Construction of AlgE-FLAG epitope insertion mutant strains.

General DNA cloning and manipulation procedures were performed as described previously (16). The E. coli strains S17.1 and S17.1(λpir) were used as plasmid hosts. All amplified DNA fragments were confirmed by DNA sequencing.

Plasmid pSM8 in which the FLAG (DYKDDDDK) epitope-coding nucleotide sequence (31) was inserted into algE was constructed using PCR. Two PCR products targeting algE gene were generated using P. fluorescens genomic DNA as the template: one with P1 and P2 (amplifying a fragment containing the end of algK and the 5′ part of algE until bp 864 of the open reading frame [ORF]) and the other one with P3 and P4 (amplifying a fragment containing the 3′ part of the gene from bp 865 of the ORF and the beginning of algG) primer pairs. A total of 24 bp encoding the FLAG epitope and the complementary sequence were inserted at the 5′ end of primers P3 and P2, respectively. To be able to confirm the correct insertion of FLAG-coding DNA sequence by PCR and restriction enzyme digestion, an Acc65I restriction site was introduced into the primer pair P2 and P3 by changing a cytosine (nucleotide 867 of algE) to a thymine; the corresponding codon still encodes a glycine. The two PCR products were then mixed in equimolar amounts and used as the template for a third PCR with the P1 and P4 primers. The resulting PCR product contained the FLAG epitope, inserted after bp 864 of algE. The purified product was then cloned into EcoRV-digested pUC128 vector, resulting in pSM7. Finally, XbaI-NsiI-digested algE-FLAG fragment (2.0 kb) was cloned into a gene replacement vector, and the new plasmid was designated pSM8.

pSM8 was then used to generate chromosomal FLAG epitope insertion variants (listed in Table 1) as described elsewhere (1). All constructed strains were verified by PCR amplification and restriction digestion.

In order to express the FLAG-tagged AlgE protein in P. fluorescens SBW25, a transposon vector, where the algE-FLAG was placed under the control of the inducible Pm-G5 promoter (32) was constructed (Table 1). This plasmid was designated pSM9 and transferred into P. fluorescens SBW25 by conjugation. Transposon insertion mutants were selected on PIA containing Am. Several transposon mutants were initially checked in order to avoid selecting recombinant strains in which the phenotype is caused by transposon insertion site and not by the expressed gene.