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Quorum-Sensing Signal Synthesis by the Yersinia pestis Acyl-Homoserine Lactone Synthase YspI
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     Department of Pharmacology, Program in Biomolecular Structure, The University of Colorado Health Sciences Center, P.O. Box 8511 MS8303, