耐盐橙色粘球菌Myxococcus fulvus HW-1社会行为对海洋生境适应的分子策略
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摘要
粘细菌是研究细菌社会行为的良好模式材料,其社会行为主要表现在滑动运动、摄食和多细胞协调发育过程,而滑动运动在细胞摄食和子实体发育过程中发挥着非常重要的作用。黄色粘球菌(Myxococcus xanthus)的滑动运动由两个独立的遗传系统调控:一个是A运动系统,主要调控独立的单个细胞运动;另一个是S运动系统,主要调控群体细胞运动。S运动系统主要包括三个组分:Ⅳ型菌毛、细胞外基质和O-抗原。黄色粘球菌的运动调控系统非常复杂,迄今为止,发现的S运动系统相关基因已超过110个。
二十世纪末以前,粘细菌被普遍认为是典型的土壤微生物,分离的陆生菌株不能够在高于1%的盐浓度条件下生长。而从1998年日本学者在海洋样品中分离到嗜盐粘细菌以来,越来越多的粘细菌从多种海洋环境中分离纯化出来。根据耐盐能力的不同,海洋粘细菌被分为两类:海洋嗜盐粘细菌和海洋耐盐粘细菌。
海洋嗜盐粘细菌主要是由日本学者分离到的,根据形态学特征和16S rDNA的分析,这些菌株被归为四个新的属。他们对海洋嗜盐粘细菌的研究主要集中在形态学分析、分类以及抗生素等代谢产物分析方面,而关于粘细菌在海洋生境中的生活模式、定殖机制及更深入的分子机制等方面一直都没有涉及。
本实验室经过多年的探索,从海洋样品中分离到了多株海洋耐盐粘细菌。前期实验中,将其中的橙色粘球菌Myxococcus fulvus HW-1菌株作为一株典型的耐盐粘细菌模式菌株,与其它耐盐粘细菌或嗜盐粘细菌对比,对其形态学、生理学和系统分类学等特征及运动和发育能力进行了详细的分析。揭示了海洋耐盐粘细菌在陆地和海洋生境下存在两种不同的生活模式;并由实验结果推测海洋耐盐粘细菌是陆生粘细菌被带到海洋生境中适应了海水环境而存活下来,即海洋耐盐橙色粘球菌M.fulvus HW-1生活在海洋生境中是适应进化的结果。并且发现,在海水条件下,海洋耐盐粘球菌M.fulvus HW-1不仅保留了双运动系统,且双运动能力尤其是S运动能力明显增强。S运动的增强可能促进细胞间的粘连,为更好的适应海洋生境提供条件,证明社会运动在海洋耐盐粘球菌M.fulvus HW-1适应海洋生境生存过程中发挥着非常重要的作用。探知自然生境下粘细菌运动系统的多样性和对环境的适应可能为我们理解粘细菌运动系统的形成和进化提供重要线索。
mariner转座因子是来自真核生物的mariner/Tc1超家族的成员,与原核生物的转座子如Tn5相比,有着多种独特的性质,如结构简单、转座作用不依赖宿主的特定因素和随机插入等,这些特性使其在自然界中广泛分布,并可在物种间水平转移。目前,已有多篇文献报道mariner转座因子可以在粘细菌中有效地转座,并且达到稳定整合,是粘细菌遗传研究的有力工具之一。
本研究以海洋耐盐橙色粘球菌M.fulvus HW-1为材料,建立了一整套适合耐盐粘球菌的分子遗传操作体系,为开展耐盐粘细菌运动和发育等细胞行为的分子遗传学研究搭建了基础平台;在此基础上,利用mariner类转座子MiniHimarl-lacZ构建了HW-1菌株的小型随机插入突变菌库,从中发现并深入研究了一个社会运动和发育相关的新基因簇mts,该结果不仅扩充了粘球菌社会运动和发育相关基因文库,弥补了在模式菌株DK1622中筛选失活后对运动仅有微弱影响基因困难的不足,为粘球菌运动基因的筛选提供了新途径和新思路,而且开启了在分子水平上研究海洋耐盐粘细菌社会行为对海洋生境适应机制的大门,为理解HW-1社会行为对海洋生境适应的分子机制提供了重要依据。
实验中,为了研究与M.fulvus HW-1适应海洋生境相关的S运动系统基因,首先将mariner类转座子MiniHimarl-lacZ电转化入M.fulvus HW-1细胞,通过随机插入突变的方法建立了M.fulvus HW-1菌株的随机突变菌库,从中筛选到一株社会运动缺陷突变株M.fulvus HL-1。利用报告基因lacZ的表达验证了突变株HL-1染色体上插入了转座子MiniHimarl-lacZ,并且发现突变基因表达时间很早、表达水平很高。
双运动能力分析的经典方法——“软硬琼脂方法”分析结果显示:突变株M.fulvus HL-1 S运动能力缺陷,A运动能力受到影响;且令人兴奋的是,该突变株在海水条件下运动能力增强的趋势明显减弱、甚至消失,证明突变株M.fulvus HL-1中的突变基因在海洋耐盐橙色粘球菌M.fulvus HW-1在海水条件下S运动能力增强行为中发挥着重要的作用。通过发育平板上发育能力的检测证明了突变株M.fulvus HL-1的子实体形成和粘孢子发育能力缺陷。分析S运动系统的重要组分细胞外基质的染料结合实验、聚集实验和扫描电镜观察实验结果充分证明了突变株M.fulvus HL-1细胞外基质数量减少,细胞粘连性降低。这些实验结果表明:突变株M.fulvus HL-1中的突变基因可能在M.fulvus HW-1社会行为(运动和发育)中、甚至在社会行为对海洋生境适应过程中发挥着非常重要的作用。
通过质粒营救法和Tail-PCR方法获得了突变株M.fulvus HL-1中转座子MiniHimarl-lacZ插入位点两侧13 kb序列。通过ORF分析软件FramePlot 3.0beta分析预测该片段编码6个功能相关的同向转录ORFs。Genbank上BLASTX比对结果表明:在黄色粘球菌Myxococcus xanthus DK1622和粘细菌中与粘球菌同亚目的另一个菌株橙色标桩菌Stigmatella aurantiaca DW4/3-1基因组上,均有该6个ORFs的高度同源基因,且中间4个ORFs及其在DK1622和DW4/3-1中的同源ORFs都与主要来自真核生物的、与细胞的粘附、运动和增殖等多种细胞行为密切相关的Thrombospondin type 3 repeat家族蛋白同源。因此该基因簇被命名为mts(意指myxobacterial thrombospondin-like),分别编码MtsA、MtsB、MtsC、MtsD、MtsE和MtsF蛋白。在突变株HL-1中,转座子MiniHimar1-lacZ插入在ORF mtsC的3'末端。结构域预测软件SMART分析结果显示:MtsA和MtsE N端均有一个穿膜结构域,MtsD和MtsE均具有通常在细胞粘连等行为中发挥着重要作用的VWA[von Willebrand factor(vWF)type A domain]结构域。信号肽分析软件SignalP分析结果表明MtsA、MtsC、MtsD、MtsE和MtsF蛋白均具有信号肽。这些分析结果与突变株HL-1的社会行为和细胞外基质等表型分析结果一致,表明Mts蛋白可能定位在细胞膜上,构成细胞外基质的一部分,参与细胞的粘连、运动和发育等社会行为。
