Journal of Bacteriology, December 2002, p . 6515-6521, Vol . 184, No . 23

Acyl-HSL signaling controls a number of bacterial processes, including virulence factor production, secondary metabolite production, and biofilm development in Pseudomonas aeruginosa (25, 27, 46) and conjugal transfer in Agrobacterium tumefaciens (10, 29, 49) . Generally, LuxR and LuxI homologs serve as signal receptors and signal generators, respectively . Depending on the system, the signal varies in acyl group length and substitution (11), and these acyl side chain differences confer signal specificity (7, 35) . Differences in acyl chain lengths are also a factor in signal permeability . Short-chain acyl-HSLs, like N-3-oxohexanoyl-HSL (3OC6-HSL) and butanoyl HSL, can diffuse freely through the cell membrane (14, 26) . While still diffusible, long-chain acyl-HSLs like the P . aeruginosa signal N-3-oxododecanoyl-HSL (3OC12-HSL) appear to partition to the cell membrane . The MexAB-OprD efflux pump and perhaps other efflux pumps can aid in 3OC12-HSL export (26) .

Rhodobacter capsulatus and Paracoccus denitrificans are members of the group of the Proteobacteria. These free-living organisms are closely related and have been well studied for reasons related to their physiological flexibility (1, 20) . We assessed their ability to synthesize acyl-HSLs by using a previously described radiotracer technique (3, 34, 38) . This allowed us to discover that R . capsulatus and P . denitrificans made a long-chain acyl-HSL that was not readily detected by any available bioassay . We show that the most abundant acyl-HSL produced by these bacteria is N-hexadecanoyl-HSL (C16-HSL) .

Because the R . capsulatus genomic sequence is available, we studied acyl-HSL signaling in this organism further . We identified luxI and luxR homologs in the R . capsulatus genome . We demonstrate that cells concentrate this hydrophobic acyl-HSL . We also show it activates production of the gene transfer agent when added to an R . capsulatus acyl-HSL synthesis mutant . Gene transfer agent is a bacteriophage-like particle that transfers random 4.5-kb fragments of host genomic DNA between R . capsulatus cells (16, 40, 47) . This type of genetic exchange is a biological process that we show is related to quorum sensing .

 
Detection and identification of R . capsulatus and P . denitrificans acyl-HSLs. Previously, discovery of acyl-HSLs has relied on bioassays, which are limited by signal specificity constraints (36) . Novel acyl-HSLs might not be detected by any of the available bioassays . We adapted an assay that detects the incorporation of ¹⁴C label into acyl-HSLs (3, 34, 38) to screen R . capsulatus, P . denitrificans, and other bacteria for acyl-HSL production regardless of whether the molecules could be readily detected in any bioassay . Late-logarithmic-phase cultures (5-ml volume) grown in methionine-free medium (RCV or SIS) were labeled for 30 to 60 min with 5 µCi of carboxy[¹⁴C]methionine, 55 mCi/mmol (American Radiochemical Company, St . Louis, Mo.) . Unless indicated, total cultures were extracted twice with an equal volume of acidified ethyl acetate (100 µl of glacial acetic acid per liter) . Extracts were combined and dried under a stream of N₂ gas . The residue was suspended in 200 µl of methanol and separated by C₁₈ reverse-phase high-performance liquid chromatography (HPLC) in a 20 to 100% methanol-in-water gradient (27) . Four milliliters of scintillation cocktail 3a70b (Research Products Inc., Mount Prospect, Ill.) was added to each of the 70 1-ml fractions collected, and radioactivity was determined by scintillation counting .

To determine C16-HSL concentrations, R . capsulatus ALS1 harboring pYP (18), a plasmid that produces ß-galactosidase in response to added C16-HSL (see Results), was used . The procedure was identical to that described for the 3OC12-HSL bioassay described elsewhere (27, 36) except for the different reporter and that cultures were grown aerobically in YPS medium for 22 h . Synthetic C16-HSL was used to generate a standard curve .

