Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.     

Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant.

The replication region of a 28-kilobase-pair (kbp) cryptic plasmid from Lactococcus lactis subsp. lactis biovar diacetylactis SSD207 was cloned in L. lactis subsp. lactis MG1614 by using the chloramphenicol resistance gene from the streptococcal plasmid pGB301 as a selectable marker. The resulting 8.1-kbp plasmid, designated pVS34, was characterized further with respect to host range, potential cloning sites, and location of replication gene(s). In addition to lactococci, pVS34 transformed Lactobacillus plantarum and, at a very low frequency, Staphylococcus aureus but not Escherichia coli or Bacillus subtilis. The 4.1-kbp ClaI fragment representing lactococcal DNA in pVS34 contained unique restriction sites for HindIII, EcoRI, XhoII, and HpaII, of which the last three could be used for molecular cloning. A region necessary for replication was located within a 2.5-kbp fragment flanked by the EcoRI and ClaI restriction sites. A 3.8-kbp EcoRI fragment derived from a nisin resistance plasmid, pSF01, was cloned into the EcoRI site of pVS34 to obtain a nisin-chloramphenicol double-resistance plasmid, pVS39. From this plasmid, the streptococcal chloramphenicol resistance region was subsequently eliminated. The resulting plasmid, pVS40, contains only lactococcal DNA. Potential uses for this type of a nisin resistance plasmid are discussed.     

Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter

An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or lacl(q) genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lac(q) improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration (c) 1993 Wiley & Sons, Inc.     

Genetic analysis of the gentamicin resistance region of pPH1JI and incorporation into a wide host range cloning vehicle

A region of the IncP plasmid pPH1JI encoding resistance to gentamicin, spectinomycin, and streptomycin was characterized by subcloning, deletion, and insertion mutagenesis. Approximate locations of these resistance determinants were established. A 1.6-kb HindIII-SphI segment of this region expresses gentamicin resistance (Gmr) in Escherichia coli when inserted into various plasmid vectors; this DNA segment encodes a polypeptide of 17.5 kDa. Incorporation of this fragment into an IncP cloning vehicle produced a Gmr wide host range vector, pRAR209, which confers levels of Gmr comparable to those expressed by pPH1JI in E. coli, Agrobacterium tumefaciens, and Rhizobium meliloti.     

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

Plasmid Stability in Pseudomonas fluorescens in the Rhizosphere

Plasmids belonging to various incompatibility (Inc) groups were introduced into the efficiently root-colonizing strain Pseudomonas fluorescens WCS365, and their stabilities in complex and minimal media and in the rhizospheres of tomato, wheat, and potato plants grown under gnotobiotic conditions without selection pressure were tested. The IncP plasmid was found to be highly unstable under all conditions tested, whereas the IncQ and IncW plasmids showed intermediate stabilities and the plasmids pVSP41 and pWTT2081, for which the Inc group is unknown, both containing the origin of replication (rep) and stability (sta) regions of the Pseudomonas aeruginosa pVS1 replicon, were stably maintained under all conditions tested. Growth experiments in which cells of strain WCS365 carrying the plasmid pWTT2081 were grown in the presence of WCS365 without the plasmid showed that the presence of pWTT2081 acts as a burden. We conclude that pVSP41 and pWTT2081 are valuable as stable vectors for the functional analysis of genes involved in root colonization, provided that control cells carry the empty vector.     

Conjugative transfer of plasmid RP1 to soil isolates of Pseudomonas fluorescens is facilitated by certain large RP1 deletions

Abstract The broad-host-range IncP plasmid RP1 could not be transferred by conjugation from Escherichia coli to Pseudomonas fluorescens strain CHA0. However, this conjugative transfer was possible with RP1 derivatives which had large deletions extending from the primase gene towards the Tra-2 region, thus lacking the kanamycin resistance gene and IS 21 . Such RP1 deletion derivatives permitted IncP cosmid mobilization to P. fluorescens CHA0 and could be used as vectors for transposon mutagenesis with a newly constructed Tn 5 derivative (carrying kanamycin and mercury resistance determinants) in strain CHA0 and another P. fluorescens soil isolate, strain S9.     