由于M.fulvus HW-1菌株的突变频率非常低,在该菌株中对mtsC基因的缺失一直没有成功。而令人欣慰的是,在同一属的菌株M.xanthus DK1622基因组上,有6个氨基酸一致性均高于85%的mts高度同源基因,无论是排列顺序还是转录方向都与M.fulvus HW-1的mts基因簇完全一致。因而利用M.xanthusDK1622中mts同源基因的功能分析来验证mts基因的功能是一条很好的途径。
M.xanthus DK1622菌株中mtsC同源基因插入失活后,突变株在软琼脂上的菌落形态与HL-1突变株一样,呈锯齿状,证明mtsC同源基因在黄色粘球菌的S运动中可能发挥着重要的作用。通过lacZ报告基因的表达分析证明了mtsC同源基因的表达情况与mtsC基因基本一致,表达水平较高,表达时间较早。这些结果充分说明用M.xanthus DK1622中mts同源基因的定点突变来验证mts基因的功能是适合的。
通过菌落扩展能力的分析比较证明了mtsC同源基因的插入失活对M.xanthus DK1622运动能力的影响远低于mtsC插入失活对M.fulvus HW-1运动能力的影响。该结果与PHX(predicted highly expressed)分析软件预测的mts及其同源基因表达水平结果一致,说明与mtsC同源基因在陆生粘球菌M.xanthusDK1622运动过程中发挥的作用相比,mtsC在海洋耐盐粘球菌M.fulvus HW-1运动过程中发挥的作用更重要,即mtsC甚至整个mts基因簇可能在M.fulvus Hw-1社会行为对海洋生境适应过程中发挥着非常重要的作用。
通过mtsC同源基因缺失突变及与已知A和S运动基因分别构建的双基因突变的分析,证明了mtsC同源基因属于S运动系统基因,在M.xanthus DK1622的S运动和发育过程中发挥着重要的作用。mts同源基因簇的缺失证明mts同源基因簇可能在M.xanthus DK1622的S运动和发育过程中发挥着重要的作用。
通过其它mts同源基因缺失的分析证明了mtsA、mtsB、mtsD、mtsE和mtsF同源基因均在M.xanthus DK1622的S运动中发挥着重要的作用;而除mtsF外,mtsA、mtsB、mtsD和mtsE同源基因也均在M.xanthus DK1622的子实体形成和粘孢子发育过程中占有重要的地位。
为了在粘球菌中对MtsB同源蛋白进行细胞定位,构建了荧光蛋白与MtsB同源蛋白融合表达菌株,但在该菌株细胞中没有检测到荧光。通过lacZ报告基因的分析显示了融合蛋白能够表达,但不能正常发射荧光。
在大肠杆菌中,异源表达了MtsB及其同源蛋白MXAN1333。通过表达细胞破碎液离心后沉淀和上清液中蛋白的分析确认了表达的融合蛋白MtsB和MXAN1333多数都呈包涵体形式。提取、纯化了融合蛋白MtsB和MXAN1333,并制备了融合蛋白MtsB和MXAN1333的多克隆抗体,为MtsB及其同源蛋白的细胞定位和表达时间谱等功能机制分析奠定了坚实的基础。
通过紫外诱变对M.fulvus HW-1进行了改造,获得了一株在液体培养基中均匀稳定分散生长诱变株M.fulvus UV684。对UV684进行了形态学特性、耐盐能力和运动能力的分析,显示了分散生长对粘球菌的营养细胞和粘孢子的形态特征、耐盐能力、A运动能力和海水条件下运动能力增强特性等基本没有影响,但可能导致子实体发育能力和S运动能力下降。通过电转化突变频率确认了HW-1经过诱变改造后适合遗传操作;显示粘球菌细胞分散生长后,对其遗传操作会更容易,为粘细菌和其它聚团生长菌株的遗传操作研究提供了线索。
Myxobacteria are Gram-negative bacteria that possess complicated multicellular social lifestyles on solid surfaces.The bacteria are model lives for the study of bacterial social behaviors.These behaviors include social gliding motility,predation and fruiting body development.Gliding motility of cells plays a crucial role during myxobacterial predatory and developmental aggregation lifestyle.The gliding,well demonstrated in Myxococcus xanthus,is controlled by two distinct motility systems: the adventurous(A)system,which controls the gliding motility of individual and isolated cells,and the social(S)system,which is essential for the swarm and aggregation movements of cells.Three cell surface components are required for S-motility,i.e.typeⅣpili(Tfp),extracellular matrix(ECM)and lipopolysaccharide (LPS)O-antigen.The mechanism of S-motility is more complicated genetically than that of flagellar motility.So far,more than 110 genes known to be essential for S-motility have been identified.
To fit the social life,myxobacteria are generally located in soil and,consequently, are common in many types of terrestrial rather than in aquatic environments,and thus, they are considered to be typical soil microbes.Moreover,no purified terrestrial myxobacteria had been confirmed to be able to grow with salt concentration of more than 1.0%.However,myxobacteria have recently been found to be able to live in many marine environments as halotolerants or low halophiles.
Halophilic myxobacteria were isolated from various marine samples in Japan,and are both phylogenetically distant and morphogenetically different from the normal terrestrial myxobacteria and thus have been classified as novel myxobacterial groups. Taxonomic characteristics,phylogenetic analysis of 16S rDNA fragments and secondary metabolites,instead of living pattern,adaptation strategies and molecular mechanism have been studied of these halophiles.
Many salt-tolerant myxobacteria have been isolated from marine samples in our laboratory.In response to changes in salinity,the salt-tolerant myxobacteria shift their growth,morphology and development characteristics.From differences in the morphogenetic characteristics between terrestrial and marine conditions,the salt-tolerant myxobacteria are thought to be the result of the adaptation of soil myxobacteria to marine conditions.Analysis of different salt-tolerant Myxococcus species or strains reveals that these myxobacteria retain the dual motion phenotypes, and the high salt-tolerant strains,including M.fulvus HW-1,even exhibit enhanced S-motility abilities in the presence of seawater.Stronger S-motility may allow closer contact between the cells and therefore provide better adaptation to the ocean conditions.Exploration and discovery of the adaptation and diversification of myxobacterial motility systems under natural conditions may provide important clues for us to understand the formation and evolution of the myxobacterial motility systems.
The mariner transposable elements of mariner/Tc1 superfamily are widely distributed in animal genomes and are especially prevalent in insects,mariner transponsable elements have a remarkable lack of host specificity.Transposition of mariner transposons almost appears to be random.These transposons are particularly valuable also because they are small and use short inverted repeats.In some papers, mariner-based transposons were reported to be active in myxobacterial strains and broadly useful as genetic tools.
In this work,the genetic manipulation methods established in M.fulvus HW-1 meet the major requirements for molecular studies on motility and development.Using the methods,a genetic screen was performed in M.fulvus HW-1 and a new locus was discovered to be required for S-motility and development in Myxococcus,which can afford new ideas and potential means for identifying new genetic loci involved in motility in myxobacteria.
