Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation

Abstract Lactobacillus plantarum ATCC 8014 was transformed with pTV1 by electroporation using a modification of a procedure described for Escherichia coli . The plasmid pTV1 which contains the pE194 replicon from Staphylococcus aureus and transposon Tn917 from Streptococcus faecalis was shown to replicate as a high copy number plasmid in L. plantarum , and the two encoded antibiotic resistance traits were expressed. Tn917 transposed with a high frequency into plasmid DNA of L. plantarum as shown by restriction enzyme analysis and Southern hybridization studies. There are no previous reports on transposition in the lactobacilli. This system may prove to be an important tool in further work on the genetics of these organisms.     

Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4

Summary A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30° C but not at 42° C, clones are isolated resistant to kanamycin at 42° C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30° C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42° C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained.About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10^-10.The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn₁ in many Gram-negative bacterial species.     

Mutagenesis using Tn917 in Bacillus licheniformis

Abstract Two plasmid vectors containing Tn 917 have been transformed into protoplasts of Bacillus licheniformis . Replication of the vectors was inhibited at high temperature. The transporon integrated efficiently into the chromosome and auxotroph formation was high (9% of transposed colonies). Most of these were argG or argH indicating a hot-spot region for integration of Tn 917 into the chromosome. However, about 0.5% of the mutants had other auxotrophies.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

Streptococcus faecalis R plasmid pJH1 contains an erythromycin resistance transposon (Tn3871) similar to transposon Tn917

The R plasmid pJH1 contains a 5.1-kilobase transposon ( Tn3871 ) that mediates inducible resistance to erythromycin. Three AvaI digestion fragments from this transposon are identical in size to and homologous with three AvaI-derived fragments from the previously described erythromycin resistance transposon Tn917 . These three DNA fragments account for greater than 90% of both transposons.     

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.     

Transposition of Tn5096 from a temperature-sensitive transducible plasmid in Streptomyces spp.

Transposon Tn5096 was inserted into a derivative of the temperature-sensitive plasmid pMT660 containing the bacteriophage FP43 pac site. The resulting plasmid, pRHB126, was transduced by FP43 into several Streptomyces species. Tn5096 transposed from pRHB126 into different sites in the genomes of Streptomyces ambofaciens, Streptomyces cinnamonensis, Streptomyces coelicolor A3(2), Streptomyces fradiae, Streptomyces griseofuscus, and Streptomyces thermotolerans.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.     

Evolution of an R plasmid from a cryptic plasmid by transposition of two copies of Tn1 in Providencia stuartii

Examination of a series of isolates of Providencia stuartii collected over an 18 month period from a chronic-care patient at Bristol Royal Infirmary revealed the emergence of resistance to carbenicillin. Resistance was mediated by a 47 kb plasmid which transferred by conjugation to a plasmid-free strain of P. stuartii but not to Escherichia coli. Carbenicillin-sensitive isolates were either plasmid-free or contained a 36 kb cryptic plasmid. Restriction endonuclease mapping of this plasmid showed it to be closely related to 32 kb and 34 kb cryptic plasmids reported previously in P. stuartii from Bristol. Mapping of the R plasmid showed it to be derived from the 34 kb cryptic plasmid by transposition of two copies of Tn1.     

A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes

Aims: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. Methods and Results: We optimized the conditions for transformation using a plasmid pUCYIT356‐1‐Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. Conclusions: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. Significance and Impact of the Study: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.     

The effect of various rec-mutations on the frequency of the Tn917 transposition in Bacillus subtilis cells

The effect of 11 rec-genes on the transposition frequency of Tn917 has been studied. Transposition frequencies in RecP, RecF15, RecB3 mutants differed from the ones in the control strains. The collection of mitomycin-sensitive mutants has been tested for transposition proficiency. The mms315 mutation decreasing the transposition frequency possess the properties of the rec mutation too.     

Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917

Plasmids pAD1 (37.8 megadaltons) and pAD2 (17.1 megadaltons) of Streptococcus faecalis strain DS16 have been mapped with restriction enzymes. The location of a hemolysin-bacteriocin determinant on the conjugative pAD1 plasmid was derived from analyses of transposon insertions. Electron microscope and hybridization analyses located Tn917(Em) and the streptomycin (Sm) and kanamycin (Km) resistance determinants on the nonconjugative pAD2 plasmid. It was shown previously that the erythromycin (Em) resistance associated with Tn917 is inducible and that transposition from pAD2 to pAD1 is also stimulated by exposure of cells to low concentrations of Em. Here we show that inducing concentrations of Em also increase the conjugative transfer potential of pAD1; this is possibly related to a mild and short-lived inhibitory stress placed on the cells before full induction of resistance. Selection of Em-resistant transconjugants arising from matings between DS16 and a plasmid-free recipient gave rise to transconjugants which primarily harbor stable pAD1::pAD2 cointegrates. A 30-min exposure of donors to Em (0.5 microgram/ml) before mating resulted in a severalfold increase in the number of such transconjugants. However, a small fraction (e.g., 3 of 40) of these Emr Smr Kmr transconjugants harbored pAD1::Tn917 and pAD2 molecules. Since we believe pAD2 is incapable of being mobilized by pAD1 without being covalently linked, it is likely that transfer in these cases involved cointegrates representing structural intermediates in the transposition of Tn917 from pAD2 to pAD1. It follows that such intermediates probably had two copies of Tn917 and readily resolved after transfer. (These cointegrates are different from the stable cointegrates which were shown to have only a single copy of Tn917; the latter are assumed not to be related to transposition.) Two variants with altered Tn917 transposition properties were derived. One of them transposed at an elevated frequency, whereas the other showed no detectabel transposition. In neither case was transposition influenced by Em exposure; however, both remained inducible for Em resistance.     

Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen

The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.     

Transposon mutagenesis in Staphylococcus epidermidis using the Enterococcus faecalis transposon Tn917

We transformed a clinical Staphylococcus epidermidis isolate with the Enterococcus faecalis transposon Tn917-carrying plasmid pTV1. Loss of plasmid replication was observed at 47 degrees C. Tn917 transposes efficiently and apparently randomly. The transposition frequency could be stimulated by erythromycin. Transposon mutagenesis in S. epidermidis provides a means for genetic study of the various virulence factors of this pathogen.     

Tn5 as a model for understanding DNA transposition

Tn5 is an excellent model system for understanding the molecular basis of DNA-mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild-type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.     

The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum

The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.     

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。     

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.