Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682A, USA1
Author for correspondence: Herbert P. Schweizer. Tel: +1 970 491 3536. Fax: +1 970 491 1815. e-mail: herbert.schweizer{at}colostate.edu
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ABSTRACT |
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Keywords: Pseudomonas, homoserine lactone, fatty acid synthesis, synthase
Abbreviations: ACP, acyl carrier protein; AHL, acyl homoserine lactone; C4-HSL, N-(butyryl)-L-homoserine lactone; Fab, fatty acid biosynthesis; HSL, homoserine lactone; SAM, S-adenosyl methionine
a Present address: Department of Microbiology, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
b Present address: VWR International, 106 Gray Road, Suite D, Indianapolis, IN 46237, USA.
c Present address: National Jewish Hospital, 1400 Jackson Street, Denver, CO 80206, USA.
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INTRODUCTION |
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Several previous studies revealed that bacterial AHLs derive their invariant homoserine lactone rings from S-adenosyl methionine (SAM) and their variable acyl chains from the cellular acyl-ACP (acyl carrier protein) pool (Hoang & Schweizer, 1999 ; Moré et al., 1996
; Parsek et al., 1999
; Val & Cronan, 1998
) (Fig. 1
). Acyl chain specificity resides in critical amino acid residues within the AHL synthase sequences (Watson et al., 2002
). The AHL synthases (LasI for 3-oxo-C12-HSL and RhlI for C4-HSL) are sufficient for catalysis of the acyl transfer and lactonization reactions (Moré et al., 1996
; Parsek et al., 1999
; Hoang & Schweizer, 1999
; Hoang et al., 1999
). P. aeruginosa culture supernatants contain 3-oxo-AHLs with various acyl chain lengths but their metabolic origins have not been elucidated. In this study, we attempted to elucidate the molecular basis for the synthesis of these 3-oxo-AHLs. Since LasI competes with NADPH-dependent ß-ketoacyl-ACP reductase, FabG, for the 3-oxo-acyl-ACP precursors for synthesis of these 3-oxo-AHLs (Fig. 1
), we reasoned that FabG activity may be a modulating factor determining acyl chain lengths in 3-oxo-AHLs. Because most Fab (fatty acid biosynthesis) enzymes, including FabG, are essential, conventional mutant analysis cannot be used to address their roles in cellular metabolism. To circumvent these problems, a complete in vitro Fab system using purified Escherichia coli Fab proteins and ACP was previously described and was shown to produce the types and distribution of acyl-ACP species found in vivo (Heath & Rock, 1996a
, b
). Since the E. coli and P. aeruginosa Fab systems are quite similar, we reasoned that an in vitro Fab-3-oxo-AHL synthesis system could be used to explore FabG activity as a factor determining acyl chain lengths of 3-oxo-AHLs. To this end, we purified the P. aeruginosa Fab proteins as hexahistidine (H6) fusion proteins and developed an in vitro Fab-AHL synthesis system by coupling them to purified LasI. Some of the observations made with the in vitro system were supported by preliminary in vivo data obtained with a conditional, temperature-sensitive fabG(Ts) mutant.
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METHODS |
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Construction of expression vectors and affinity purification of proteins.
The coding sequences for the individual enzymes were PCR amplified from PAO1 genomic DNA utilizing Taq polymerase and previously described conditions (Hoang & Schweizer, 1999 ; Hoang et al., 1998
). The general strategy involved the use of a forward primer that incorporated an NdeI restriction site at the start codon of the respective gene and a reverse primer that incorporated a BamHI restriction site after the stop codon of the same gene (Table 1
). The gel-purified (QIAquick gel extraction kit; Qiagen) PCR fragments were digested with NdeI/BamHI and then ligated between the same sites of pET-15b (Novagen). Since fabG contained a BamHI site, the reverse primer incorporated a BglII site, which allowed subcloning into the BamHI site of pET-15b. Standard molecular biological techniques were used (Sambrook & Russell, 2001
). Subcloning into pET-15b yielded the expression vectors pPS837 (FabB), pPS980 (FabG), pPS998 (FabH) and pPS937 (FabZ). For FabA, the PCR fragment was first cloned into the TA cloning vector pGEM-T (Promega) to yield pPS847. An NdeIBamHI fragment derived from this plasmid was then subcloned between the same sites of pET-15b (Novagen) to yield the FabA expression vector pPS848. For expression of the resulting proteins with NH2-terminal hexahistidine (H6) tags, the plasmids were transformed into BL21(DE3) (Novagen). Screening of H6-Fab protein-expressing transformants, cell lysis and purification of the soluble fusion proteins on Ni2+ agarose affinity columns (Qiagen) was performed as previously described (Hoang et al., 1999
), except for FabD. Since FabD eluted from the columns with 40 mM imidazole, washing of the column was done with 30 bed vols buffer with 20 mM imidazole.
