A chimeric vector for efficient chromosomal modification in Enterococcus faecalis and other lactic acid bacteria

Aim: To construct a chimeric vector named pBVGh for quickly generating gene modifications in Enterococcus faecalis. Methods and Results: The constructed plasmid pBVGh carries the pG⁺host replicon (a thermosensitive (TS) derivative of pWV01), allowing a simple generation of mutants by growing colonies first at the permissive temperature and then switching the culture to the nonpermissive temperature. Additionally, this vector facilitates the screening of mutants by a rapid colorimetric blue‐white discrimination of plasmid‐free bacteria. Conclusions: The pBVGh vector allows a straightforward inactivation or modification of target genes as well as a fast selection of enterococcal mutant strains. Significance and Impact of the Study: The broad range of the TS replicon utilized in this plasmid permits the easy establishment and the efficient generation of food‐grade mutant strains in Ent. faecalis and several other Gram‐positive bacteria.     

A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes.

A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori+ RepA- vector pORI19, containing lacZ alpha and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA+ helper strain Escherichia coli EC101. The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA-), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363 (pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal- mutant at 30 degrees C resulted in excision of the integrated plasmid and restoration of the ability of ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal+ colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.     

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.     

Isolation and characterization of a Clo DF13::Tn901 plasmid mutant with thermosensitive control of DNA replication.

After nitrosoguanidine mutagenesis, a mutant Escherichia coli strain harboring the Clo DF13::Tn901 plasmid pJN03 was isolated that is thermosensitive (Ts) for growth at 43 degrees C. The mutation responsible for this thermosensitive phenotype resides on the pJN03 plasmid genome. Cells harboring the pJN03 cop-1(Ts) plasmid mutant showed a large increase in plasmid copy number at 43 degrees C accompanied by an increase in the synthesis of plasmid-specified gene products like cloacin DF13 and beta-lactamase. The pJN03 cop-1(Ts) mutant showed uncontrolled plasmid DNA replication at the nonpermissive temperature. Analysis of plasmid deletions showed that the mutation is located in the Clo DF13 map interval from 0 to 12% or 29 to 45%. This implies that native cloacin DF13 and the Clo DF13-specified polypeptides B, C, D, E, and G are not involved in the pleiotropic phenotype of the plasmid mutant pJN03 cop-1(Ts).     

Novel Escherichia coli mutant, dnaR, thermosensitive in initiation of chromosome replication.

A newly isolated Escherichia coli mutant thermosensitive in DNA synthesis had an allele named dnaR130, which was located at 26.3 minutes on the genetic map. The mutant was defective in initiation of chromosome replication but not in propagation at a high temperature. This mutant was capable of growing in the absence of the rnh function at the high temperature by means of a dnaA-independent replication mechanism. In the mutant exposed to the high temperature, an oriC plasmid was able to replicate, although at a lower rate than at the low temperature. The plasmid replication at the high temperature depended on the dnaA function essential for the initiation of replication from oriC. The mutant lacking the rnh function persistently maintained the oriC plasmid at the high temperature in a dnaA-dependent manner. Thus, the dnaR function was required for initiation of replication of the bacterial chromosome from oriC but not the oriC plasmid. This result reveals that a dnaR-dependent initiation mechanism that is dispensable for oriC plasmid replication operates in the bacterial chromosome replication.     

Unmarked gene modification in Streptococcus mutans by a cotransformation strategy with a thermosensitive plasmid

Unmarked gene modifications are desirable for various genetic analyses; however, they have been difficult to construct without selectable markers. We describe here a new genetic method for constructing unmarked mutants in Streptococcus mutans. The desired mutant allele is first constructed and introduced into the strain by cotransformation with pGhost4, a thermosensitive plasmid that replicates in several low G+C Gram positive bacteria. With this method, we have modified two different loci with high frequency by insertion or deletion in S. mutans. Because pGhost4 contains a broad host range thermosensitive replicon, this method can be applied to any transformable low G+C Gram positive bacteria, including oral streptococci.     

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.     

A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis

A series of variants of transposon Tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. A thermosensitive replication mutant of the broad-host-range transmissible plasmid R388 was also constructed for use as a suicide vector for the delivery of transposable elements.     

Filamentation promotes F'lac loss in Escherichia coli K12.

The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.     

Cloning and nucleotide sequence of a gene upstream of the eaeA gene of enterohemorrhagic Escherichia coli O157:H7

Abstract Conjugative, thermosensitive shuttle plasmids capable of transfer from Escherichia coli to Mycobacterium smegmatis were constructed. They contain both an E. coli replicon and a thermosensitive derivative of the pALS000 mycobacterial replicon. Using a temperature shift protocol, the conjugative plasmid, pJAZll was used to deliver the transposon Tn 611 from E. coli into the chromosome of M. smegmatis . Analysis of transconjugants revealed the random insertion of the transposon.     

Origin and direction of in vitro replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids.

The origin of replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids was located by cloning BamHI restriction fragments into vector plasmid pAT153 and a derivative plasmid, pAT2. Selection was made for plasmid maintenance in a polA mutant. Direction of replication was determined by in vitro replication of plasmid DNA in the presence of radiolabeled deoxynucleotide.     

