1 Departamento de Biología Molecular (Unidad asociada al CIB, CSIC), Universidad de Cantabria, C. Herrera Oria s/n, E-39011 Santander, Spain
2 Cubist Pharmaceuticals (UK) Ltd, 545 Ipswich Road, Slough SL1 4EQ, UK
3 Department of Molecular Microbiology, BioCentrum-DTU, DK-2800 Lyngby, Denmark
4 Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria
5 Centro de Investigaciones Biológicas (CIB), CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
6 Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany
Correspondence
Fernando de la Cruz
delacruz{at}unican.es
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ABSTRACT |
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A detailed report of the nuclear magnetic resonance assays is available as supplementary data with the online version of the paper.
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INTRODUCTION |
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Previous studies demonstrated the potential for inhibiting bacterial conjugation by the addition of chemical compounds. Ou & Reim (1976) studied the inhibitory effect of 1,10-phenanthroline, a zinc-chelating agent, on F plasmid transfer. Michel-Briand & Laporte (1985)
showed the inhibitory effect of nitrofurans in conjugation of plasmids belonging to six different incompatibility groups. However, nitrofurans cause a general disruption of bacterial DNA, and thus behaved as non-specific inhibitors. Mandi & Molnar (1981)
demonstrated that the major tranquillizer chlorpromazine is able to inhibit conjugal transfer of plasmid F. Chlorpromazine is a cationic amphipathic molecule that, by insertion into the inner leaflet of the membrane lipid bilayer, induces modifications of the membrane topology. It also produces a general stress response (Conter et al., 2002
) and reduces growth, so its inhibitory effect might have been caused by a general disturbance of cell physiology. Nalidixic acid or coumermycin inhibit conjugal transfer of plasmids F and R64 (Hooper et al., 1989
) but, again, their target (DNA gyrase) is not a conjugation-specific protein, but a general factor in DNA metabolism. These studies demonstrated that it is generally possible to inhibit plasmid transfer, although none of the compounds assayed targeted specifically the conjugative transfer machinery. In general, the lack of a fast, automated screening method that allows large-scale compound testing has hindered the search for conjugation inhibitors. An additional hindrance has been the difficulty in discriminating between compounds that inhibit conjugation by affecting bacterial metabolism and those that exert their action on the conjugation system per se.
In this work we developed an automated high-throughput conjugation assay that discriminates between true conjugation inhibitors and substances that diminish conjugation due to perturbations in cell growth or bacterial physiology. We then used this assay to search for specific conjugation inhibitors within a chemical library of bacterial and fungal extracts. We found that unsaturated fatty acids, a common component of those extracts, are inhibitors of plasmid conjugation. The discovery of efficient conjugation inhibitors can be useful for at least two reasons: (i) they can help us to understand the mechanisms of conjugation and type IV protein secretion and (ii) they may prove useful in controlling the spread of antibiotic resistance.
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METHODS |
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Construction of pSU2007 : : Tnlux.
A DNA segment containing the bioluminescence operon luxCDABE from Photorhabdus luminescens was excised from plasmid pSB395 (Winson et al., 1998a, b
) by EcoRI/PstI digestion. The resulting DNA (containing the lux operon ribosome-binding site but not its promoter) was cloned into the expression vector pUHE-24, downstream of a strong artificial Lac promoter (Plac), yielding plasmid pSM1979 (Deuschle et al., 1986
). This promoter is repressible by LacI. Plasmid pSM1979 was digested with XhoI/PstI and the fragment containing lux, now preceded by Plac, was inserted in vector pLow2 cut with SalI/PstI, in order to have the Plac-lux construction flanked by NotI sites. The resulting plasmid, pSM1981, was cut with NotI and inserted into the unique NotI site in pUT-miniTn5 (Gmr) (de Lorenzo et al., 1990
). The pUT construction was mobilized with helper plasmid pRK600 (de Lorenzo & Timmis, 1994
) into Pseudomonas putida R1, harbouring plasmid pSU2007, a kanamycin-resistant derivative of plasmid R388 (Martínez & de la Cruz, 1988
). Resulting transconjugants were subsequently used as donors in a mating experiment with E. coli. A mutant derivative of pSU2007 was thus identified where the Tnlux insertion into the plasmid does not interfere with conjugative functions. The resulting tagged plasmid (pSU2007-Tnlux) exhibited the same conjugation frequency as the parental plasmids pSU2007 and R388.
