一个植物乳杆菌隐蔽质粒的序列分析

[目的]分离植物乳杆菌(Lactobacillus plantarum LP101)中的质粒,并对其序列进行分析。[方法]从植物乳杆菌LP101中提取质粒并行双酶切,纯化后与载体pet22b进行连接,电转化到大肠杆菌DH5α中。将测序后得到的质粒片段进行拼接,对拼接成功的质粒进行序列分析。[结果]从植物乳杆菌LP101中分离得到一个隐蔽质粒p LP101,测序结果显示该质粒大小为3 496 bp,碱基G+C含量为38.2%,编码了一个移动蛋白和一个复制蛋白。BLAST结果显示该质粒的核苷酸序列与干酪乳杆菌(L.casei)中的质粒p SMA23相似性在99%以上。[结论]分离得到了植物乳杆菌LP101中的一个隐蔽质粒p LP101,推定其复制方式为滾环复制,属于滚环复制p C194家族成员。     

一个植物乳杆菌新质粒的序列分析与归类

[目的]分离鉴定植物乳杆菌PC518的质粒并分析滚环复制p C194家族复制起点特征。[方法]从植物乳杆菌PC518中提取质粒,HindⅢ单酶切后克隆测序,然后用反向PCR方法验证质粒序列的完整性。使用DNAMAN V6. 0软件和MEGA X软件对43个p C194家族质粒的复制起点序列和复制蛋白进行比对分析。[结果]分离得到一个3 325 bp的新质粒p LP325。43个p C194家族质粒复制起点中:24个在nick上、下游均有反向重复序列,12个只在nick上游有反向重复序列,4个只在下游有反向重复序列。复制蛋白的聚类与复制起点中反向重复序列的位置是对应的。[结论]p LP325的复制方式推定为滚环复制,属于p C194家族。p C194家族复制起点的bind以反向重复序列为特征,位于nick上游或下游。     

植物乳杆菌PC518质粒pLP224的发现以及pMV158家族质粒的保守性和多样性分析

【背景】植物乳杆菌含有丰富的天然质粒,分析这些质粒的序列特征有利于分析质粒所携带的遗传信息。【目的】分析从植物乳杆菌PC518分离的新质粒pLP224,聚类分析其所属家族质粒的保守性与多样性。【方法】提取植物乳杆菌PC518的质粒,酶切后构建质粒DNA文库,测序和BLAST鉴定文库中的新序列;通过反向PCR完成质粒全序列测定,注释新质粒;使用进化树软件MEGA X构建质粒的Rep蛋白进化树,并分析结合序列的变化。【结果】从植物乳杆菌PC518分离出一个质粒pLP224,大小为1 766 bp,其中(G+C)mol%含量为41.39%,与已知质粒的最大序列相似性为86.85%。推定其复制方式为滚环复制,属于pMV158家族成员。17个pMV158家族质粒的Rep蛋白分析表明：pMV158家族质粒的Rep蛋白进化距离越近,其dso位点的结合序列相似性越高,进化距离越远则其序列相似性越低。【结论】 pLP224是pMV158家族的新成员。pMV158家族质粒在dso位点的切开序列上保守,在结合序列上多样。其Rep蛋白随结合序列变化而不同。这种差异有利于pMV158家族不同成员在同一宿主的共存,是家族成员持续存在并稳定进化的基础。     

苏云金芽胞杆菌拟步行甲亚种YBT_1765中稳定质粒pBMB175的克隆和功能分析

从苏云金芽胞杆菌拟步行甲亚种YBT_1765中克隆得到一个大小约152 kb的质粒pBMB175,构建了该质粒的限制性图谱,通过功能验证,将其最小的复制区定位在一个1151bp的片段上。分析了包含有这个复制区的一个大小为4152 bp的核苷酸序列,该片段包含有3个编码框（ORF1、OFR2和ORF3）。氨基酸序列同源性比较发现, ORF1(767AA)与UvrD_旋促酶、重组酶RecD和RecB家族具有20%～30%的相似性;ORF2（149AA）没有发现与任何已知序列具有同源性;ORF3（83AA）与pGI3中一个未知功能的蛋白（ORF7）具有34%的相似性。通过缺失及序列比较分析推测ORF2可能编码一种新的复制蛋白。因此pBMB175的复制类型可能属于一类新的复制家族。利用最小复制区构建的重组质粒在无抗生素选择压力下可稳定遗传40多代,具备构建稳定遗传质粒载体的潜力。     