First of all,to identify the S-motility genes related to the adaptation of M.fulvus HW-1 to ocean conditions,we screened S-motility deficient mutants following random insertion of the transposon MiniHimar1-lacZ into genomic DNA.From more than 2000 insertion mutants,we identified a mutant,named M.fulvus HL-1,showed significant reduction in colony expansion on a 0.3%agar surface,which is indicative of an S-motility defect.Activity analysis ofβ-galactosidase indicated that the mutated gene in HL-1 can early express and express at high level.
The mutant HL-1 was assessed for motility phenotypes using standard methods, and the phenotypes suggest that the mutant HL-1 is defective in social motility and partially A motile.Interestingly,the effect of seawater on swarming ability was significantly decreased by the mutation.These results suggest that mutated gene(s)are involved in or responsible for the enhancement of surface translocation in response to the presence of seawater.The mutant HL-1 was assessed for developmental ability on TPM starvation media,and the results indicate that the mutant HL-1 is also significantly defective in developmental aggregation and sporulation.Liquid colorimetric assay,agglutination assay and electron microscopy detection revealed less extracellular matrix on the cell surface of HL-1 and that the mutant cells exhibited less cohesion than the wild type cells.These results demonstrate that the mutated gene is probably due to the requirements for adaptation to the marine conditions in HW-1.
An upstream 6.3 kb segment and a downstream 6.7 kb segment flanking the insertion were obtained by plasmid rescue method and Tail-PCR amplifications. Using programs FramePlot 3.0beta,the 13 kb segment was predicted to contain six open reading frames,which likely form a gene cluster.Blastx against the GenBank database revealed that the sequences are significantly homologous to the corresponding ORFs from M.xanthus DK 1622 and Stigmatella aurantiaca DW4/3-1. Most of these ORFs are predicted to be putative type 3 thrombospondin.Thus,the gene cluster was designated mts for myxobacterial thrombospondin-like proteins (MtsA-F).The MiniHimarl-lacZ insertion interrupted the codon for Tyr359,located 83 residues from the C-terminus of the predicted MtsC.MtsA and MtsE possess transmembrane regions at their N-termini,as assessed by SMART.SignalP-HMM program predicted that MtsA,MtsC,MtsD,MtsE and MtsF contain signal peptides. Bioinformatics analysis of the predicted mts,together with the phenotypes of the mutant,suggested that the Mts proteins are probably involved in the construction of cell surface matrix for S-motility and development.
Genetic manipulation of HW-1 is difficult.We were not able to mutagenize the strain by deletion using the plasmid pBJ113.Instead,an insertion of mtsC homologue was performed in M.xanthus strains DK1622.The mutant produced colony with similar edge to that of HL-1,indicating that mtsC homologue is probablely required for motility in DK1622.The expression of mtsC homologue was also similar to that of mtsC in HW-1.
Interestingly,the effect of the insertion in mtsC homologue on motility in M. xanthus seems to be not as strong as the insertion in mtsC in M.fulvus HW-1.These results corresponding to E(g)values of the predicted mts suggested that the mrs products play a more important role on S-motility in M.fulvus HW-1 than their homologues do in M.xanthus DK1622,probably due to the requirements for adaptation to the marine conditions.
We made an in-frame deletion of mtsC homologue in DK1622(A~+S~+),DK1217 (A~-S~+)and DK10410(A~+S~-)to determine its function.The results indicated that mtsC homologue is involved in S-motility and required for development in M.xanthus. Also,we made a complete deletion of the sequence of MXAN1332～MXAN1337 from DK1622,and the results indicated that the whole mts homologues are likely to be involved in S-motility and required for development in M.xanthus.
Then,in-frame deletion of each mts homologue except mtsC homologue was performed in DK1622,the phenotypes of these mutants indicated that mtsA,mtsB and mtsD～mtsF homologues are involved in S-motility and mtsA,mtsB,mtsD and mtsE homologues are required for development in M.xanthus.
We were not able to identify the localization of MtsB homologue by expressing a MtsB homologue-fluorescent protein fusion protein.Thus,MtsB and MXAN1333 were hetero expressed in the host strain E.coli BL21(DE3),respectively.The fusion proteins MtsB and MXAN1333 were both extracted from inclusion bodies formed in the host strain and purified with Ni~(2+)column with a His-tag Multi-clone antibodies were produced with the purified MtsB and MXAN1333 as the antigens,respectively.
The strain HW-1 was induced by UV radiation and a mutant UV684 was obtained. Compared with the parent,UV684 showed completely dispersed growth in liquid medium and exhibited high transformation/tranposition efficiency as 10~5-10~6 CFU/μg DNA.On the other hand,the phenotypic characteristics,including salt-tolerant growth, morphology,fruiting-body formation and gliding motility,were analyzed and compared with the parent strain HW-1.The results indicated that the fruiting body formation and S-motility of UV684,to some extent,was affected in UV mutagenesis, but no difference was observed between the two strains in the size and shape of vegetative cells and myxospores,the salt-tolerant growth capacity,and the motilities increasing in the presence of seawater.So the mutant UV684 can be studied as an excellent parent in these unchanged unique characteristics by molecular genetic manipulation.