Conjugation assays.
For the plate-mating procedure (Llosa et al., 1991), a 200 µl mixture of equal volumes of donor and recipient cultures, both in stationary phase, was centrifuged and placed onto a GS Millipore filter (0·22 µm pore size) on top of an LB-agar plate for 1 h at 37 °C. Bacteria were washed from the filter and plated on selective media.
For the lux-monitored conjugation procedure, donor cells contained plasmid pSU2007 : : Tnlux and pUC18 : : lacIq (so expression of the lux operon was completely repressed and donor bacteria were non-luminescent). Upon conjugation, pSU2007 : : Tnlux, but not pUC18 : : lacIq, moves to the recipient cell, resulting in expression of luminescence in transconjugants. Luminescence was measured as arbitrary light units (ALU) using either an image-capturing system (Chemidoc; Bio-Rad) or a microplate luminometer (either Fluoroskan Ascent from Thermolab Systems, or Victor2 from Perkin Elmer).
For high-throughput conjugation assays in the presence or absence of potential inhibitors, 96-well microtitre plates were used. Extracts were dissolved in ethanol/DMSO (75 : 25, v/v) and 10 µl aliquots dispensed into wells prior to addition of 300 µl molten LB agar. Plates were stored at 4 °C overnight prior to use to allow for diffusion of the compounds. To initiate mating, 10 µl of a 1 : 1 (v/v) mixture of donor and recipient bacteria in stationary phase was inoculated on the surface of individual wells. Conjugation was allowed to proceed for 3 h at 37 °C.
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RESULTS |
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The principle of the conjugation detection assay relies on the production of visible light. pSU2007 : : Tnlux, a derivative of plasmid R388, contains a lux operon under the control of a lac promoter. lux expression in conjugative donor cells is repressed by the lac repressor LacI carried in a co-resident and non-mobilizable multicopy plasmid (pUC18 : : lacIq). Upon conjugation, pSU2007 : : Tnlux, but not pUC18 : : lacIq, is transferred to recipient cells; thus light is produced exclusively in transconjugant cells. Donor cells produce relatively low amounts of light (0·1 ALU) when compared to the transconjugant population after a 1 h mating period (18 ALU). This difference implies more than a 2-log enhancement in light production, so inhibition of the process can be easily quantified.
The assay was optimized by analysing a series of variables. First, different E. coli strains were tested as donor and recipients. CSH53 showed optimal results (the peak of light was more stable, more reproducible and of longer duration). Other strains (e.g. DH5) produced more intense peaks, but light production adversely affected cell growth. The kinetics of plasmid transmission was analysed by following light production versus time (Fig. 1
). Light emission increased significantly over donor strain levels from about 40 min and reached a maximum at about 500 min, before cells entered stationary phase. The maximal difference in light emission between donor strain and conjugation mixture ranged between 2·5 and 3 logs.
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To obtain proof of principle that light production can be reduced in response to conditions limiting conjugation, we used CSH53 recipient bacteria carrying plasmid pSU5024, which contains the R388 entry-exclusion gene (eex) cloned in expression vector pET3A. The entry-exclusion protein is a small polypeptide able to prevent conjugal transfer of R388 when residing in a recipient cell. No light emission (<0·1 ALU) was observed in the conjugation assay when recipient bacteria expressed this protein (data not shown). This experiment demonstrated that the automated conjugation assay can be also used for the screening of genetic conditions affecting conjugation.
Screening a validation set of 224 reference chemicals
As a first application for the automated conjugation assay we tested a set of 224 common chemicals (Table 2). The assay was carried out as described in Methods, with compounds added at 62·5 µg ml1 final concentration. In order to distinguish compounds affecting conjugation from those that affected either cell growth or the process of light production, a control assay was developed, which was run in parallel to the primary assay for each of the tested compounds. In the control assay, CSH53 cells containing pSU2007 : : Tnlux (but not pUC18 : : lacIq) were grown for 2 h in the presence of the potential inhibitor, and light production was measured. Any condition affecting cell growth, plasmid stability, lux expression or the light production reaction would be detected as a decrease in light production in the control assay. The control assay was validated by the use of several antibiotics including inhibitors of replication (bleomycin, coumermycin), transcription (rifampicin, actinomycin), protein synthesis (tetracycline, chloramphenicol) and compounds affecting membrane integrity (polymixin, patulin), among others. A compound was considered a conjugation inhibitor if it reduced light production by 95 % or more in the primary assay, but caused a reduction of less than 50 % in the control assay. All measurements were carried out at least in triplicate. When the 224 compounds in the validation plate were screened (Table 2
), and compounds positive in the control assay were discarded, only two (oleic and linoleic acids) were found to be true conjugation inhibitors.