To identify acyl-HSLs, we extracted late-logarithmic-phase culture fluid with acidified ethyl acetate . The acyl-HSLs were purified from extracts of LB-grown cultures by C₁₈ reverse-phase HPLC as described above . The purified material was analyzed by chemical ionization mass spectrometry (CI-MS) with a VG Trio-1 quadropole mass spectrometer with methane as the reagent gas . We restricted our isolation of material for mass spectrometry to material from the culture fluid . As described in the Results, this represented about half of the total for each of the acyl-HSLs . This facilitated purification because the bulk of the cellular lipids were not included in the extract . Alternatively, identification was done by coelution of radiolabeled material from whole cultures (cells plus culture fluid) with chemically synthesized acyl-HSLs .

Acyl-HSL association experiments. To determine if the long-chain acyl-HSLs are associated with cells, we compared the amount of radiolabeled acyl-HSL associated with the cell-free culture fluid and cell pellet for R . capsulatus, P . denitrificans, and, as a control, V . fischeri . Radiolabeling was performed as described above except that prior to extraction, the cultures were centrifuged (10,000 x g, 4°C), and the culture fluid was separated from the cell pellet . The cell pellet was resuspended in 5 ml of fresh medium (the original culture volume) . Acyl-HSLs from the culture fluid and suspended cell pellet were extracted and separated by HPLC as described above . The total amounts of [¹⁴C]3OC6-HSL (fraction 10), [¹⁴C]C16-HSL (fractions 65 to 67), and [¹⁴C]C14-HSL (fractions 60 to 62) in the culture fluid and cell pellet were determined by scintillation counting .

Acyl-HSLs. C16-HSL was synthesized in a manner similar to that described elsewhere (5) . Hexadecanoic acid (Sigma Chemical Company, St . Louis, Mo.) was mixed with L-homoserine lactone hydrochloride in dry dichloromethane with activation by dicyclohexylcarbodiimide and 1-hydroxybenzotriazol . The crude synthetic product was washed with 5% NaHCO₃, and the C16-HSL was purified by C₁₈ reverse-phase HPLC . As shown by CI-MS, the product showed the expected molecular mass . All other synthetic acyl-HSLs were purchased from Aurora Biosciences .

Identification and genetic analysis of R . capsulatus acyl-HSL synthase. We performed a Blast search of the R . capsulatus genome (http://ergo.integratedgenomics.com/ERGO; Integrated Genomics, Chicago, Ill.) for translation products showing similarity to the Rhodobacter sphaeroides CerI protein (accession number AAC46022) . One putative R . capsulatus gene, open reading frame (ORF) RRC03805, coded for a polypeptide that showed significant similarity to CerI and was adjacent to a gene coding for a probable LuxR-type regulatory protein, ORF RRC03806 . A 2.7-kb fragment of R . capsulatus SB1003 chromosomal DNA containing ORF RRC03805 was amplified with the Expand long-template PCR system (Boehringer Mannheim) and the following primers: forward, 5'-CAATTGGGCTACCGCCGTCTGAACCG-3', and reverse, 5'-TCTAGACGGGTCCGATCCGCGGACGG-3' .

The PCR fragment was used to create pGtaI-1 with the Original TA cloning kit (Invitrogen, Carlsbad, Calif.) . A 2-kb SmaI fragment of pHP45Sp (containing the spectinomycin resistance [Sp^r] cassette) was cloned into a unique PshAI site of pGtaI-1, disrupting RRC03805 . This construct was designated pGtaIKO . A pGtaIKO MfeI-XbaI fragment containing the inactivated RRC03805 gene was ligated to EcoRI- and XbaI-digested pJP5603 to create pJPGtaIKO, which contains the Sp^r cassette 178-bp downstream of the predicted translational start codon of RRC03805 . The Sp^r cassette in pJPGtaIKO is flanked by 1.4 kb of upstream and 1.3 kb of downstream R . capsulatus DNA . E . coli S17-1 pir was used to mobilize pJPGtaIKO into R . capsulatus SB1003 (28) . We selected spectinomycin-resistant colonies and screened for a kanamycin-sensitive mutant . One mutant, ALS1, contained an Sp^r cassette insertion in RRC03805, as shown by Southern blot analysis with RRC03805 and Sp^r cassette probes .

For complementation studies, pBBRGtaI was constructed by cloning the RRC03805 ORF into the SalI and EcoRI sites of pBBRMCS-2, a broad-host-range plasmid (15) . The resulting plasmid was introduced into R . capsulatus ALS1 by conjugation as described above . A kanamycin-resistant exconjugant was selected for subsequent studies .