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Protein concentrations were determined using the Bradford dye-binding assay (Bio-Rad) and BSA as the standard. Proteins were analysed by electrophoresis on 0·1% SDS-10% polyacrylamide gels (SDS-PAGE) (Makowski & Ramsby, 1993 ) and visualized by staining with Coomassie Brilliant Blue R-250 (Chen et al., 1993
).
Complementation assays.
The coding sequences for the H6-tagged FabA, FabB and FabD proteins were subcloned into the broad-host-range vector pUCP21T (Schweizer et al., 1996 ) on BamHIXbaI fragments. Subcloning between the BamHI and XbaI sites of pUCP21T placed the H6-Fab coding sequences in the correct transcriptional orientation with respect to the lac promoter contained on this cloning vector and yielded pPS1013 (H6-FabA), pPS1025 (H6-FabB) and pPS1019 (H6-FabD). To test for expression of functional H6-FabA and H6-FabB proteins, pPS1013 and pPS1025 were transformed (Hoang et al., 1998
) into strain PAO191 (fabA) and PAO192 (fabB) (Hoang & Schweizer, 1997
), respectively. Since FabA and FabB are required for unsaturated fatty acid synthesis, PAO191 and PAO192 will not grow at 42 °C unless supplemented with oleic acid or complemented with either a FabA- or FabB-expressing plasmid. Complementation was therefore scored as the ability to grow at 42 °C on RB medium without oleate supplementation (Hoang & Schweizer, 1997
). To test for expression of a functional H6-FabD protein, pPS1019 was transformed into the fabD(Ts) mutant PAO204 (Kutchma et al., 1999
). Successful complementation was scored as the ability of the transformants to grow on LB plates at 42 °C. In all instances, strains were transformed with pUCP21T as a negative control.
Reconstitution of the Fab-AHL pathway and extraction of 3-oxo-acyl-HSLs.
Complete reactions (total volume 500 µl) contained buffer [10 mM Tris/HCl (pH 7·4), 330 mM NaCl, 15%, w/v, glycerol, 0·7 mM DTT, 2 mM EDTA, 25 mM MgSO4, 0·1 mM FeSO4] (Moré et al., 1996 ), 2 µg ACP, 1 µg each FabA, FabB, FabD, FabH, FabI and FabZ, 0·5 µg FabG, 5 µg LasI, 0·25 mM SAM, 0·08 mM acetylCoA, 0·8 mM malonyl-CoA and 0·6 mM each NADH and NADPH (substrates and cofactors were obtained from Sigma). Reactions were incubated at 37 °C for 1 h and extracted three times with 250 µl ethyl acetate. Extracted AHLs were dried by rotary vacuum evaporation and resuspended in 20 µl acetonitrile. For detection of fractions containing AHLs, 510 µl each fraction was spotted on a C18 reverse-phase TLC plate (Whatman) and the plates were dried at 37 °C for 15 min before being overlaid with the detection strain. For TLC analysis of AHL fractions, the plates were developed in 60% methanol in water (v/v) and then dried for 20 min at 37 °C prior to being overlaid with the detection strain.
Detection, identification and quantification of AHLs.
