Abstract

BackgroundNontypeable 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.ResultsA 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.ConclusionThe 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.

Highlights

  • 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

  • Nontypeable Haemophilus influenzae (NTHi) is a gramnegative bacterium, which is a major cause of otitis media [1,2]

  • Construction of a non-polar mutant in the NTHi pilA gene Mutations have been engineered into the E. coli chromosome using the lambda phage recombinase to catalyze the site-specific insertion of a PCR amplicon containing 50 bp homology arms and a cassette flanked by FRT (FLP recombinase target) sites, replacing the gene of interest [12,13]

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Summary

Introduction

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 have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi. Nontypeable Haemophilus influenzae (NTHi) is a gramnegative bacterium, which is a major cause of otitis media [1,2]. We previously demonstrated that NTHi produce Tfp under defined environmental conditions. These Tfp are responsible for twitching motility, Tfp-mediated adherence and contribute to biofilm development [4,5,6]. PilD is predicted to be the prepilin peptidase These genes are in an operon, which necessitates the construction of non-polar mutants in order to carefully define the role of each gene product

Methods
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Conclusion

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