二十世纪末以前,粘细菌被普遍认为是典型的土壤微生物,分离的陆生菌株不能够在高于1%的盐浓度条件下生长。而从1998年日本学者在海洋样品中分离到嗜盐粘细菌以来,越来越多的粘细菌从多种海洋环境中分离纯化出来。根据耐盐能力的不同,海洋粘细菌被分为两类:海洋嗜盐粘细菌和海洋耐盐粘细菌。
海洋嗜盐粘细菌主要是由日本学者分离到的,根据形态学特征和16S rDNA的分析,这些菌株被归为四个新的属。他们对海洋嗜盐粘细菌的研究主要集中在形态学分析、分类以及抗生素等代谢产物分析方面,而关于粘细菌在海洋生境中的生活模式、定殖机制及更深入的分子机制等方面一直都没有涉及。
本实验室经过多年的探索,从海洋样品中分离到了多株海洋耐盐粘细菌。前期实验中,将其中的橙色粘球菌Myxococcus fulvus HW-1菌株作为一株典型的耐盐粘细菌模式菌株,与其它耐盐粘细菌或嗜盐粘细菌对比,对其形态学、生理学和系统分类学等特征及运动和发育能力进行了详细的分析。揭示了海洋耐盐粘细菌在陆地和海洋生境下存在两种不同的生活模式;并由实验结果推测海洋耐盐粘细菌是陆生粘细菌被带到海洋生境中适应了海水环境而存活下来,即海洋耐盐橙色粘球菌M.fulvus HW-1生活在海洋生境中是适应进化的结果。并且发现,在海水条件下,海洋耐盐粘球菌M.fulvus HW-1不仅保留了双运动系统,且双运动能力尤其是S运动能力明显增强。S运动的增强可能促进细胞间的粘连,为更好的适应海洋生境提供条件,证明社会运动在海洋耐盐粘球菌M.fulvus HW-1适应海洋生境生存过程中发挥着非常重要的作用。探知自然生境下粘细菌运动系统的多样性和对环境的适应可能为我们理解粘细菌运动系统的形成和进化提供重要线索。
mariner转座因子是来自真核生物的mariner/Tc1超家族的成员,与原核生物的转座子如Tn5相比,有着多种独特的性质,如结构简单、转座作用不依赖宿主的特定因素和随机插入等,这些特性使其在自然界中广泛分布,并可在物种间水平转移。目前,已有多篇文献报道mariner转座因子可以在粘细菌中有效地转座,并且达到稳定整合,是粘细菌遗传研究的有力工具之一。
本研究以海洋耐盐橙色粘球菌M.fulvus HW-1为材料,建立了一整套适合耐盐粘球菌的分子遗传操作体系,为开展耐盐粘细菌运动和发育等细胞行为的分子遗传学研究搭建了基础平台;在此基础上,利用mariner类转座子MiniHimarl-lacZ构建了HW-1菌株的小型随机插入突变菌库,从中发现并深入研究了一个社会运动和发育相关的新基因簇mts,该结果不仅扩充了粘球菌社会运动和发育相关基因文库,弥补了在模式菌株DK1622中筛选失活后对运动仅有微弱影响基因困难的不足,为粘球菌运动基因的筛选提供了新途径和新思路,而且开启了在分子水平上研究海洋耐盐粘细菌社会行为对海洋生境适应机制的大门,为理解HW-1社会行为对海洋生境适应的分子机制提供了重要依据。
实验中,为了研究与M.fulvus HW-1适应海洋生境相关的S运动系统基因,首先将mariner类转座子MiniHimarl-lacZ电转化入M.fulvus HW-1细胞,通过随机插入突变的方法建立了M.fulvus HW-1菌株的随机突变菌库,从中筛选到一株社会运动缺陷突变株M.fulvus HL-1。利用报告基因lacZ的表达验证了突变株HL-1染色体上插入了转座子MiniHimarl-lacZ,并且发现突变基因表达时间很早、表达水平很高。
双运动能力分析的经典方法——“软硬琼脂方法”分析结果显示:突变株M.fulvus HL-1 S运动能力缺陷,A运动能力受到影响;且令人兴奋的是,该突变株在海水条件下运动能力增强的趋势明显减弱、甚至消失,证明突变株M.fulvus HL-1中的突变基因在海洋耐盐橙色粘球菌M.fulvus HW-1在海水条件下S运动能力增强行为中发挥着重要的作用。通过发育平板上发育能力的检测证明了突变株M.fulvus HL-1的子实体形成和粘孢子发育能力缺陷。分析S运动系统的重要组分细胞外基质的染料结合实验、聚集实验和扫描电镜观察实验结果充分证明了突变株M.fulvus HL-1细胞外基质数量减少,细胞粘连性降低。这些实验结果表明:突变株M.fulvus HL-1中的突变基因可能在M.fulvus HW-1社会行为(运动和发育)中、甚至在社会行为对海洋生境适应过程中发挥着非常重要的作用。
通过质粒营救法和Tail-PCR方法获得了突变株M.fulvus HL-1中转座子MiniHimarl-lacZ插入位点两侧13 kb序列。通过ORF分析软件FramePlot 3.0beta分析预测该片段编码6个功能相关的同向转录ORFs。Genbank上BLASTX比对结果表明:在黄色粘球菌Myxococcus xanthus DK1622和粘细菌中与粘球菌同亚目的另一个菌株橙色标桩菌Stigmatella aurantiaca DW4/3-1基因组上,均有该6个ORFs的高度同源基因,且中间4个ORFs及其在DK1622和DW4/3-1中的同源ORFs都与主要来自真核生物的、与细胞的粘附、运动和增殖等多种细胞行为密切相关的Thrombospondin type 3 repeat家族蛋白同源。因此该基因簇被命名为mts(意指myxobacterial thrombospondin-like),分别编码MtsA、MtsB、MtsC、MtsD、MtsE和MtsF蛋白。在突变株HL-1中,转座子MiniHimar1-lacZ插入在ORF mtsC的3'末端。结构域预测软件SMART分析结果显示:MtsA和MtsE N端均有一个穿膜结构域,MtsD和MtsE均具有通常在细胞粘连等行为中发挥着重要作用的VWA[von Willebrand factor(vWF)type A domain]结构域。信号肽分析软件SignalP分析结果表明MtsA、MtsC、MtsD、MtsE和MtsF蛋白均具有信号肽。这些分析结果与突变株HL-1的社会行为和细胞外基质等表型分析结果一致,表明Mts蛋白可能定位在细胞膜上,构成细胞外基质的一部分,参与细胞的粘连、运动和发育等社会行为。
由于M.fulvus HW-1菌株的突变频率非常低,在该菌株中对mtsC基因的缺失一直没有成功。而令人欣慰的是,在同一属的菌株M.xanthus DK1622基因组上,有6个氨基酸一致性均高于85%的mts高度同源基因,无论是排列顺序还是转录方向都与M.fulvus HW-1的mts基因簇完全一致。因而利用M.xanthusDK1622中mts同源基因的功能分析来验证mts基因的功能是一条很好的途径。