A. tumefaciens reporter strain NTL4/pZLR4 was grown at 30 °C for 48 h in M9 medium (Miller, 1992 ) with 1 mM MgSO4, 0·1 mM CaCl2, 0·6% glucose and 30 µg gentamicin ml-1 (Shaw et al., 1997
). Cells were harvested and resuspended in warm (
45 °C) fresh M9 medium with 0·4% agar, 1 mM MgSO4, 0·1 mM CaCl2, 0·6% glucose and 40 µg X-Gal ml-1. This suspension was used immediately to overlay the TLC plates. The presence of AHLs was usually evident by the appearance of blue spots after incubation at room temperature for 3648 h. Synthetic 3-oxo-C12-HSL, and bacterial-derived 3-oxo-C8-HSL and 3-oxo-C6-HSL were included as standards. The latter two were extracted from 10 ml stationary-phase clarified culture supernatants of A. tumefaciens strain NT1/pTiC58
accR or Erw. carotovora strain EC14, respectively, using a previously described method (Shaw et al., 1997
). The concentrations of 3-oxo-C12-HSL were estimated utilizing the Esc. coli reporter strain MG4/pKDT17 (lasR+ lasBlacZ) as previously described (Schaefer et al., 2000
) and by using a dilution series of synthetic 3-oxo-C12-HSL to establish a standard curve. For determination of HSL levels in the supernatants of the fabG(Ts) mutant ts-67, its parental strain 4 and the ts-67R1 revertant of strain ts-67, the strains were grown in LB medium until the cultures reached an optical density of
1·6 (600 nm). The pH in the cultures was monitored to avoid excess alkalinization of the medium since AHLs are very unstable at alkaline pH values (Schaefer et al., 2000
). Aliquots (1 ml) were harvested by centrifugation. The supernatants were extracted three times with 1 ml acidified ethyl acetate (ethyl acetate containing 0·1 ml glacial acetic acid per litre), dried and suspended in 200 µl acidified ethyl acetate. For detection of fractions containing 3-oxo-HSLs, 10 µl each fraction was spotted on a C18 reverse-phase TLC plate. The plates were processed as described above and then overlaid with the A. tumefaciens detection strain.
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RESULTS AND DISCUSSION |
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Dehydratases. Of the two dehydratases, only FabZ was essential for AHL formation but not FabA. This is probably due to the fact that FabZ is mostly required in the initial cycles since its E. coli counterpart has greatest affinity for C4C8 ß-hydroxyacyl-ACP intermediates, but can use substrates with longer acyl chains (Heath & Rock, 1996a ). In contrast, E. coli FabA acts preferably on C10C14 ß-hydroxyacyl-ACP intermediates.
Reductants. Exclusion of NADH led to detectable AHL production but at much reduced levels. Since NADH is the reductant preferred by FabI (Hoang & Schweizer, 1999 ), this result indicates that FabI can utilize NADPH but that this step becomes rate-limiting in the absence of NADH.
When the known Fab inhibitors cerulenin, triclosan, diazoborine and thiolactomycin were added to the reaction mixture, only cerulenin and triclosan efficiently inhibited 3-oxo-AHL formation at the concentration tested (50 µM). For unknown reasons, at the same concentrations, thiolactomycin and diazoborine had little effect but from other experiments we suspected that these two antimicrobials, which are not available commercially, had lost much of their activities during storage (data not shown).
Nature of AHL molecules synthesized in vitro
TLC analysis (Fig. 4) was used to identify AHL species contained in representative positive reactions shown in Fig. 3
. The analysis showed that reactions containing all essential components of the Fab-3-oxo-AHL synthesis system almost exclusively yielded 3-oxo-C12-HSL, and only minute amounts of shorter chain 3-oxo-AHLs were discernible. Conversely, in the absence of NADPH but presence of NADH, LasI synthesized hardly any 3-oxo-C12-HSL but larger amounts of 3-oxo-C10-HSL and 3-oxo-C8-HSL, and lesser amounts of 3-oxo-C6-HSL (lane labelled -NADPH). Since 3-oxo-C8-HSL is the cognate A. tumefaciens AHL, its spot size is not indicative of a higher quantity of 3-oxo-C8-HSL relative to the other 3-oxo-AHLs, but rather indicates a better response to its native AHL. According to the pathway model (Fig. 1
), LasI and FabG compete for 3-oxo-acyl-ACP substrates from the Fab system. Although FabG can utilize NADH, NADPH is its preferred cofactor and in its absence the FabG-catalysed reduction step becomes rate limiting, leading to accumulation of shorter chain 3-oxo-acyl-ACPs. This now enables LasI to compete for the shorter-chain 3-oxo-acyl-ACP substrates and use them for synthesis of the corresponding shorter chain 3-oxo-AHLs. These results also proved that LasI alone is sufficient for synthesis of the shorter chain 3-oxo-AHLs found in P. aeruginosa culture supernatants.
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ACKNOWLEDGEMENTS |
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Received 17 July 2002;
revised 4 September 2002;
accepted 11 September 2002.