Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli

We have constructed two promoter-selection vectors based upon the broad-host-range plasmid pRO1614. pQF40 (6 kb) contains a promoterless tetA gene downstream from a large multiple cloning site while pQF26 (5.4 kb) possesses a promoterless cat cartridge. The latter vector displayed a copy number of 13 in Pseudomonas aeruginosa and 39 in Escherichia coli. When promoter sequences derived from the Pseudomonas phage phi PLS27 were cloned into pQF26, high levels of chloramphenicol-acetyltransferase were detected in P. aeruginosa. In E. coli the activity was approximately one-third that in P. aeruginosa when corrections were made for the plasmid copy number.     

Alielic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.     

Cloning vectors, derived from a naturally occurring plasmid of Pseudomonas savastanoi, specifically tailored for genetic manipulations in Pseudomonas

A minimal replicon of 1.8 kb isolated from a 10-kb plasmid of Pseudomonas savastanoi, pPS10, has been used to obtain a collection of small vectors specific for Pseudomonas (P. savastanoi, P. aeruginosa and P.putida). In addition, shuttle vectors that can be established both in Pseudomonas and Escherichia coli have been constructed by adding a pMB9 replicon. The vectors permit cloning of DNA fragments generated by a variety of restriction enzymes using different antibiotic resistance markers for selection and offer the possibility to screen recombinants by insertional inactivation. This cloning system can be used to establish recombinant plasmids in Pseudomonas either at low or high copy number. pPS10 derivatives are compatible with other Pseudomonas vectors derived from broad-host-range replicons of the incompatibility groups P1, P4/Q and W. Introduction and expression of the iaaMH operon in a P. savastanoi mutant deficient in the production of indoleacetic acid has been achieved using one of these vectors.     

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas

Host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. They comprise restriction-negative host strains of Pseudomonas aeruginosa and P. putida and new cloning vectors derived from the high-copy-number, broad-host-range plasmid RSF1010, which are stably maintained in a wide range of Gram-negative bacteria. These plasmids contain EcoRI, SstI, HindIII, XmaI, XhoI, SalI, BamHI, and ClaI insertion sites. All cloning sites, except for BamHI and ClaI, are located within antibiotic-resistance genes' insertional inactivation of these genes during hybrid plasmid formation provides a readily scored phenotypic change for the rapid identification of bacterial clones carrying such hybrids. One of the new vector plasmids is a cosmid that may be used for the selective cloning of large DNA fragments by in vitro lambda packaging. An analogous series of vectors that are defective in their plasmid-mobilization function, and that exhibit a degree of biological containment comparable to that of current Escherichia coli vector plasmids, are also described.     

Escherichia-Pseudomonas shuttle vectors derived from pUC18/19

Two new broad-host-range plasmid vectors, pUCP18 and pUCP19, which are stably maintained in Escherichia coli and Pseudomonas aeruginosa have been constructed. The plasmids are based on the E. coli pUC18 and pUC19 vectors and possess all their features: (i) convenient direct screening of recombinants; (ii) versatile multiple cloning site; (iii) use as sequencing and expression vectors; (iv) small size; and (v) intermediate to high copy number.     

A Tn7-based broad-range bacterial cloning and expression system

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.     

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.     

Characteristics of derivatives of the plasmid RP4 in a broad range of hosts with altered properties of maintenance and inheritance

Hydroxylamine-induced mutants of the plasmid pPD6 (8.4 kb) were isolated which are resistant to high doses of tetracycline. One of the plasmids studied--pPD21 is a multicopy mutant, another one, pPD12 is a dimeric form of the pPD6 plasmid. The pPD12 plasmid is very unstable, its derivative, pPD13 spontaneous mutant acquiring stability but not the ability to resolve DNA multimeric forms into monomeric forms. Multicopy bireplicon pPD619 plasmid was constructed by joining in vitro pPD6 and pUC19 plasmids. Removing the replicon pUC19 from the bireplicon plasmid gives a new low-copy plasmid pPD620. All of the plasmids constructed were mobilized by the conjugative pRK2013 plasmid into the strains of Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens. The pPD6 plasmid and its derivatives can be used as cloning vectors.     

Development of broad-host-range vectors for expression of cloned genes in Pseudomonas

The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas. We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites. These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid. From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E. coli and Pseudomonas. These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P. putida strain. Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation.     

Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.     

Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.     

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.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Cloning of genes degrading 3-chlorobenzoate from Pseudomonas putida strain 87]

The ability of Pseudomonas putida strain 87 to catabolize 3-chlorobenzoate was shown to be mediated by genes of pBS109 plasmid. The plasmid may be transferred by conjugation into P. aeruginosa PAO2175. It seems possible that the pBS109 plasmid codes for pyrocatechase II specific for halogenated catechol, but not catechol. The genes specifying utilization of 3-chlorobenzoate from pBS109 plasmid were cloned in the 5.5 kb BgIII fragment by using broad-host cloning system. The resulting pBS110 plasmid was transferred into P. putida, which results in utilization of 3-chlorobenzoate by transconjugants.     

Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase.

A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis. The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA. Upon transformation, they can be used to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type. An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M. maripaludis. The beta-galactosidase gene from Escherichia coli was expressed to approximately 1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M. maripaludis genomic library.     

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.     

Expression of the phospholipase C gene of Pseudomonas aeruginosa in Escherichia coli and Pseudomonas

The plc gene for phospholipase of Pseudomonas aeruginosa, able to be transcribed only from its own promoter, has been introduced into Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida cells in the recombinant plasmid pPMS21 of a wide host range. The expression of plc gene in all recipient cells has been shown to be phosphate regulated. The fact emphasizes the identity of pho-regulation systems in Escherichia coli and Pseudomonas cells. The level of phospholipase activity is similar in Pseudomonas putida and Pseudomonas aeruginosa under the conditions of the gene derepression, while in Escherichia coli cells the level does not exceed 10% of activity registered in Pseudomonas cells.