, 1999). In contrast to LipA, LipC is expressed only in very low amounts and a physiological function has not yet been assigned to this enzyme. Transcription of the lipC gene is repressed

by the products of two pilus biogenesis genes, pilX and pilY1 (Alm et al., 1996; Martinez et al., 1999), suggesting that LipC function may Selleck MK 2206 affect either type IV pilus biogenesis or pilus-dependent cellular functions. The strains and plasmids used are listed in Table 1. For mutant construction, the lipC gene was isolated by PCR amplification using Pfu-DNA-polymerase (Fermentas) and the LipCup1 (5′-GTAATGGCGTGCGCCGGGAGCC CAAC-3′) and LipCdwn1 (5′-CGCGAACAGGAGGTGA TATCCAGGTGCCATTAG-3′) primers. The resulting 1.1-kb fragment

was ligated into the EcoRV-digested cloning vector pUCPSK. The resulting plasmid pUCPLipC carrying the lipC gene under Plac control was digested with BamHI and HindIII, and the resulting lipC fragment was cloned into BamHI/HindIII-digested pSUP202 (Simon et al., 1983), yielding pSUPLipC. GSK2118436 mw The lipC gene was disrupted by exchange of an internal SalI fragment of lipC in pSUPLipC by a Gmr cassette taken from SalI-digested pWKR202 ligated into SalI sites of pSUPLipC. For gene replacement by homologous recombination, the resulting plasmid pSUPLipCGM was transferred to P. aeruginosa PAO1 by diparental mating using Escherichia coli S17.1 (Simon et al., 1983) and transconjugants were selected on Gm-containing Luria–Bertani (LB) agar. Resulting clones were tested by contra-selection on Cm-containing agar to ensure Farnesyltransferase the loss of vector sequences. The integration of the disrupted lipC allele

was confirmed by Southern blot and PCR. For Southern blot analysis, SmaI-digested chromosomal DNA of the resulting clones was probed with the Gm cassette and the lipC PCR fragment. For PCR analysis, internal primers of the Gm cassette and the lipC flanking primers LipCup1 and LipCdwn1 were used and the resulting products were analysed in terms of their length on agarose gels (data not shown). For complementation, the lipC gene was PCR-amplified using Pfu-DNA-polymerase and primers LipCup2 and LipCdwn2 (5′-GGAGTCTCGCATATGAACAAGAA CAAGACG-3′; 5′-GTAGGATCCAGGTGATATCCAGGTGC CATTAG-3′), which were designed to add an NdeI site overlapping the lipC translational startcodon and a BamHI site downstream of lipC. The resulting 1.1-kb fragment was digested with the respective enzymes and ligated to the NdeI-/BamHI-sites of pET22b (Novagen). The resulting plasmid pET22bLipC was digested with BglII and BamHI and a fragment containing the pET22b ribosomal-binding site and the T7 promoter preceding the lipC gene was ligated to BamHI-digested pBBL7, which is a derivative of pBBR1MCS containing the lipA/lipH operon of P.