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Only one of the 52 selected fractions (Mollisia ventosa-Fx14) inhibited conjugal transfer of the three plasmids R388, R1 and RP4. Most fractions were active against R388 and R1, but not against RP4. They were almost always late fractions of the corresponding extracts and in several cases chemical fingerprinting showed them to contain unsaturated fatty acids, principally linoleic acid. Four of these extracts were selected for further work because they contained active early fractions, which could contain different types of compounds. The five prioritized extracts and their active fractions are shown in Table 3. They were refermented and the corresponding extracts fractionated for chemical fingerprinting. The main results are shown also in Table 3
. As can be seen, only two compounds were found to be pure in the analysed fractions. Several fractions contained linoleic acid. Fraction Sistotrema semanderi-Fx21 contains an uncommon unsaturated fatty acid, and was interesting because it could represent a novel structure. The rest of the fractions (including Mollisia ventosa-Fx14) were complex in composition, and were not analysed further.
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The MICs of the unsaturated fatty acids were also determined. Oleic and linoleic acids had MIC98 values of about 400 µM, while the MIC of DHCA was 70 µM (data not shown). At a final concentration of 2xMIC98, R388 transfer frequency decreased by a factor of 20 with oleic acid, while 200-fold and 350-fold reductions were observed for the polyunsaturated fatty acids linoleic acid and DHCA respectively.
Finally we tested the spectrum of plasmids inhibited by linoleic acid and DHCA (Table 4). As expected, the compounds strongly inhibited conjugation mediated by R388 and the F-plasmid derivative pOX38. The effect of DHCA was always at least twice as pronounced as that of linoleic acid. As expected also, there was no inhibition of plasmid RP4 transfer. Consistent with that observation, plasmid R6K (most similar to RP4) was not inhibited either. All these results are consistent with the notion that unsaturated fatty acids may be targeting the plasmid Dtr systems, which are in common between R388 and F, but distantly related to those of RP4 and R6K (Francia et al., 2004
). This notion was reinforced by the observation that mobilization of plasmid CloDF13, which requires the R388 Mpf system for its transmission, was not inhibited when a donor strain contained both plasmids. In this situation, R388 was inhibited while CloDF13 was not (Table 4
). The Dtr components as a site of action for these substances became less likely, however, when their effect on conjugative transfer of plasmid pKM101 (whose Dtr is more similar to R388 and F than to RP4) was compared and no inhibition was observed (Table 4
).
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DISCUSSION |
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The lux-based conjugation assay we developed is well-suited for this purpose. Light production can be measured continuously in real time. Conjugation frequencies can be estimated directly and the assay is sensitive and easily automated. We used it to screen the NatChem compound library, a collection of 12 000 fungal and bacterial extracts. During assay validation it was discovered that linoleic and oleic acids (cis-unsaturated C18 fatty acids) were true inhibitors of conjugation mediated by plasmid R388. These compounds were also present in many extracts of the NatChem library, as fungi often produce them in high amounts. This was unfortunate, because many NatChem hits were produced by unsaturated fatty acids, largely precluding the finding of additional classes of compounds.
Nevertheless, NatChem library screening allowed us to discover an atypical fatty acid that was a bona fide inhibitor of R388-mediated conjugation. Its structure, solved by NMR, revealed dehydrocrepenynic acid. DHCA is also a C18, cis-unsaturated (9,12,14) fatty acid, with the peculiarity of having a triple bond between C-12 and C-13. The inhibitory potential and spectrum of DHCA were essentially the same as those of oleic and linoleic acids. Saturated short- and long-chain fatty acids, or other organic compounds related to them, showed no significant inhibitory effect, so we believe that a carboxylic group, chain length and position of the double bonds are essential features of this class of conjugation inhibitors.
