and the receptor(ftsY,PHZ_c0131)are present.The twin arginine motif translocation(Tat) pathway accomplishes the secretion of folded cofactor-containing proteins across the membrane.Of the 5 known Tat proteins,TatABC are present.13 proteins coding a set of typeⅡsecretion system are aligned tightly with each other to form an operon. There are 3 sets of typeⅣsecretion system,one of which is in the plasmid.Therefore, it is possible for any exported proteins to translocate across the membrane no matter which pathway they utilize.
The genome of P.zucineum contains machineries to adhering and subsequently invading host cells.We identified 1 pili biosynthesis gene(pilA)and two operons related to pili biosynthesis located in chromosome.
The genome also has fundamental compositions against oxidative stress, including superoxide dismutases,and hydroperoxide reductase,etc.In addition,the compositions of GSH-redox cycling,a major defense system against intracellular oxidative stress is also present.
To survive in the intracellular environment,P.zucineum encodes various proteins to utilize the host iron source.The genome has one ABC type siderophore transporter system and 60 TonB-dependent(2 in the plasmid)which could uptake iron-siderophore complexes.
Among all these proteins,35.4%was predicted to encode exported proteins, most of which(totally 735,19.0%of the proteome and 53.3%of all the exported proteins)utilize uncleavable transmembrane helices.In addition,typeⅠsignal peptides(499,12.9%of the proteome and 36.2%of the exported proteins), lipoproteins(101,2.6%of the proteome and 7.3%of the exported proteins)were also identified.Four proteins contain typeⅣsignal peptides and 12 proteins which exported by Tat pathway were detected as well.
There are 25 COGs in NCBI and P.zucineum contains 20 of them.Among them, the most abundant group is "function unknown".Based on our exported protein prediction and the characteristics of facultative intracellular bacterium and its "Zipper" invasion mode,some categories of COGs are more important than the others. These categories are cell wall/membrane/envelope biogenesis;inorganic ion transport and metabolism;amino acid transport and metabolism;lipid transport and metabolism; signal transduction mechanisms;intracellular trafficking,secretion,and vesicular transport.
The genome of HLK1~T encodes various types of exported proteins which may play an important role in the interaction with the host cells and facilitate the strain to invade the latter.
引文
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6. Aslam Z, Im, W. T., Ten, L. N., et al. Phenylobacterium koreense sp. nov., isolated from South Korea. Int J Syst Evol MicrobioL 2005; 55(Pt 5): 2001-2005.
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13. Delcher AL, Harmon, D., Kasif, S., et al. Improved microbial gene identification with Glimmer. Nucleic Acids Res. 1999; 27(23): 4636-4641.
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16. Frishman D, Mironov, A., Mewes, H. W., et al. Combining diverse evidence for gene recognition in completely sequenced bacterial genomes. Nucleic Acids Res. 1998; 26(12): 2941-2947.
17. Tatusov RL, Natale, D. A., Garkavtsev, I. V., et al. The COG database: new developments in phylogentic classification of proteins from complete genomes. Nucleic Acids Res. 2001; 29(1): 22-28.
18. Shawn Lewenza JLG, Fiona S. L. Brinkman, Robert E. W. Hancock. Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen. Genome Research. 2005; 15: 321-329.
19. Bendtsen JD, Nielsen, H., von Heijne, G, BRunak, S. Inmproved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004; 340: 782-795.
20. Juncker AS, Willenbrock, H., von Heijne, G, Brunak, S., Nielsen, H., Krogh, A. Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 2003; 12: 1652-1662.
21. Kall L, Krogh, A., Sonnhammer, E. L. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 2004; 338: 1027-1036.
22. Krogh A, Larsson, B., von Heijne, G., Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001; 305: 567-580.
23. Salzberg S, Delcher, A., Kasif, S., et al. Microbial gene identification using interpolated Markov models. Nucleic Acids Res. 1998; 26(2): 544-548.
24. Mikonnen M, Vuoristo, J., Alatossava, T. Ribosome binding site consensus sequence of Lactobacilus delbrueckii. FEMS Microbiol. Lett. 1994; 116: 315-320.
25. Ely BG, C. J. Use of pulsed-field-gradient gel electronphoresis to construct a physical map of the Caulobacter crescentus genome. Gene. 1988; 68: 323-333.
26. Ryan KR, Judd, E. M., Shapiro, L. The CtrA response regulator essential for Caulobacter crescentus cell-cycle progression requires a bipartite degradation signal for temporally controlled proteolysis. J Mol BioL 2002; 324:443-455.
27. Skerker JM, Prasol, M. S., Perchuk, B. S., et al. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PloS Biol. 2005; 3(10): 1770-1788.
28. Master SS, Springer, B., Sander, P., et al. Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages. Microbiology. 2002; 148(Pt 10): 3139-3144.
29. Ramos JL, Martinez-Bueno, M., Molina-Henares, A. J., et al. The TetR family of transcriptional repressors. Microbiol Mol Biol Rev. 2005; 69(2): 326-356.