Assessment of gene transfer agent transcription and production. Plasmid pYP, which contains the gene transfer agent promoter, orfg1, and an in-frame translational fusion of the gene transfer agent structural gene orfg2 to lacZ (18), was introduced into R . capsulatus strains SB1003 and ALS1 by conjugation . Exconjugants were grown phototrophically in YPS at 30°C, and ß-galactosidase activity was measured in stationary-phase cells (22 h) (46) . Where indicated, synthetic C16-HSL (2 µM) was added to cultures . As a control, similar experiments were performed with pYNP, which contains a promoterless orfg2::lacZ fusion (18) . With pYNP, ß-galactosidase activity was less than 0.1 U .

Transducing particle production was measured as gene transfer agent-mediated transfer of a rifampin resistance (Rif^r) marker from the gene transfer agent donor strains (SB1003 and ALS1) to the Rif^s gene transfer agent recipient strain (B10) as described previously (18) . R . capsulatus SB1003 and ALS1 were grown as described above, and synthetic C16-HSL (2 µM) was added to cultures where indicated .

We screened R . capsulatus and P . denitrificans for acyl-HSL synthesis with the radiotracer assay . These were both organisms in which we had failed to detect acyl-HSLs with a variety of bioassays (31; unpublished results) . P . denitrificans incorporated ¹⁴C label into a product that was ethyl acetate extractable and was eluted with approximately 95% methanol in our C₁₈ reverse-phase HPLC (Fig . 1B) . R . capsulatus appeared to make two acyl-HSLs, one that coeluted with the P . denitrificans product and an additional product that was eluted with 90% methanol in water (Fig . 1A) . These acyl-HSLs were more hydrophobic than any previously described naturally occurring acyl-HSLs . Our results demonstrate one significant advantage of the nonbiological detection approach, that acyl-HSLs exhibiting little or no activity in existing bioassays can be detected .

 
Identification of R . capsulatus and P . denitrificans acyl-HSLs. The R . capsulatus product from fraction 60 coeluted with synthetic tetradecanoyl-HSL (C14-HSL), but the identities of the more hydrophobic compounds produced by R . capsulatus and P . denitrificans (fractions 65 and 66) were unknown . The material in fractions 65 and 66 was purified from P . denitrificans culture fluid as described in Materials and Methods and analyzed by CI-MS, which showed a quasimolecular (M + H)⁺ ion with an m/z of 340 (Fig . 2A) . There were also peaks at 102 and 143 m/z, corresponding to homoserine lactone and the homoserine lactone ring with two of the acyl carbons, respectively, which are characteristic peaks found in CI-MS spectra of acyl-HSLs (36) . The spectrum was consistent with the conclusion that the compound in fractions 65 and 66 was C16-HSL . As a confirmation, we chemically synthesized C16-HSL . Synthetic C16-HSL showed an elution profile identical to that of the P . denitrificans material and had a CI-MS spectrum similar to that of the P . denitrificans material (Fig . 2B) . It was not possible to obtain sufficiently pure material from R . capsulatus for further analysis, but we conclude that the R . capsulatus material that coeluted with C16-HSL in HPLC is also C16-HSL .

 
R . capsulatus acyl-HSL synthase gene. We chose to study C16-HSL production further in R . capsulatus, an organism for which the genome has been sequenced . We identified an ORF, RRC03805, that encoded a product that showed 26% identity (43% similarity) to the R . sphaeroides CerI and contained each of the nine completely conserved amino acid residues found in acyl-HSL synthases (Fig . 3A) (24) .

 
To verify that RRC03805 coded for an acyl-HSL synthase, we created an RR03805 SPr insertion mutant, R . capsulatus ALS1 (Table 1) . Strain ALS1 did not synthesize C16-HSL or C14-HSL (Fig . 4A), and we detected no other acyl-HSLs . This suggested that the RRC03805 gene codes for an acyl-HSL synthase . Unlike the R . sphaeroides cerI mutant, which overproduces exopolysaccharide (31), R . capsulatus ALS1 had no obvious phenotype . When the RRC03805 mutation in strain ALS1 was complemented with pBBRGtaI, acyl-HSL synthesis was restored (Fig . 4A) . Interestingly, E . coli containing pBBRGtaI synthesized C14-HSL, additional putative acyl-HSLs (fractions 57 to 58), but not C16-HSL (Fig . 4B) . To our knowledge, this appears to be the first acyl-HSL synthase that fails to direct the synthesis of all cognate acyl-HSL molecules in E . coli .