苏云金芽胞杆菌拟步行甲亚种YBT-1765中稳定质粒pBMB175的克隆和功能分析

从苏云金芽胞杆菌拟步行甲亚种YBT_1765中克隆得到一个大小约15.2kb的质粒pBMB175,构建了该质粒的限制性图谱,通过功能验证,将其最小的复制区定位在一个1151bp的片段上。分析了包含有这个复制区的一个大小为4152bp的核苷酸序列,该片段包含有3个编码框(ORF1、OFR2和ORF3)。氨基酸序列同源性比较发现,ORF1(767AA)与UvrD_旋促酶、重组酶RecD和RecB家族具有20%～30%的相似性;ORF2(149AA)没有发现与任何已知序列具有同源性;ORF3(83AA)与pGI3中一个未知功能的蛋白(ORF7)具有34%的相似性。通过缺失及序列比较分析推测ORF2可能编码一种新的复制蛋白。因此pBMB175的复制类型可能属于一类新的复制家族。利用最小复制区构建的重组质粒在无抗生素选择压力下可稳定遗传40多代,具备构建稳定遗传质粒载体的潜力。     

高温链霉菌质粒pTSC2的测序分析和滚环复制方式的鉴定

从猪粪堆肥中分离到一株编号为X3-3的可以在50℃高温生长的链霉菌菌株,该菌株含有一个约7kb的环型质粒pTSC2。【目的】克隆、测序和分析pTSC2,以及鉴定质粒的复制方式。【方法】利用分段克隆和引物延伸获得pTSC2的全序列,利用多序列比对寻找复制元件rep、dso和sso,利用中性转移和Southern杂交检测复制中间体。【结果】克隆和测序获得了全长为7516bp的pTSC2序列,预测编码8种蛋白,其中4种蛋白与链霉菌滚环复制的质粒pIJ101中负责复制和接合转移的蛋白非常相似。pTSC2的复制元件rep、dso和sso也与pIJ101的相似。克隆、转化变铅青链霉菌ZX7以及高温链霉菌2C证明了rep和dso为复制所必需元件。Southern杂交检测到pTSC2复制过程中积累了大量的单链DNA。【结论】高温链霉菌质粒pTSC2以滚环方式进行复制。这是首次在高温链霉菌中克隆和测序质粒,以及鉴定其复制方式。     

植物乳杆菌编码复制起始蛋白天然质粒的系统进化分析

【目的】探究复制起始蛋白(Replication initiation protein,Rep)是否可以作为天然质粒系统进化关系研究的分子标记。【方法】以植物乳杆菌天然质粒编码的Rep为研究对象,通过构建Rep系统进化树详细分析和讨论这些质粒的系统进化关系。【结果】植物乳杆菌45个编码Rep天然质粒可以划分为5个进化关系紧密的家族和1个独立进化质粒p G6302,家族1-4质粒可以进一步划分为10个进化关系更近的亚家族类群,因此这些质粒可能起源于6个祖先质粒。【结论】Rep氨基酸序列显示了适度的保守性和变异性,是植物乳杆菌编码Rep质粒进化研究理想的分子标记,为植物乳杆菌天然质粒系统进化研究提供了一种简单、有效的分析方法和标准,并为植物乳杆菌或其他乳酸菌天然质粒系统进化研究提供了分子水平的佐证和依据。     

溶藻弧菌质粒pVAE259全序列测定与分子生物学特征分析

【目的】获得溶藻弧菌环状质粒pVAE259全序列,分析其分子生物学特征并探索该质粒可能具备的功能。【方法】使用酶切、克隆测序的方法获得pVAE259的全序列,利用软件分析DNA序列和可能的编码蛋白,推测质粒的生物学信息。【结果】pVAE259为闭合环状质粒,全长6,075 bp,GC含量为42.16%。在NCBI中比对发现pVAE259与Vibriosp.41隐蔽性质粒pPS41具有较高的相似性。我们在序列中找到一个oriT位点,另外全序列的4118-5494 bp推测为质粒复制区域。pVAE259中存在7个氨基酸序列长度大于100的开放式阅读框(ORF):ORF1-ORF7。其中ORF1编码蛋白属于释放酶超级家族(Relaxase Super-family)蛋白,在NCBI数据库中它与大肠杆菌(Escherichia coli)的MobA-like蛋白最相似;ORF2编码蛋白属于复制酶超级家族(Replicase Super-family),它与嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)的复制蛋白RepA最相似;ORF5与伸长盐单胞菌(Halomonas elongata)质粒pHE1的转移蛋白MobC相似。【结论】根据上述结果及相关文献分析,pVAE259可能是具有转移能力的质粒,该质粒是否影响宿主菌的表型性状还不清楚。     