炭疽芽胞杆菌mntA基因缺失突变株的构建及鉴定

目的:通过同源重组的方法敲除炭疽芽胞杆菌减毒AP422株的mntA基因,使菌株进一步减毒,用于构建新的疫苗候选株。方法:利用PCR方法扩增mntA基因上下游同源臂后与温敏质粒连接,构建打靶载体,并转化炭疽芽胞杆菌减毒AP422株;利用抗生素和温度2种选择压力实现同源重组,敲除目标基因mntA,然后利用Cre-LoxP系统去除抗性筛选标记,得到无抗性标记的缺失突变株,并利用PCR和Western印迹等方法对重组菌进行系统鉴定,最后分析突变株的生物学性状。结果:敲除了AP422株的mntA基因,获得了无抗性标记的缺失突变株,突变株的生存竞争能力比原始菌株明显减弱。结论:突变株获得了进一步减毒,可用于构建新的疫苗候选株。     

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.     

Characterization of the Streptomyces plasmid pVE1

pVE1 (11.0 kb) was isolated from Streptomyces venezuelae ATCC 14585 and characterized. pVE1 has a broad host range and an apparent copy number of about 100 during exponential growth, rising to 1500 during stationary phase. It is a pock-forming, self-transmissible fertility factor which promotes chromosome recombination in S. lividans at frequencies of about 0.1% per total parents. A detailed restriction map for 15 enzymes was determined. Genes for ThioR (thiostrepton resistance), NeoR (neomycin resistance), and pBR322 derivatives were inserted into pVE1 and the resulting plasmids were analyzed for self-transmissibility and stability. The plasmid has an essential region of approximately 2.5 kb and a region of 1.0-3.6 kb required for conjugation. NeoR and ThioR vectors were constructed with unique HindIII, PvuI, BamHI, EcoRI, BglII, and EcoRV sites available for insertion of foreign DNA.     

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2

The gene coding for recA in the oral pathogen Actinobacillus actinomycetemcomitans SUNY 465 was cloned and sequenced. The DNA sequence coded for a 352-amino acid protein that was homologous to RecA of a variety of bacterial species. A derivative of a non-replicating mobilizable plasmid was constructed for directed mutagenesis in A. actinomycetemcomitans. A recA-deficient strain of A. actinomycetemcomitans was developed by homologous recombination of an internal recA fragment contained on the mobilizable suicide vector. The recA mutant strain was more sensitive to UV radiation and showed a reduced recombinatorial proficiency than the isogenic parent strain. These data suggest that recA of A. actinomycetemcomitans SUNY 465 is involved in the repair of DNA damage caused by UV irradiation and homologous recombination as determined for other bacteria.     

Recombinant plasmid conferring proline overproduction and osmotic tolerance

A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.     

Recombinant plasmid conferring proline overproduction and osmotic tolerance.

A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.     

Regulation of transfer of the Enterococcus faecalis pheromone-responding plasmid pAD1: temperature-sensitive transfer mutants and identification of a new regulatory determinant, traD.

The enterococcal, conjugative, cytolysin plasmid pAD1 confers a mating response to the peptide sex pheromone cAD1 secreted by plasmid-free strains of Enterococcus faecalis. Cells carrying pAM714, a pAD1::Tn917 derivative with wild-type conjugation properties, were mutagenized with ethyl methanesulfonate to obtain variants that were induced (in the absence of pheromone) to transfer plasmid DNA upon shifting from 32 to 42 degrees C. Of 31 such mutants generated, the results of analyses of 7 are presented in detail. All seven strains were thermosensitive in the E. faecalis host FA2-2; colony morphology, clumping, and DNA transfer correlated well with each other at the two temperatures. In the nonisogenic host E. faecalis OG1X, however, only one derivative (pAM2725) exhibited correlation of all three traits at both temperatures. Three (pAM2700, pAM2703, and pAM2717) clumped and had colonies characteristic of pheromone-induced cells at 32 degrees C but transferred plasmid DNA at a higher frequency only at the elevated temperature. The other three (pAM2708, pAM2709, and pAM2712) were derepressed at both temperatures for all three characteristics. Four of the mutations, including that of pAM2725, mapped within the traA determinant, whereas two mapped identically in a previously unnoted open reading frame (designated traD) putatively encoding a short (23-amino-acid) peptide downstream of the inhibitor peptide determinant iad and in the opposite orientation. One mutant could not be located in the regions sequenced. Studies showed that the traA and traD mutations could be complemented in trans with a DNA fragment carrying the corresponding regions.     

构建无抗性标记双拷贝透明质酸合成酶基因工程菌

【目的】探讨一种构建无抗生素选择标记链球菌透明质酸合成酶基因工程菌的方法。【方法】PCR扩增链球菌透明质酸合成酶操纵子hasABC基因,用温度敏感载体pJR700构建携带hasABC基因重组质粒pXL32;电转化pXL32质粒到马链球菌兽疫亚种感受态细胞,卡那霉素(Kanamycin,kan)平板37℃培养筛选重组子,在不含kan液体培养基中30℃传代,37℃平板分离挑取抗生素敏感菌落,RT-PCR检测工程菌染色体hasABC基因表达,Bitter-Muir法测定菌体透明质酸含量。【结果】在无抗生素选择压力条件下,获得透明质酸产率提高34%的马链球菌兽疫亚种透明质酸合成酶基因工程菌。【结论】用pJR700温度敏感载体系统,构建能提高透明质酸产量的无抗生素选择标记的基因工程菌是可行的。