Herbert P. Schweizer

Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa.

The nucleotide sequence of the 1.9-kb PstI fragment from pRO1614, that allows stable maintenance of pMB1 (ColE1)-based cloning vectors in Pseudomonas, was determined. This fragment encodes a putative origin of replication (ori), a replication-controlling protein, and the C terminus of the Tn3 beta-lactamase-encoding gene. Improved versions of the broad-host-range plasmid vectors, pUCP18 and pUCP19, were constructed by deletion of nonessential DNA or replacement of nonessential DNA with an antibiotic-resistance cassette.

AN IMPROVED SYSTEM FOR GENE REPLACEMENT AND XYLE FUSION ANALYSIS IN PSEUDOMONAS AERUGINOSA

A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ alpha allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

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.

Allelic 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 PA01 and should allow the exchange of any DNA segment into any non‐essential regions of the P. aeruginosa chromosome.

Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5)

Previous work from our laboratory has shown that cosmid clone pFVl00, containing a 26 kb insert, is able to restore O‐antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi‐rough (SR) mutants, AK14O1 and rd7513, resulted in O‐antigen expression, indicating that pFV100 may contain an O‐polymerase (rfc) gene. pFV.TK6, a subclone of pFVl00 that contains a 5.6 kb chromosomal insert, was able to complement O‐antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O‐antigen expression in AK14O1. A 2.0 kb Xhol‐HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross‐reactive O2‐O5‐O16‐O18‐O20 serogroup, suggesting that these serotypes may share a common O‐polymerase gene. In functional studies of the rfc gene, the PAOl (serotype O5) chromosomal rfc was mutated using a gene‐replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O‐polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane‐spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.

Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes

Two plasmids, X1918 and Z1918, were constructed which contain the promoter-less xylE and lacZ reporter genes flanked symmetrically by the multiple cloning site (MCS) from pUC19. These cassettes can easily be derived from the multicopy plasmid, pUC1918.

Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa

Several new broad-host-range vectors for the construction of protein fusions to the Escherichia coli lacZ gene have been developed. In all of the constructs, a multiple cloning site (MCS) containing unique restriction sites is located upstream of lac operon segments whose lacZ genes lack translational start signals. Some of the vectors (pPZ10, pPZ20 and pPZ30) also contain transcriptional terminators upstream of the MCS. The new vectors allow the fusion of genes to lacZ in all translational reading frames. Due to a higher copy number they allow direct screening in E. coli for weakly expressed foreign promoters. Their usefulness for gene analysis in Pseudomonas aeruginosa was demonstrated by construction and expression of a regA::lacZ-encoded protein fusion.

Utilization of a mim-Dlac transposable element to create an α-complementation and regulated expression system for cloning in Pseudomonas aeruginosa

A lac-based alpha-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophage D3112-based mini-Dlac transposable element, containing the lacIq-regulated lacZ delta M15 gene next to a selectable marker, was constructed. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, and derivatives containing randomly inserted chromosomal copies of the mini-Dlac element were obtained. Transformation of the PAO1::mini-Dlac transductants with the broad-host-range vector, pUCP19, led to the formation of blue colonies on indicator medium in the presence of inducer. In contrast, transformants harboring the pUCP19 derivative pCDO, containing the catechol-2,3-dioxygenase (C23O)-encoding xylE gene under lac promoter control, were white on the same medium. Expression of xylE was tightly controlled by single-copy mini-Dlac-encoded lac repressor and in induced cultures was increased more than 100-fold over that observed in uninduced cultures. The usefulness of the system for molecular cloning in P. aeruginosa was demonstrated by ligating size-fractionated PAO1 chromosomal fragments into pUCP19, followed by transformation of the newly isolated PAO1::mini-Dlac host. All randomly chosen white colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone. The functionality of the system was also shown in another frequently studied strain, PA103.

A novel phosphate-regulated expression vector in Escherichia coli

The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn -glycerol-3-phosphate transport system in Escherichia coli

SummaryThe gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.