Long-chain fatty acids are actively transported into the E. coli cytoplasm by a highly specific system (Black & DiRusso, 2003). FadL is an outer-membrane protein that binds long-chain fatty acids with high specificity and facilitates their transfer across the membrane. Translocated fatty acids cross the periplasmic space and the inner membrane via an unknown mechanism, although there is some evidence of a H+/fatty acid cotransporter (fatty acid transport depends on membrane potential). Most transported fatty acids are activated by FadD, an inner-membrane-associated acyl-CoA synthetase, and directed to
-oxidation. A small proportion of the exogenous fatty acids are incorporated directly into the phospholipid production system by the acyl-acyl carrier protein synthetase. This implies that most of the oleic, linoleic and DHCA acids supplied as inhibitors will be degraded when they enter the bacterial cell. A small amount, however, will reach the phospholipid pool and perhaps cause a change in lipid composition of the membranes. Although we have no evidence for the mechanism of action of these compounds, some hypotheses can be suggested. A culture medium highly enriched in fatty acids may cause a perturbation in the general physiology of E. coli (in osmotic pressure control, membrane potential or energetic balance) that impairs conjugation as a pleiotropic effect. However, and perhaps significantly, only certain conjugative plasmids are affected by these compounds (i.e. F and R388) while others are not inhibited (RP4). Thus, a general metabolic disturbance as the cause of inhibition is unlikely.
Alternatively, unsaturated fatty acids may directly target the conjugation machinery. It is known that unsaturated fatty acids can affect the function of some proteins associated with the bacterial membrane. For instance, DnaA, an ATPase involved in chromosome replication initiation in E. coli, specifically binds phospholipids containing oleic acid. This interaction is essential to stimulate DnaA ADP/ATP exchange, and inhibitors of unsaturated fatty acid synthesis produce a sharp decrease in DnaA activity that can be restored by providing exogenous oleic acid (Yung & Kornberg, 1988). Since bacterial conjugation requires the active participation of at least three membrane ATPases (TrwB, TrwD and TrwK in the case of R388), it is possible that phospholipids containing unsaturated fatty acids have a more direct role in the mechanism of conjugation by specifically interacting with these proteins. In fact, preliminary biochemical data from our laboratory indicate that the traffic ATPase TrwD is inhibited by unsaturated fatty acids in a non-competitive manner (C. Machón and others, unpublished).
Our results have shown that unsaturated fatty acids inhibit conjugation mediated by plasmids F and R388, while conjugation mediated by plasmids pKM101, RK6 and RP4 is insensitive to these compounds. The absence of an effect of linoleic acid and DHCA on R388-mediated mobilization of plasmid CloDF13 suggests that the main target of these compounds is the DNA transfer replication (Dtr) machinery. Plasmid CloDF13, like most mobilizable plasmids, uses its own Dtr machinery but takes advantage of the R388-built transport channel (the so-called Mpf system) to achieve its own transfer. The fact that it is not inhibited by DHCA while R388 transfer (from the same donor cell) is inhibited, suggests that the target is more likely to be a component of the Dtr system than the Mpf system. The closer relationship between the Dtr modules of R388 and F as compared to RP4 supports this possibility. However, plasmid pKM101 also shows a close degree of homology to the R388 and F Dtr systems, while its conjugal transfer was not affected by unsaturated fatty acids. Although we propose this obviously simplistic working hypothesis, we are aware that the mechanism underlying the inhibition might be more subtle or complex. At present, the mode of action of the inhibitors is simply unknown. Our current research aims to identify the molecular targets of unsaturated fatty acids in R388-mediated conjugation and characterize their interactions in the hope of a rational design of better inhibitors.
The availability of a robust and sensitive high-throughput conjugation assay will allow massive screening of compound libraries in new searches for compounds affecting bacterial conjugation and type IV protein secretion. The assay can also be used for analysis of libraries of bacterial mutants in a search for genes affecting conjugation (or type IV secretion) and its regulation using a variety of plasmids or host strains. All of this will result in better knowledge of the mechanisms and physiological control of bacterial conjugation and, perhaps, in the discovery of compounds that may help us to control the spread of antibiotic resistance.
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ACKNOWLEDGEMENTS |
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Received 24 May 2005;
revised 11 July 2005;
accepted 20 July 2005.
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