Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology

Abstract

Background: Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi.

Results: A cassette containing a spectinomycin resistance gene and an rpsL gene flanked by FRT sites was constructed. A PCR amplicon containing 50 base pairs of DNA homologous to the 5' and 3' ends of the gene to be disrupted and the cassette was generated, then recombineered into the target NTHi gene, cloned on a plasmid, using the lambda recombination proteins expressed in E. coli DY380. Thus, the gene of interest was replaced by the cassette. The construct was then transformed into a streptomycin resistant NTHi strain and mutants were selected on spectinomycin-containing growth media. A plasmid derived from pLS88 with a temperature sensitive replicon expressing the FLP recombinase gene under the control of the tet operator/repressor was constructed. This plasmid was electroporated into the NTHi mutant at the permissive temperature and FLP expression was induced using anhydrotetracycline. The recombinase recognizes the FRT sites and eliminates the antibiotic cassette by site-specific recombination, creating the unmarked non-polar mutation. The plasmid is cured by growth of cells at the restrictive temperature.

Conclusion: The products of the genes in the NTHi pilABCD operon are required for type IV pilus biogenesis and have a role in transformation. We demonstrated the utility of our methodology by the construction of a non-polar pilA mutation in NTHi strain 2019 and complementation of the mutation with a plasmid containing the pilA gene. Utilization of this approach allowed us to readily generate unmarked non-polar mutations in NTHi genes.

Figure 1

Generation of a PCR amplicon for insertion/deletion mutagenesis of the pilA gene. In Panel A, the pilA gene from NTHi strain 2019 is shown. Homology arm 1 (H1) contains 50 base pairs of DNA 5' to and including the ATG start codon of pilA. Homology arm 2 (H2) contains the complement of the last 21 base pairs of the pilA gene and 29 base pairs immediately downstream of the pilA gene. In Panel B, the template region of pRSM2832 is shown. Primer H1P1 contains the sequence from H1 and 20 base pairs of DNA homologous to the 5' end of the cassette (P1). Primer H2P2 contains the sequence from H2 and 20 base pairs of DNA homologous to the 3' end of the cassette. PCR amplification of pRSM2832 with primers H1P1 and H2P2 (primers 5 and 6) yielded the product shown in Panel C.

Figure 2

Construction of a non-polar pilA mutant. The PCR amplicon from Figure 1 is shown in Panel A. The cloned pilABCD region of pRSM2855 is shown in Panel B. The amplicon was electroporated into E. coli DY380(pRSM2855) and the lambda recombinase genes were induced by temperature shock. Spectinomycin-resistant clones were selected. The insert region of plasmid pRSM2857 is shown in Panel C. Plasmid pRSM2857 was linearized and transformed into NTHi strain 2019 rpsL; spectinomycin-resistant clones were isolated. NTHi strain 2019 rpsLΔpilA::spec-rpsL_Ng was saved, then transformed with pRSM2947 at the permissive temperature; kanamycin resistant clones were isolated. Expression of the FLP recombinase resulted in the loss of the spectinomycin resistance gene-rpsL_Ng cassette; growth at 37°C resulted in the loss of the plasmid. The pil region of NTHi strain 2019 rpsLΔpilA is depicted in Panel D.

Figure 3

Map of plasmid pRSM2947. The plasmid contains: the FLP recombinase gene under the control of the tet regulatory system; a temperature sensitive replicon suitable for use in NTHi; a ColE1 origin that is functional in E. coli and a kanamycin resistance marker.