A new simple method for introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria by FLP/FRT recombination

Abstract

Background: For the disruption of a target gene in molecular microbiology, unmarked mutagenesis is preferable to marked mutagenesis because the former method raises no concern about the polar effect and leaves no selection marker. In contrast to naturally competent bacteria, there is no useful method for introducing an unmarked mutation into a large gene of non-competent bacteria. Nevertheless, large genes encoding huge proteins exist in diverse bacteria and are interesting and important for physiology and potential applications. Here we present a new method for introducing an unmarked mutation into such large genes of non-competent Gram-negative bacteria.

Results: Two gene replacement plasmids, pJQFRT and pKFRT/FLP, were constructed to apply the FLP/FRT recombination system to introduce an unmarked mutation into a large gene of non-competent Gram-negative bacteria. In our methodology, pJQFRT and pKFRT/FLP are integrated into the upstream and the downstream regions of a target gene, respectively, through homologous recombination. The resultant mutant has antibiotic resistance markers, the sacB counter-selection marker, flp recombinase under the control of the tetR regulator, and identical FRT sites sandwiching the target gene and the markers on its chromosome. By inducing the expression of flp recombinase, the target gene is completely deleted together with the other genes derived from the integrated plasmids, resulting in the generation of an unmarked mutation. By this method, we constructed an unmarked mutant of ataA, which encodes the huge trimeric autotransporter adhesin (3,630 aa), in a non-competent Gram-negative bacterium, Acinetobacter sp. Tol 5. The unmarked ataA mutant showed the same growth rate as wild type Tol 5, but lost the adhesive properties of Tol 5, similar to the transposon-inserted mutant of ataA that we generated previously.

Conclusions: The feasibility of our methodology was evidenced by the construction of an unmarked ataA mutant in the Tol 5 strain. Since FLP/FRT recombination can excise a long region of DNA exceeding 100 kb, our method has the potential to selectively disrupt much larger genes or longer regions of gene clusters than ataA. Our methodology allows the straightforward and efficient introduction of an unmarked mutation into a large gene or gene cluster of non-enterobacterial Gram-negative bacteria.

Figure 1

Plasmids constructed to introduce an unmarked mutation into a large gene of non-competent bacteria. (A, B) Multiple cloning sites (MCS) of pJQ200sk and pK18mob were substituted with that of pLOI2224, generating pJQFRT and pKFRT, respectively. The pJQFRT plasmid contains a single FRT site adjacent to a multiple cloning site; p15A origin, a replication origin of E. coli; oriT, origin of transfer; SacB, a counter-selection marker; and Gm^R, a gentamicin resistance marker. The arrows indicate the primers used in PCR to amplify the substitute MCS. The nucleotide sequences of these primers are shown in Table 2. (C) A cassette containing tetR-Ptet promoter and flp recombinase amplified by PCR from pFT-A was ligated with the inverse-PCR product of pKFRT. The resultant pKFRT/FLP plasmid contains a single FRT site adjacent to a multiple cloning site; TetR-FLP, flp recombinase gene under the control of the tetR regulation system; Km^R, a kanamycin resistance marker; oriT, origin of transfer; and ColE1 origin, a replication origin of E. coli.

Figure 2

Scheme for the unmarked deletion of a large gene by FLP/FRT recombination. The plasmid pJQFRT with the insertion of the upstream region of the target gene is integrated into the host chromosome by homologous recombination. Next, the plasmid pKFRT/FLP with the insertion of the downstream region of the target gene is integrated into the host chromosome by homologous recombination. As a result, the target gene is sandwiched between the two integrated plasmids. The expression of flp is induced by adding anhydrotetracycline, and then the target region is excised together with the integrated plasmids bracketed by the two FRT sites, leaving a single FRT site.

Figure 3

Construction of an unmarked mutant of ataA from Acinetobacter sp. Tol 5. (A) Genetic organization around ataA in Acinetobacter sp. Tol 5 and its derivative mutants obtained by plasmid integration and FLP/FRT recombination. The arrows indicate the primers used in PCR analysis for the confirmation of the constructs. (B) PCR confirmation of plasmid integration and the deletion of ataA in the Tol 5 derivatives. Chromosomal DNA was extracted as a template for PCR from Tol 5 and its derivatives (G4, G4K1, and 4140). PCR analyses were performed by using three different primer sets: P1 (AtaAupstF2) + P2 (FRT-SP6R), P3 (FRT-leftF) + P4 (AtaAdwstR2), and P1 + P4. The nucleotide sequences of these primers are shown in Table 2.

Figure 4

Plating tests to confirm the presence or excision of the selection markers. Wild type Acinetobacter sp. Tol 5 (Tol 5 WT), the plasmid-integrated mutants Tol 5 G4 (G4) and Tol 5 G4K1 (G4K1), and the unmarked ataA mutant Tol 5 4140 (4140) were streaked on BS (Control), BS containing 100 μg/ml gentamicin (Gm), BS containing 100 μg/ml gentamicin and 100 μg/ml kanamycin (Gm + Km), and BS containing 5% sucrose (5% sucrose) plates, and incubated with a supply of toluene as a carbon source.

Figure 5

Examination of the phenotype of the unmarked mutant Tol 5 4140. (A) Immunodetection of AtaA using an anti-AtaA antiserum against whole cell lysates prepared from Tol 5 WT and the 4140 mutant. (B) Growth curve of Tol 5 WT and the 4140 mutant in LB medium at 28°C, with shaking at 115 rpm. Data are expressed as the mean and SD obtained from 3 independent cultures. (C) Adhesion of Tol 5 WT and the 4140 mutant to a polystyrene surface. The photograph indicates the stained cells adhering to a 48-well plate. Data are expressed as the mean and SEM (n = 3). Statistical significance, *P < 0.01. (D) Autoagglutination assay of Tol 5 WT and the 4140 mutant by the tube-settling assay. The photographs indicate test tubes after a 3-h incubation without agitation. Data are expressed as the mean and SEM (n = 3). Statistical significance, *P < 0.001.