M.xanthus DK1622菌株中mtsC同源基因插入失活后,突变株在软琼脂上的菌落形态与HL-1突变株一样,呈锯齿状,证明mtsC同源基因在黄色粘球菌的S运动中可能发挥着重要的作用。通过lacZ报告基因的表达分析证明了mtsC同源基因的表达情况与mtsC基因基本一致,表达水平较高,表达时间较早。这些结果充分说明用M.xanthus DK1622中mts同源基因的定点突变来验证mts基因的功能是适合的。
通过菌落扩展能力的分析比较证明了mtsC同源基因的插入失活对M.xanthus DK1622运动能力的影响远低于mtsC插入失活对M.fulvus HW-1运动能力的影响。该结果与PHX(predicted highly expressed)分析软件预测的mts及其同源基因表达水平结果一致,说明与mtsC同源基因在陆生粘球菌M.xanthusDK1622运动过程中发挥的作用相比,mtsC在海洋耐盐粘球菌M.fulvus HW-1运动过程中发挥的作用更重要,即mtsC甚至整个mts基因簇可能在M.fulvus Hw-1社会行为对海洋生境适应过程中发挥着非常重要的作用。
通过mtsC同源基因缺失突变及与已知A和S运动基因分别构建的双基因突变的分析,证明了mtsC同源基因属于S运动系统基因,在M.xanthus DK1622的S运动和发育过程中发挥着重要的作用。mts同源基因簇的缺失证明mts同源基因簇可能在M.xanthus DK1622的S运动和发育过程中发挥着重要的作用。
通过其它mts同源基因缺失的分析证明了mtsA、mtsB、mtsD、mtsE和mtsF同源基因均在M.xanthus DK1622的S运动中发挥着重要的作用;而除mtsF外,mtsA、mtsB、mtsD和mtsE同源基因也均在M.xanthus DK1622的子实体形成和粘孢子发育过程中占有重要的地位。
为了在粘球菌中对MtsB同源蛋白进行细胞定位,构建了荧光蛋白与MtsB同源蛋白融合表达菌株,但在该菌株细胞中没有检测到荧光。通过lacZ报告基因的分析显示了融合蛋白能够表达,但不能正常发射荧光。
在大肠杆菌中,异源表达了MtsB及其同源蛋白MXAN1333。通过表达细胞破碎液离心后沉淀和上清液中蛋白的分析确认了表达的融合蛋白MtsB和MXAN1333多数都呈包涵体形式。提取、纯化了融合蛋白MtsB和MXAN1333,并制备了融合蛋白MtsB和MXAN1333的多克隆抗体,为MtsB及其同源蛋白的细胞定位和表达时间谱等功能机制分析奠定了坚实的基础。
通过紫外诱变对M.fulvus HW-1进行了改造,获得了一株在液体培养基中均匀稳定分散生长诱变株M.fulvus UV684。对UV684进行了形态学特性、耐盐能力和运动能力的分析,显示了分散生长对粘球菌的营养细胞和粘孢子的形态特征、耐盐能力、A运动能力和海水条件下运动能力增强特性等基本没有影响,但可能导致子实体发育能力和S运动能力下降。通过电转化突变频率确认了HW-1经过诱变改造后适合遗传操作;显示粘球菌细胞分散生长后,对其遗传操作会更容易,为粘细菌和其它聚团生长菌株的遗传操作研究提供了线索。
Myxobacteria are Gram-negative bacteria that possess complicated multicellular social lifestyles on solid surfaces.The bacteria are model lives for the study of bacterial social behaviors.These behaviors include social gliding motility,predation and fruiting body development.Gliding motility of cells plays a crucial role during myxobacterial predatory and developmental aggregation lifestyle.The gliding,well demonstrated in Myxococcus xanthus,is controlled by two distinct motility systems: the adventurous(A)system,which controls the gliding motility of individual and isolated cells,and the social(S)system,which is essential for the swarm and aggregation movements of cells.Three cell surface components are required for S-motility,i.e.typeⅣpili(Tfp),extracellular matrix(ECM)and lipopolysaccharide (LPS)O-antigen.The mechanism of S-motility is more complicated genetically than that of flagellar motility.So far,more than 110 genes known to be essential for S-motility have been identified.
To fit the social life,myxobacteria are generally located in soil and,consequently, are common in many types of terrestrial rather than in aquatic environments,and thus, they are considered to be typical soil microbes.Moreover,no purified terrestrial myxobacteria had been confirmed to be able to grow with salt concentration of more than 1.0%.However,myxobacteria have recently been found to be able to live in many marine environments as halotolerants or low halophiles.