Prevotella intermedia native plasmid can be mobilized by an Escherichia coli conjugal IncP plasmid

We have determined the nucleotide sequence of a small Prevotella intermedia cryptic plasmid, pYHBi1, which consisted of sequences that were highly homologous to the amino acid sequence of the replication and mobilization proteins found in related organisms. We have also demonstrated that chimeric plasmids derived from this P. intermedia native plasmid can be mobilized between Escherichia coli strains by using a broad-host-range E. coli conjugative plasmid, IncP plasmid RP4. The results suggest that pYHBi1 possesses gene(s) responsible for conjugal transfer.     

Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression

Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.     

Development of a system vector-host in methylotrophic bacteria

Cloning of methylotropic and other Gram negative bacteria's genes was performed using vectors derived from IncP4 plasmids. Plasmids, such s RSF1010 are 8.8 kb in length, have a high copy number and broad host range and can be mobilized efficiently by a number of conjugative plasmids. IncP4 plasmids have relatively few restriction enzyme's targets suitable for cloning. In this paper the construction of versatile and special purpose IncP4 vectors available for cloning DNA into broad range of bacterial species are described. The seria of versatile vectors involves the transposon containing plasmid and two-replicon vectors.In genetic construction of special vector for direct cloning of restriction fragments the genetic regulation elements of Tn 1 were used. On the base of IncP4 replicon special vectors for construction of bank genes (cosmids) and the vectors for cloning of regulation sequence were also constructed.     

Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxydans

Abstract: Conjugal transfer of a series of incompatibility group P and Q plasmids has been studied in the acetic acid bacterium, Gluconobacter oxydans ssp. suboxydans . Transfer frequencies for the IncP/Q vectors ranged from 10⁻⁵−10⁻⁹ exconjugants per recipient cell. It was found in the case of the IncP vector, pRK290, that Bgl II insert constructs displayed increased conjugal transfer frequencies over pRK290 per se, the parent plasmid. A gentamycin-resistant encoding pRK290 vector which was constructed offers considerable potential as a versatile gene delivery system for Gluconobacter . The lactose transposon, Tn951, was used as a model to examine heterologous gene expression in G. oxydans ssp. suboxydans . The expression level of Tn951 encoded β-galactosidase in this strain was found to be less than 5% of that found in the parent Escherichia coli strain, JC3272.     

A family of shuttle vectors for lactobacilli and other gram-positive bacteria based on the plasmid pLF1311 replicon

A set of broad-host-range single-replicon shuttle vectors for cloning nucleotide sequences in gram-positive bacteria (lactobacilli, enterococci, lactococci, bacilli, etc.) was created. The vectors are based on the cryptic plasmid pLF1311 from Lactobacillus fermentum VKM 1311 belonging to a family of the sigma-type pE194-like plasmids. The vectors can replicate in gram-positive bacteria and Escherichia coli. They are stable in many gram-positive bacteria, have small sizes, and allow the selection of recombinants on media with X-Gal. The vectors that contain the region of initiation of the conjugal transfer of plasmid RP4 belonging to the incompatibility group IncP alpha can be mobilized in a great number of bacteria using a helper plasmid from E. coli but not from gram-positive bacteria.     

Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element.

