The acyl-HSLs used in this study, N-butanoyl-L-homoserine lactone, (C4-HSL);
N-hexanoyl-L-homoserine lactone, (C6-HSL); N-octanoyl-L-homoserine lactone, (C8-HSL); N-decanoyl-L-homoserine lactone, (C10-HSL);
N-dodecanoyl-L-homoserine lactone, (C12-HSL); N-(3-oxobutanoyl)-L-homoserine lactone, (3-oxo-C4-HSL); N-(3-oxohexanoyl)-L-homoserine lactone, (3-oxo-C6-HSL);
N-(3-oxooctanoyl)-L-homoserine lactone, (3-oxo-C8-HSL);
N-(3-oxodecanoyl)-L-homoserine lactone, (3-oxo-C10-HSL);
N-(3-oxododecanoyl)-L-homoserine lactone, (3-oxo-C12-HSL) and N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL) were synthesized as described by Chhabra et al., (1993). Each compound was purified to homogeneity by semipreparative HPLC, and its structure was confirmed by MS and proton nuclear magnetic resonance spectroscopy (see Camara et al., 1998 for a review). Stock solutions at 10 mM in acetonitrile (far-UV grade) were diluted into the growth medium to give the stated concentrations.
Purification and characterization of acyl-HSLs
Spent supernatants (1 liter) from stationary-phase cultures of S. marcescens SS-1 and E.
coli JM109 transformed with the recombinant plasmid (both grown in M9 minimal growth medium) were extracted three times with dichloromethane (700:300
supernatant:dichloromethane). The dried extract was reconstituted in 50 µl acetonitrile, and then samples were subjected to analytical and preparative thin-layer chromatography (TLC) and preparative HPLC. Tentative identification of acyl-HSLs was made by comparing the Rf values of the positive sample spots with those of synthetic standards. For preparative TLC, samples were separated as described above and the silica matrix at the relevant Rf was collected. Acyl-HSLs were extracted from the TLC matrix three times with 2 ml of acetone and evaporated to dryness. For preparative HPLC, samples were separated by using a Kromasil KR100-5C8 (250 by 8 mm) reverse-phase column (Hichrom, England) with an isocratic mobile phase of 70%
(vol/vol) acetonitrile in water at a flow rate of 2 ml per min and monitored at 210 nm.
Fractions showing activity in the CV026 reporter assay were pooled and rechromatographed by using 60% (vol/vol) acetonitrile in water; the procedure was repeated, using a final chromatographic separation employing 35% (vol/vol) acetonitrile in water. Active fractions with the same retention times were pooled and analyzed by mass spectrometry (MS) on a V.G.70-SEQ instrument (Fisons Instruments, VG Analytical, England). Samples were ionized by positive-ion fast atom bombarbment (FAB), and the molecular ion (M+1 H) peaks recorded by FAB-MS were further analyzed by tandem MS (MS-MS).
Assay of nuclease and surfactant production
Nuclease activity was detected using the DNase microplate assay following the protocol of Chen et al., (1992). Qualitative assay of surfactant production was performed using the drop collapsing method (Lindum et al., 1998). Semi-quantitative assay of biosurfactant was performed using a TLC based assay as described by Matsuyama et al.,
(1992).
Construction of S. mar cescens SS-1 spnI and spnR deletion insertion mutants
PCR primers (Table 3) were designed to introduce specific HindIII sites into spnI and spnR respectively for subsequent insertion of HindIII digested Ω (Sm r) gene cassette (Prentki and Krisch, 1984). Primer pairs SI-1/SI-2 (for spnI) and SR-7/SR-8 (for spnR) were used to amplify the 5’-region (approximately 400 bp) of the gene to be inactivated.
PCR products were T-cloned into pCR2.1 (Stratagene, UK), sub-cloned as an XbaI/BamHI fragment into pZero-2 (Invitrogen, Nederland) and excised as a XbaI/HindIII fragment. A second PCR product encompassing the 3’-region of the gene to be inactivated (approximately 400 bp) was generated using primer pairs SI-3/SI-4 (spnI) and SR-5/SR-6 (spnR), T-cloned into pCR2.1 and excised as an HindIII/EcoRI fragment. The 2 kb Sm-resistant Ω DNA fragment was excised from pHP45Ω (Prentki and Krisch, 1984) as a HindIII fragment. The three DNA fragments were ligated together with XbaI/EcoRI digested suicide vector pUT-mini-Tn5-Km (de Lorenzo and Timmis 1994). The resultant pUT-spnI::Sm and pUT-spnR::Sm vectors were selected as conferring streptomycin resistance upon the permissive E. coli strain CC118 and verified by restriction enzyme mapping. For gene inactivation mutagenesis by homologous recombination, the respective plasmids were transferred from the permissive host strain E. coli S17-1 λpir to S. marcescens SS-1 by conjugation and the transonjugants were spread on LB plates with streptomycin (100 µg/ml) and tetracycline (13 µg/ml). The mutants with double cross over events were selected by CV026 T-streaking (spnI::Sm) or by selecting mutants with fast-sliding phenotype followed by PCR screening (spnR::Sm). Southern hybridization using the PCR
amplified spnI gene (SI-1 and SR-6 as primers) or spnR gene (SR-8 and SI-3 as primers) as the probe respectively was performed. In a BamHI digestion of chromosomal DNA from the parent strain, a single band corresponding to the predicted size of 7.4 kbp hybridized to the probe. In the SS-1 ∆I mutant, a 9 kbp and a 0.4 kbp band hybridized (data not shown). In the SS-1 ∆R mutant a band of 0.8 kbp and a band of 8.6 kbp hybridized (data not shown). The data confirmed that a double-crossover event had taken place and the new strains were designated SS-1∆I and SS-1 ∆R strains.
Acknowledgements
The authors are grateful to whomever for funding.
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