30. Schumann W. Regulation of the heat shock response in Escherichia coli and Bacillus subtilis. J Biosci 1996; 21(2): 133-148.
31. Feder ME, Hofmann, G. E. Heat-shock proteins, molecular chaperons, and the stress response: evolutionary and ecological physiology. Annu Rev PhysioL 1999; 61: 243-282.
32. Roop RM, Bellaire, B. H., Valderas, M. W., et al. Adaptation of the Brucellae to their intracellular niche. Mol Microbiol. 2004; 52(3): 621-630.
33. Miller RA, Britigan, B. E. Role of oxidants in microbial pathophysiology. Clin Microbiol Rev. 1997; 10(1): 1-18.
34. Nathan C, Shiloh, M. U. Reactive oxygen and introgen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA. 2000; 97(16): 8841-8848.
35. Ratledge C, Dover, L. G. Iron metabolism in pathogenic bacteria. Annu Rev Microbiol. 2000; 54: 881-941.
36. Tatusov RL, Koonin, E. V., Lipman, D. J. A genomic perspective on protein families. Science. 1997; 278: 631-637.
1.von Heijine G.The signal peptide.J.Membr.Biol.1990;115(3):195-201.
2.Pugsley AP,Francetic,O.,Sauvonnet,O.M.,et al.Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria-a review.Gene.1997;192(1):13-19.
3.Bendtsen JD,Nielsen,H.,von Heigine,G.,et al.Improved prediction of signal peptides:SignalP 3.0.J.Mol.Biol.2004;340(4):783-795.
4.Bendtsen JD,Nielsen,H.,Widdick,D.,et al.Prediction of twin-arginine signal peptides.BMC Bioinformatics.2005;6:167-175.
5.Berks BC.Acoomon export pathway for proteins binding complex redox cofactors? Mol.Microbiol.1996;22:393-404.
6.Cristobal S,de Gier,J.W.,Nielson,H.,et at.Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.EMBO J.1999;18:2982-2990.
7.Krough A,Larsson,B.,von Heijine,G.,et al.Predicting transmembrane protein topology with a hidden Markov model:application to complete genomes.J.Mol.Biol.2001;305(3):567-580.
8.Moller S,Croning,M.D.,Apweiler,R.Evalution of methods for the prediction of membrane spanning regions.Bioinformatics.2001;17(7):646-653.
9.Kall L,Krogh,A.,Sonnhammer,E.L.L.A combined transmembrane topology and signal peptide prediction method.J.Mol.Biol. 2004;338(5): 1027-1036.
10. Jacoboni I, Martelli, P. L, Fariselli, P. et al. Prediction of the transmembrane regions of b-barrel membrane proteins with a neural network-based predictor. Protein Sci. 2001; 10:779-787.
11. Martelli PL, Fariselli, P., Krough, A., et al. A sequence-profile-based HMM for predicting and discriminating b-barrel membrane proteins. Bioinformatics. 2002;18:S46-S53.
12. Junker AS, et al. Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 2003;12:1652-1662.
13. Gardy JL, et al. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insignts gained from comparative proteome analysis. Bioinformatics. 2005; 18:298-305.
14. Nair R, Rost, B. Inferring sub-cellular localization through antomated lexical analysis. Bioinformatics. 2002;18:S78-S86.
2. Zhang K, Han, W. D., Zhang, R., et al. Phenylobacterium zucineum sp. nov., a facultative intracellular bacterium isolated from a human erythroleukemia cell line K562. Systematic and applied microbiology. 2007; 30:207-212.
3. Lingens F, Blecher, R., Blecher, H., et al. Phenylobacterium immobile gen. nov., sp. nov., a gram-negative bacterium that degrades the herbicide chloridazon. Int J Syst Bacteriol. 1985; 35:26-39.
4. Kanso S, Patel, B. K. Phenylobacterium lituiforme sp. nov., a moderately thermophilic bacterium from a subsurface aquifer, and emended description of the genus Phenylobacterium. Int J Syst BacterioL 2004; 54(Pt 6): 2141-2146.
5. Tiago I, Mendes, V., Pires, C, et al. Phenylobacterium falsum sp. nov., an Alphaproteobacterium isolated from a nonsaline alkaline groundwater, and emended description of the genus Phenylobacterium. Syst Appl Microbiol. 2005; 28(4): 295-302.
6. Aslam Z, Im, W. T., Ten, L. N., et al. Phenylobacterium koreense sp. nov., isolated from South Korea. Int J Syst Evol MicrobioL 2005; 55(Pt 5): 2001-2005.
7. Isberg RR, Barnes, P. Subversion of integrins by enteropathogenic Yersinia. J Cell Sci. 2001; 114(1): 21-28.
8. Levy L, Ji, B. The mouse foot-pad technique for cultivation of Mycobacterium leprae. Lepr Rev. 2006; 77: 5-24.
9. Von Heijne G Signal sequences: The limits of variation. J. Mol. Biol. 1985; 184:99-105.
10. Lory S. Leader peptideses of type IV prepilins and related proteins. Austin, TX: R. G. Landes Co.; 1994: Pages.
11. Cristobal S, de Gier, J. W., Nielson, H. et al. Competition between Sec- and TAT- pathway protein translocation in Escherichia coli. EMBO Journal. 1999; 18(11): 2982-2990.