发根农杆菌及其应用

农杆菌是一类侵染性非常广泛的Ｇ-土壤杆菌,农杆菌质粒介导的基因转移系统是植物基因工程中比较完善与有效的基因转移方法。在众多的转基因植物中有80%是由农杆菌介导转化的,但其中大部分是有根癌农杆菌Ｔｉ质粒介导法获得的。根癌农杆菌含有的Ｔｉ质粒,能诱导被侵染的植物细胞形成肿瘤,即诱发冠瘿瘤。发根农杆菌含有Ｒｉ质粒,侵染植物后能诱发植物细胞产生毛发状根,即发根瘤。近年来的研究发现发根农杆菌Ｒｉ质粒介导法比Ｔｉ质粒法具有一定优越性而被广泛重视,而且由其转化获得转基因植物和生物有效成分的报道愈来愈多,本文主要讨论发根农…     

植物乳杆菌LY-78乳酸氧化酶基因克隆与生物信息学分析

[目的]对植物乳杆菌LY-78乳酸氧化酶基因(lox)进行克隆及生物学信息学分析。[方法]PCR克隆lox基因,测序后采用多种生物学信息软件预测该基因对应蛋白的理化性质和结构特征。[结果]植物乳杆菌LY-78的乳酸氧化酶基因全长1 101 bp,编码366个氨基酸,其蛋白分子量38 730.1 Da,p I 5.58,亲水性非分泌蛋白。该蛋白氨基酸序列系统进化树分析表明,该基因序列保守性较高,可以反映近缘物种的亲缘关系;具有保守的α-羟基酸脱氢酶结合结构域。[结论]为揭示植物乳杆菌LY-78乳酸氧化酶的生理功能及在苯乳酸代谢机理中的作用提供了理论依据。     

Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.     

Characterization of pLP18, a novel cryptic plasmid of Lactobacillus plantarum PC518 isolated from Chinese pickle

A cryptic plasmid of Lactobacillus plantarum PC518 isolated from Chinese pickle, designated pLP18, was sequenced and characterized. It is a 1806-bp circular molecule with a G+C content of 37.5%. Sequence analysis of pLP18 revealed three putative open reading frames (ORFs), in which ORF1 contained conserved motifs of pMV158-family Rep proteins and showed 60% similarity with the Rep protein of pPSC22, a member of rolling-circle replication (RCR) pMV158 family. The double strand origin (dso) of pMV158 family and the single strand origin A (ssoA) located upstream of the rep gene. The putative cop and rnaII genes were predicted to be regulatory genes controlling copy number of pLP18. The results of Southern hybridization suggested that pLP18 replicate via the RCR mechanism. Furthermore, the relative copy number of pLP18 was estimated to be about 24 copies per chromosome equivalent by quantitative PCR.     

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.     

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.     

Complete nucleotide sequence of plasmid pST-III from Lactobacillus plantarum ST-III

The complete nucleotide sequence of the 53,560-bp plasmid pST-III from Lactobacillus plantarum ST-III has been determined. The plasmid contains 42 predicted protein-coding sequences, and the functions of 34 coding sequences could be assigned. Homology analysis for the replication protein and the typical features of the origin of replication suggested that pST-III replicates via the theta-type mechanism. Among the predicted genes, we identified a kdp gene cluster (a high-affinity K(+)-transport system) for the first time in the Lactobacillus genus and a system for osmolyte transport. Analysis of the plasmid-encoded functions and the plasmid-cured experiment showed that the genes of pST-III could serve for the niche adaptations of L. plantarum ST-III and make significant contributions to its viability under hyperosmotic conditions. Furthermore, the relative copy number of pST-III was determined to be 6.79±1.55 copies per cell.     

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp cremoris P8-2-47

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered (copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.