Halophilic myxobacteria were isolated from various marine samples in Japan,and are both phylogenetically distant and morphogenetically different from the normal terrestrial myxobacteria and thus have been classified as novel myxobacterial groups. Taxonomic characteristics,phylogenetic analysis of 16S rDNA fragments and secondary metabolites,instead of living pattern,adaptation strategies and molecular mechanism have been studied of these halophiles.
Many salt-tolerant myxobacteria have been isolated from marine samples in our laboratory.In response to changes in salinity,the salt-tolerant myxobacteria shift their growth,morphology and development characteristics.From differences in the morphogenetic characteristics between terrestrial and marine conditions,the salt-tolerant myxobacteria are thought to be the result of the adaptation of soil myxobacteria to marine conditions.Analysis of different salt-tolerant Myxococcus species or strains reveals that these myxobacteria retain the dual motion phenotypes, and the high salt-tolerant strains,including M.fulvus HW-1,even exhibit enhanced S-motility abilities in the presence of seawater.Stronger S-motility may allow closer contact between the cells and therefore provide better adaptation to the ocean conditions.Exploration and discovery of the adaptation and diversification of myxobacterial motility systems under natural conditions may provide important clues for us to understand the formation and evolution of the myxobacterial motility systems.
The mariner transposable elements of mariner/Tc1 superfamily are widely distributed in animal genomes and are especially prevalent in insects,mariner transponsable elements have a remarkable lack of host specificity.Transposition of mariner transposons almost appears to be random.These transposons are particularly valuable also because they are small and use short inverted repeats.In some papers, mariner-based transposons were reported to be active in myxobacterial strains and broadly useful as genetic tools.
In this work,the genetic manipulation methods established in M.fulvus HW-1 meet the major requirements for molecular studies on motility and development.Using the methods,a genetic screen was performed in M.fulvus HW-1 and a new locus was discovered to be required for S-motility and development in Myxococcus,which can afford new ideas and potential means for identifying new genetic loci involved in motility in myxobacteria.
First of all,to identify the S-motility genes related to the adaptation of M.fulvus HW-1 to ocean conditions,we screened S-motility deficient mutants following random insertion of the transposon MiniHimar1-lacZ into genomic DNA.From more than 2000 insertion mutants,we identified a mutant,named M.fulvus HL-1,showed significant reduction in colony expansion on a 0.3%agar surface,which is indicative of an S-motility defect.Activity analysis ofβ-galactosidase indicated that the mutated gene in HL-1 can early express and express at high level.