12. Chaddock AM, Mant, A., Karnauchov, I., Brink, S., Herrmann, R. G, Klosgen, R. B., Robinson, C. A new type of signal peptide: Central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase. EMBO J. 1995; 14:2715-2722.
13. Delcher AL, Harmon, D., Kasif, S., et al. Improved microbial gene identification with Glimmer. Nucleic Acids Res. 1999; 27(23): 4636-4641.
14. Suzek BE, Ermolaeva, M. D., Schreiber, M., et al. A probabilistic method for identifying start codons in bacterial genomes. Bioinformatics. 2001; 17(12): 1123-1130.
15. Lewin B. Genes VI. Oxford: Oxford University Press; 1997: Pages.
16. Frishman D, Mironov, A., Mewes, H. W., et al. Combining diverse evidence for gene recognition in completely sequenced bacterial genomes. Nucleic Acids Res. 1998; 26(12): 2941-2947.
17. Tatusov RL, Natale, D. A., Garkavtsev, I. V., et al. The COG database: new developments in phylogentic classification of proteins from complete genomes. Nucleic Acids Res. 2001; 29(1): 22-28.
18. Shawn Lewenza JLG, Fiona S. L. Brinkman, Robert E. W. Hancock. Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen. Genome Research. 2005; 15: 321-329.
19. Bendtsen JD, Nielsen, H., von Heijne, G, BRunak, S. Inmproved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004; 340: 782-795.
20. Juncker AS, Willenbrock, H., von Heijne, G, Brunak, S., Nielsen, H., Krogh, A. Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 2003; 12: 1652-1662.
21. Kall L, Krogh, A., Sonnhammer, E. L. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 2004; 338: 1027-1036.
22. Krogh A, Larsson, B., von Heijne, G., Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001; 305: 567-580.
23. Salzberg S, Delcher, A., Kasif, S., et al. Microbial gene identification using interpolated Markov models. Nucleic Acids Res. 1998; 26(2): 544-548.
24. Mikonnen M, Vuoristo, J., Alatossava, T. Ribosome binding site consensus sequence of Lactobacilus delbrueckii. FEMS Microbiol. Lett. 1994; 116: 315-320.
25. Ely BG, C. J. Use of pulsed-field-gradient gel electronphoresis to construct a physical map of the Caulobacter crescentus genome. Gene. 1988; 68: 323-333.
26. Ryan KR, Judd, E. M., Shapiro, L. The CtrA response regulator essential for Caulobacter crescentus cell-cycle progression requires a bipartite degradation signal for temporally controlled proteolysis. J Mol BioL 2002; 324:443-455.
27. Skerker JM, Prasol, M. S., Perchuk, B. S., et al. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PloS Biol. 2005; 3(10): 1770-1788.
28. Master SS, Springer, B., Sander, P., et al. Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages. Microbiology. 2002; 148(Pt 10): 3139-3144.
29. Ramos JL, Martinez-Bueno, M., Molina-Henares, A. J., et al. The TetR family of transcriptional repressors. Microbiol Mol Biol Rev. 2005; 69(2): 326-356.
30. Schumann W. Regulation of the heat shock response in Escherichia coli and Bacillus subtilis. J Biosci 1996; 21(2): 133-148.
31. Feder ME, Hofmann, G. E. Heat-shock proteins, molecular chaperons, and the stress response: evolutionary and ecological physiology. Annu Rev PhysioL 1999; 61: 243-282.
32. Roop RM, Bellaire, B. H., Valderas, M. W., et al. Adaptation of the Brucellae to their intracellular niche. Mol Microbiol. 2004; 52(3): 621-630.
33. Miller RA, Britigan, B. E. Role of oxidants in microbial pathophysiology. Clin Microbiol Rev. 1997; 10(1): 1-18.
34. Nathan C, Shiloh, M. U. Reactive oxygen and introgen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci USA. 2000; 97(16): 8841-8848.
35. Ratledge C, Dover, L. G. Iron metabolism in pathogenic bacteria. Annu Rev Microbiol. 2000; 54: 881-941.
36. Tatusov RL, Koonin, E. V., Lipman, D. J. A genomic perspective on protein families. Science. 1997; 278: 631-637.
1.von Heijine G.The signal peptide.J.Membr.Biol.1990;115(3):195-201.
2.Pugsley AP,Francetic,O.,Sauvonnet,O.M.,et al.Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria-a review.Gene.1997;192(1):13-19.
3.Bendtsen JD,Nielsen,H.,von Heigine,G.,et al.Improved prediction of signal peptides:SignalP 3.0.J.Mol.Biol.2004;340(4):783-795.
4.Bendtsen JD,Nielsen,H.,Widdick,D.,et al.Prediction of twin-arginine signal peptides.BMC Bioinformatics.2005;6:167-175.
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