Mobilization of the pACYC184 plasmid by hybrid pAS8-121 delta plasmids in Escherichia coli cells

The plasmid pACYC184 is shown to be mobilized for conjugal transfer in Escherichia coli cells by the deleted (Tn7-TcR) derivatives of the hybrid conjugative plasmid pAS8-121 (RP4-Co1E1). Both the mobilized and mobilizing plasmids are autonomously inherited by the recipient cells when the mobilizing plasmid carries single copy of IS8 (the plasmid pAS8-121 delta 16). Cointegrates pAS8-121 delta 16D:: ::pACYC184 are found in the recipient cells with pACYC184 being inserted between two repeats of IS8 if the derivate plasmid pAS8-121 delta 16D having the duplication of IS8 is used to mobilize pACYC184 for conjugal transfer. The insertion of pACYC184 between IS8 repeats in the plasmid pAS8-121 delta 16D eliminates the plasmid ability to be inserted with high frequency into the chromosome of the phototrophic bacterium R. sphaeroides 2R. The cointegrate pAS8-121 delta 16D:: pACYC184 is stable but can be resolved during the transformation deriving the plasmid pACYC184:: IS8. The latter may be used as a probe for isolation and analysis of IS8 DNA sequences and for constructing the vectors on the basis of pACYC184.     

Transformation of colonial Escherichia coli on solid media

We report that colonial Escherichia coli cells on various solid media can develop modest genetic competence. Using an on-filter culture system, we found that E. coli colonies on CaCl2-containing agar were transformed in the presence of plasmid DNA. Interestingly, transformation also occurred on LB-agar, various moist foods and even on H2O-agar. These results suggest that some populations of colonial E. coli in various environments could become transformable regardless of the surrounding Ca2+ concentration.     

Recombinant plasmids capable of integration into the chromosome of the cyanobacterium Anacystis nidulans R2

Recombinant plasmids, series pIAB and pIAH, have been constructed by insertion of BamHI or HindIII chromosomal fragments from Anacystis nidulans R2 into the tet gene of plasmid pACYC184. Plasmids pIAB and pIAH are stably maintained in Escherichia coli cells and transfer the CmR marker in transformation of Anacystis nidulans. Blot hybridization technique has shown the formation of CmR clones in transformation to result from integration of plasmid pACYC184 with the chromosome of cyanobacterium.     

细菌自然转化的分子机制研究进展

自然环境中,具备自然转化能力的细菌可以自发地从外界获取DNA,从而获得新的遗传性状。为能够自然地被转化,细菌需首先建立一个被称作感受态的生理状态并在此状态下表达DNA摄取和加工相关的基因。DNA摄取基因的表达产物可组装一个能将外源DNA摄入细胞质的蛋白复合物。在细胞质中,进入的DNA可同基因组DNA发生同源重组或建立成一个独立的质粒。一般DNA摄入细胞的过程可分为两个阶段,即从外部基质到细胞周质和跨细胞内膜的转运。近年来,包括作者在内的研究人员发现大肠杆菌中存在新的自然质粒转化模式。本文将首先综述近年来细菌自然转化的分子机制,随后简要介绍大肠杆菌中独特的自然质粒转化模式。     

Cloning and expression of Thiobacillus versutus cryptic plasmid pTAV-1 DNA in Escherichia coli

pTAV-1 is an approximately 100 kb Thiobacillus versutus cryptic plasmid. pTAV-1 DNA was cloned in Escherichia coli. Nine recombinant plasmids containing pTAV-1 DNA inserted into the EcoRI restriction site of pACYC184 were constructed. The origin of DNA inserts was confirmed by Southern blot hybridization. The expression of mixotrophic T. versutus plasmid genes was demonstrated in E. coli.     

Spot-transformation with plasmids

Summary A method is described which allows transformation of bacterial cells on the surface of agar plates. It is suitable for transferring large numbers of different plasmids, such as gene libraries, into a new genetic background. One nanogram of plasmid DNA is sufficient for transformation.     

Localization of a genetic region involved in McrB restriction by Escherichia coli K-12.

A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9. The resultant hybrid plasmid was shown to complement the mcrB mutation of E. coli K802. The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184. These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.     

一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Cloning of the phospho-β-galactosidase gene in Escherichia coli from lactose-negative mutants of Streptococcus mutans isolated following random mutagenesis with plasmid pVA891 clone banks

Abstract In order to mutagenize Streptococcus mutans a marker rescue plasmid, pVA891, was employed. The plasmid was ligated with Sau 3AI digested chromosomal DNA fragments from S. mutans GS-5IS3 and the resultant plasmids were amplified in Escherichia coli . These plasmids were then randomly integrated into the chromosome of strain GS-5IS3 following transformation. Lactose-negative transformants were isolated as white colonies on lactose-BTR-Xgal agar plates containing erythromycin. Six lactose-negative mutants representing three different chromosomal sites of integration were isolated from about eight thousand transformants. Mutant chromosomal DNA fragments flanking the plasmids were recovered by a marker-rescue method in E. coli and exhibited phospho-β-galactosidase activity.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.     

Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ～9 kDa and ～30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5)-10(-6), suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.     

DNA conformation of the region preceding the beta-lactamase promoter of pUC19 which is required for efficient transcription.

The upstream region of the beta-lactamase promoter of Escherichia coli plasmid pUC19 has a DNA curvature (bent DNA). This region was replaced with another sequence by using randomly synthesized oligonucleotides. Among the reconstructed plasmids, a plasmid which could endow E. coli cells with the strongest resistance to ampicillin on plates was selected. Nucleotide sequence and DNA conformation of the altered region was investigated.     

Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation

An electroporation procedure for the plasmid-mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O-1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug-resistance genes on the vector plasmid were expressed in the Vibrio strains.     

Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences

The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells. Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.     

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.