The mutant HL-1 was assessed for motility phenotypes using standard methods, and the phenotypes suggest that the mutant HL-1 is defective in social motility and partially A motile.Interestingly,the effect of seawater on swarming ability was significantly decreased by the mutation.These results suggest that mutated gene(s)are involved in or responsible for the enhancement of surface translocation in response to the presence of seawater.The mutant HL-1 was assessed for developmental ability on TPM starvation media,and the results indicate that the mutant HL-1 is also significantly defective in developmental aggregation and sporulation.Liquid colorimetric assay,agglutination assay and electron microscopy detection revealed less extracellular matrix on the cell surface of HL-1 and that the mutant cells exhibited less cohesion than the wild type cells.These results demonstrate that the mutated gene is probably due to the requirements for adaptation to the marine conditions in HW-1.
An upstream 6.3 kb segment and a downstream 6.7 kb segment flanking the insertion were obtained by plasmid rescue method and Tail-PCR amplifications. Using programs FramePlot 3.0beta,the 13 kb segment was predicted to contain six open reading frames,which likely form a gene cluster.Blastx against the GenBank database revealed that the sequences are significantly homologous to the corresponding ORFs from M.xanthus DK 1622 and Stigmatella aurantiaca DW4/3-1. Most of these ORFs are predicted to be putative type 3 thrombospondin.Thus,the gene cluster was designated mts for myxobacterial thrombospondin-like proteins (MtsA-F).The MiniHimarl-lacZ insertion interrupted the codon for Tyr359,located 83 residues from the C-terminus of the predicted MtsC.MtsA and MtsE possess transmembrane regions at their N-termini,as assessed by SMART.SignalP-HMM program predicted that MtsA,MtsC,MtsD,MtsE and MtsF contain signal peptides. Bioinformatics analysis of the predicted mts,together with the phenotypes of the mutant,suggested that the Mts proteins are probably involved in the construction of cell surface matrix for S-motility and development.
Genetic manipulation of HW-1 is difficult.We were not able to mutagenize the strain by deletion using the plasmid pBJ113.Instead,an insertion of mtsC homologue was performed in M.xanthus strains DK1622.The mutant produced colony with similar edge to that of HL-1,indicating that mtsC homologue is probablely required for motility in DK1622.The expression of mtsC homologue was also similar to that of mtsC in HW-1.
Interestingly,the effect of the insertion in mtsC homologue on motility in M. xanthus seems to be not as strong as the insertion in mtsC in M.fulvus HW-1.These results corresponding to E(g)values of the predicted mts suggested that the mrs products play a more important role on S-motility in M.fulvus HW-1 than their homologues do in M.xanthus DK1622,probably due to the requirements for adaptation to the marine conditions.
We made an in-frame deletion of mtsC homologue in DK1622(A~+S~+),DK1217 (A~-S~+)and DK10410(A~+S~-)to determine its function.The results indicated that mtsC homologue is involved in S-motility and required for development in M.xanthus. Also,we made a complete deletion of the sequence of MXAN1332～MXAN1337 from DK1622,and the results indicated that the whole mts homologues are likely to be involved in S-motility and required for development in M.xanthus.
Then,in-frame deletion of each mts homologue except mtsC homologue was performed in DK1622,the phenotypes of these mutants indicated that mtsA,mtsB and mtsD～mtsF homologues are involved in S-motility and mtsA,mtsB,mtsD and mtsE homologues are required for development in M.xanthus.
We were not able to identify the localization of MtsB homologue by expressing a MtsB homologue-fluorescent protein fusion protein.Thus,MtsB and MXAN1333 were hetero expressed in the host strain E.coli BL21(DE3),respectively.The fusion proteins MtsB and MXAN1333 were both extracted from inclusion bodies formed in the host strain and purified with Ni~(2+)column with a His-tag Multi-clone antibodies were produced with the purified MtsB and MXAN1333 as the antigens,respectively.
The strain HW-1 was induced by UV radiation and a mutant UV684 was obtained. Compared with the parent,UV684 showed completely dispersed growth in liquid medium and exhibited high transformation/tranposition efficiency as 10~5-10~6 CFU/μg DNA.On the other hand,the phenotypic characteristics,including salt-tolerant growth, morphology,fruiting-body formation and gliding motility,were analyzed and compared with the parent strain HW-1.The results indicated that the fruiting body formation and S-motility of UV684,to some extent,was affected in UV mutagenesis, but no difference was observed between the two strains in the size and shape of vegetative cells and myxospores,the salt-tolerant growth capacity,and the motilities increasing in the presence of seawater.So the mutant UV684 can be studied as an excellent parent in these unchanged unique characteristics by molecular genetic manipulation.
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