Bacteroides-Escherichia coli shuttle vectors containing a nonmobilizable pBR322 derivative and either pBFTM10 (pDP1, pCG30) or pB8-51 (pEG920) were mobilized by IncP plasmid R751 or pRK231 (an ampicillin-sensitive derivative of RK2) between E. coli strains and from E. coli to Bacteroides recipients. IncI alpha R64 drd-ll transferred these vectors 1,000 times less efficiently than did the IncP plasmids. pDP1, pCG30, and pEG920 could be mobilized from B. uniformis donors to both E. coli and Bacteroides recipients by a conjugative Bacteroides Tcr (Tcr ERL) element which was originally found in a clinical Bacteroides fragilis strain (B. fragilis ERL). However, the shuttle vector pE5-2, which contains pB8-51 cloned in a restriction site that prevents its mobilization by IncP or IncI alpha plasmids, also was not mobilized at detectable frequencies from Bacteroides donors by the Tcr ERL element. The mobilization frequencies of pCG30, pDP1, and pEG920 by the Tcr ERL element in B. uniformis donors to E. coli recipients was about the same as those to isogenic B. uniformis recipients. Transfer of the shuttle vectors from B. uniformis donors to E. coli occurred at the same frequencies when the matings were done aerobically or anaerobically. Growth of the B. uniformis donors in tetracycline (1 microgram/ml) prior to conjugation increased the mobilization frequencies of the vectors to both E. coli and Bacteroides recipients 50 to 100 times.     

The replication initiator operon of promiscuous plasmid RK2 encodes a gene that complements an Escherichia coli mutant defective in single-stranded DNA-binding protein.

The amino acid sequence of the 13-kDa polypeptide (P116) encoded by the first gene of the trfA operon of IncP plasmid RK2 shows significant similarity to several known single-stranded DNA-binding proteins. We found that unregulated expression of this gene from its natural promoter (trfAp) or induced expression from a strong heterologous promoter (trcp) was sufficient to complement the temperature-sensitive growth phenotype of an Escherichia coli ssb-1 mutant. The RK2 ssb gene is the first example of a plasmid single-stranded DNA-binding protein-encoding gene that is coregulated with replication functions, indicating a possible role in plasmid replication.     

Specific-purpose broad-host-range vectors

Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 were constructed and characterized. Vector pAYC30 was constructed by insertion in vivo into the genome of RSF1010 the Hgr transposon Tn501, originating from the plasmid pVS1 of Pseudomonas aeruginosa. Plasmids with inserts of PstI or SacI fragments may be selected by inactivation of genes sul and aph, respectively. The cloning at unique site SalGI leads to the appearance of HgCl2--sensitive transformants. Versatile cloning vector pAYC1 consists of two replicons, RSF1010 and plasmid pMZ7, a derivative of R6K. The constructed plasmid is 16.9 kb in length and determines resistance to five drugs. Two promoter-probe broad-host-range vectors, pAYC36 and pAYC37, were obtained by replacing a small segment from the DNA sequence of the aph gene promoter of previously described plasmid pAYC32 with the polylinker from plasmid pUC19. Therefore, vector plasmids retained the intact gene aph (Smr); however, they have Sms phenotype because of the insertional inactivation of the promoter. The genetic structure of promoter-probe vectors allows one to select clones, containing hybrid plasmids with an active promoter for gene aph expression.     

Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer.

Transfer genes of the IncP plasmid RP4 are grouped in two separate regions, designated Tra1 and Tra2. Tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells. To provide information relevant to understanding the function of Tra2 gene products, the nucleotide sequence of the entire RP4 Tra2 region is presented here. Twelve open reading frames were identified in the Tra2 core region, being essential for intraspecific Escherichia coli matings. Predicted sizes of 11 of the 12 Tra2 polypeptides could be verified by expression in E. coli. Based on hydropathy plot analysis, most of the Tra2 open reading frames encode proteins that may interact with membranes. Interestingly, six of the predicted Tra2 gene products exhibited significant sequence similarities to gene products encoded by the VirB operon of the Agrobacterium Ti plasmid. VirB proteins are thought to function in the formation of a transmembrane structure that mediates the passage of T-DNA molecules from bacteria into plant cells. Because of this analogy and the hydropathy of Tra2 gene products, we assume that the DNA transfer machineries acting in bacterial conjugation and T-DNA transfer are structurally and functionally similar. Therefore, the data presented here, support the hypothesis that Ti vir and IncP tra genes evolved from a common ancestor. This suggestion is favored by previous findings of sequence similarities between the IncP and Ti DNA transfer system.