Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria

Abstract

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.

FIG. 1.

Tool kit for use with genes from gram-negative bacteria. Shown are maps of vectors for manipulating bacterial genes by using S. cerevisiae-based homologous recombination. Vector maps are shown as linearized at the first base pair and are not shown to scale. Constructs are clustered based upon suggested utility. sacB, Bacillus subtilis levansucrase gene for counterselection; oriT, origin of conjugal transfer; T1T2, E. coli rrnB transcriptional terminators; lacZα, lacZα with multicloning site driven by the lactose promoter; ColE1, high-copy-number variants of the narrow-host-range ColE1 origin of replication; p15a, narrow-host-range, low-copy origin of replication; aacC1, gentamicin resistance determinant from Tn1696; URA3, orotidine-5′-phosphate decarboxylase gene from S. cerevisiae; CEN/ARSH, low-copy yeast replication and segregation machinery; kanMX, kanamycin/G418 resistance cassette from pUG6; P_oriV-rep, pRO1600 broad-host-range replicon; bla, ampicillin and carbenicillin resistance determinant; 2μm, yeast episomal plasmid replicon; P_BAD-araC, arabinose-inducible promoter system from E. coli; RK2-trfA, minimal replicon from the extremely-broad-host-range RK2 plasmid; GFP, bright and stable GFPmut3 variant. Plasmid request information can be found at http://www.dartmouth.edu/∼gotoole/vectors.html.

FIG. 2.

P. aeruginosa clpP deletion and clpX disruption mutants have altered surface-associated phenotypes. (A) Overnight cultures of P. aeruginosa strain PA14 (wild type [wt]) and isogenic ΔclpP and clpX::pRMQS84 strains were spotted on swarming agar plates and incubated at 37°C for 24 h (4, 38). Swarming in the wt strain is indicated by the tendril-like structures originating from the point of inoculation and spreading across the agar surface. The absence of ClpP and ClpX correlated with a reduction in surface motility. (B) ΔclpP mutants exhibit increased biofilm formation under flow conditions in M63 medium supplemented with arginine. Shown here are photomicrographs of wt, ΔclpP, and clpX::pMQ84 biofilms 24 and 48 h after inoculation.

FIG. 3.

P_BAD-driven expression in Pseudomonas species. (A) Aliquots of P. aeruginosa cultures bearing pMQ78 grown overnight in LB with ampicillin and various concentrations of l-arabinose were viewed with either phase-contrast microscopy (left) or epifluorescent microscopy (right). Micrographs of GFP fluorescence were taken with identical exposure times. No fluorescence was detected in the absence of l-arabinose or in cells containing an identical vector without GFP (pMQ72 [not shown]). Bar = 10 μm. (B) LapD immunoblot of membrane fractions from P. fluorescens. (Lane 1) Wild-type samples exhibit the predicted 71-kDa LapD protein. (Lane 2) Anti-LapD antibodies do not react with samples from a ΔlapD strain. (Lane 3) P. fluorescens bearing pMQ72 plus lapD exhibits relatively high levels of LapD in the presence of 0.2% l-arabinose (grown in M63 plus 0.2% glucose plus 0.5% Casamino Acids) and (lane 4) very little LapD in the absence of l-arabinose. (Lane 5) Fractions with pMQ72+lapDΔC-term exhibited a predicted 45-kDa protein in the presence of 0.2% l-arabinose. (C) Thin-layer chromatographic analysis of P. aeruginosa. The wild type and the rhlB::kanMX mutant were grown in PPGAS with 100 mM l-arabinose for 24 h at 30°C as reported previously (4). The arrow indicates dirhamnolipids. (Lane 1) Wild type (wt) grown with vector alone (V) (pMQ78). (Lane 2) Wild type grown with a plasmid-borne rhlB-GFPmut3 fusion construct (B-gfp) under the control of the P_BAD promoter (pRMQS96). (Lane 3) rhlB::kanMX mutant bearing the vector alone. (Lane 4) rhlB::kanMX mutant bearing the P_BAD-rhlB-GFPmut3 plasmid. These data show that the rhlB::kanMX mutant is deficient in formation of dirhamnolipids and that this mutation is complemented by an RhlB-GFPmut3 fusion protein.

FIG. 4.

Yeast recombination facilitates the construction of multiple pieces of DNA. (A and B) Schematics depicting the recombination of four PCR-based amplicons into a gapped vector in a single step. (A) pMQ87 that has been digested with SmaI (gapped) and the four PCR-based amplicons: 1, xylR repressor-T5X promoter; 2, a 5′ region of GFP_uvr with two mutations added to the primer; 3, a 3′ region of GFP_uvr with two mutations added to the primer; 4, a 3HA tag. (B) Schematic diagram representing the recombinant product which has a modified GFP-3HA allele under the control of a xylose-regulated promoter (no. 1 to 4) all upon a yeast-E. coli shuttle vector. (C) Phase-contrast and epifluorescent micrographs of E. coli strains bearing either pMQ87 (no GFP) or pRMQS106 (GFP-3HA). Images were taken with identical exposure times. Magnification, ×400. Bar = 10 μm. (D) Immunoblots of lysates of E. coli cells bearing pMQ80 (P_BAD-GFP), pMQ87 (vector alone), or pRMQS106 (P_T5X-GFP-3HA). Blots were probed with either anti-GFP or anti-HA antiserum. Cells were grown overnight in the presence of gentamicin and 0.4% glucose and then pelleted and diluted at 1:20 in LB supplemented with gentamicin and either 40 mM l-arabinose for the pMQ80 culture (GFP) or 0.4% (vol/vol) d-xylose for pMQ87 (vector) and pRMQS106 (GFP-3HA). Diluted cultures were grown for 3 h at 37°C before aliquots were taken for analysis. Lysates of cells bearing two independent pRMQS106 constructs are shown.