 
A Blast search of the R . capsulatus translation products with the R . sphaeroides CerR (AAC47021) revealed three related polypeptides, RRC03806, RRC04617, and RRC02401 . These translation products were 26%, 21%, and 23% identical (37%, 31%, and 36% similar) to CerR, respectively . Two of the three, RRC03806 and RRC04617, contained all seven of the most highly conserved residues found among LuxR family members (Fig . 3B) (42) . RRC02401 possessed only three of the seven conserved residues, but did show similarity to other LuxR-related proteins along what is thought to be the acyl-HSL binding region (Fig . 3B) . RRC03806 was 53 bp upstream of the acyl-HSL synthase, and the two genes were in the same orientation . The ORFs encoding the other two LuxR-like proteins were unlinked .

C16-HSL induction of R . capsulatus gene transfer agent. The R . capsulatus genome harbors a 15-kb cluster of genes that code for production of a phage-like particle known as the gene transfer agent (17, 18) . Each gene transfer agent particle can deliver approximately 4.5 kb of randomly packaged genomic DNA to a recipient cell, where recombination may occur (40, 47) . Because gene transfer agent production is maximal in stationary phase (18, 41), when cell densities are high, and because a gene transfer system in another bacterium has been reported to be controlled by quorum sensing (10, 49), we tested whether the gene transfer agent is regulated by C16-HSL .

First, we examined transcription of a gene transfer agent structural gene . Plasmid pYP (Table 1), which carries the gene transfer agent gene orfg2 fused to lacZ (18), was introduced into the wild-type and acyl-HSL synthase mutant R . capsulatus strains . Measurements of lacZ-encoded ß-galactosidase activity indicated that orfg2 expression was reduced sevenfold in the acyl-HSL synthase mutant compared to the wild-type strain (Table 2) . Addition of C16-HSL restored wild-type ß-galactosidase activity to the mutant (Table 2) . This sevenfold reduction in ß-galactosidase activity was observed in early, mid-, and late logarithmic phase and in stationary phase (data not shown) . Second, we assessed the number of gene transfer agent particles produced by the wild-type and acyl-HSL synthase mutant R . capsulatus strains . In agreement with the lacZ reporter data, gene transfer agent production was fivefold less in the acyl-HSL-deficient strain, and the number of transductants was increased by addition of C16-HSL (Table 2) . These data indicate that gene transfer agent production is controlled by C16-HSL .

 
Both C16-HSL and C14-HSL activated gene transfer agent gene expression in a concentration-dependent manner (Fig . 5) . The half-maximal response occurred at 2.5 to 5 nM C16-HSL and 7.5 to 10 nM C14-HSL . This is consistent with information about other acyl-HSL systems, in which responses generally require nanomolar concentrations of signals (14, 27, 49) . C16-HSL appeared to be the preferred signal for gene transfer agent gene expression (Fig . 5) . It is clear from the available R . capsulatus genomic sequencing data (assembled into nine contigs) that the acyl-HSL synthase gene and the gta genes are not tightly linked; the minimum distance between them is 570 kbp (www.integratedgenomics.com) .

 
Concentrations and locations of long-chain acyl-HSLs in R . capsulatus cultures. One limitation of the ¹⁴C-labeled acyl-HSL assay is that it does not provide information on how much of an acyl-HSL accumulates during culture growth (34) . Thus, we needed to perform bioassays to determine the absolute level of C16-HSL . Our discovery that a gene transfer agent structural gene-lacZ fusion is expressed in response to C16-HSL (Fig . 5) provided us with a bioassay tool (see Materials and Methods) . Cell-free late-logarithmic-phase R . capsulatus and P . denitrificans culture fluids contained approximately 325 to 390 nM and 350 to 400 nM C16-HSL, respectively . Unlike the case with V . fischeri, for example, where almost all of the 3OC6-HSL is found in the culture supernatant fluid (14), a significant fraction of C14 and C16-HSL remained with the cells .

Ethyl acetate extracts of P . denitrificans cell pellets contained 48 to 50% of the total ¹⁴C-labeled C16-HSL . Extracts of R . capsulatus cells contained 33 to 34% and 49 to 52% of the total ¹⁴C-labeled C16-HSL and putative ¹⁴C-labeled C14-HSL signals, respectively . For comparison, we measured the fraction of 3OC6-HSL in V . fischeri cells . With V . fischeri, 2 to 4% of the total ¹⁴C-labeled 3OC6-HSL remained with cells, consistent with a previous report (14) . These data suggest that the long-chain acyl-HSLs produced by P . denitrificans and R . capsulatus are concentrated in cells . Presumably, they partition with the membranes, where they may be unavailable as signals .

We assume that the bulk of the cellular acyl-HSLs was associated with the membranes . The implications of this are that although there would be significant levels of acyl-HSLs in membranes, the cytoplasmic concentrations might be similar to the environmental concentrations . Thus, the acyl-HSLs could still serve a signaling function . Short-chain acyl-HSLs, like 3OC6-HSL and butanoyl HSL, are freely diffusible and not concentrated in cells (14, 26) . Longer-chain signals like 3OC12-HSL in P . aeruginosa are concentrated in cells, and an efflux pump has been shown to aid 3OC12-HSL export (26) .

We studied acyl-HSL production by R . capsulatus further because of the availability of the genome sequence for this organism . We identified a single ORF that showed significant sequence similarity with LuxI family members, and we showed that this ORF directed the synthesis of acyl-HSLs . Insertion of an Sp^r cassette in this ORF generated a strain that did not produce acyl-HSLs . Introduction of a wild-type copy of this gene into the R . capsulatus ALS1 mutant or into E . coli resulted in acyl-HSL synthesis . In the complemented R . capsulatus mutant, the acyl-HSLs produced were identical to those produced by the wild-type strain, but E . coli did not produce C16-HSL . To our knowledge, this is the first example of an I gene-containing recombinant E . coli that does not produce the native acyl-HSL . Adjacent to this acyl-HSL synthase gene is a gene coding for a LuxR homolog . However, there are other LuxR homologs in the R . capsulatus genome database, and we do not know if the adjacent gene is a transcription factor that responds to C16-HSL .

Although we have not identified the receptor experimentally, we have shown that C16-HSL can serve as an environmental signal for activation of gene transfer agent structural genes in R . capsulatus . This demonstrates that even though C16-HSL is concentrated in cells, it is a signal for gene expression when added externally . Of course, it is possible that long-chain acyl-HSLs play an additional role in R . capsulatus and P . denitrificans membranes .

The R . capsulatus long-chain acyl-HSL induced gene transfer agent gene expression five- to sevenfold, whereas expression of gene transfer agent and flagellar genes was reduced 10-fold or more in cckA and ctrA mutants (16) . It will be interesting to evaluate the possibility of connections between the cckA/ctrA system and the acyl-HSL-dependent regulation described here . Perhaps there are independent but overlapping signals that cooperatively induce maximal production of gene transfer agent .

This work was supported by grants from the W . M . Keck Foundation, the National Institutes of Health (GM59026), the National Science Foundation (MCB 9808308), and the Natural Sciences and Engineering Research Council (Canada) . A.L.S . was supported by U.S . Public Health Service Training Grant T32-AI07343 .

Present address: W . M . Keck Microbial Communities & Cell Signaling Laboratory, Kewalo Marine Laboratories, Pacific Biomedical Research Center, University of Hawaii-Manoa, Honolulu, HI 96813 .

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What Is Genetic Engineering?, What Is Functional Genomics?, What Is Bioremediation?, What Is Prokaryote?, What Is Environmental Microbiology?, o, Bacteriology, a, Microbes, e, Bacteria, a, Microorganisms, r, Bacterium, n, Fermentations, c, Growth media, n, Escherichia coli, i, S. cerevisiae, n, Ps. putida, i, Staphylococcus, r, Escherichia coli, s, Staphylococcus, i, Enterobacteriacea, a, Pseudomonas aeruginosa, r, Staphylococcus, e, Yeasts, a, Penicillin, r, Bacillus, c, Serratia, a, Halomonas, e, Escherichia coli, e, Streptococci, n, Culture medium, s, Microbial, a, Lactococci




 

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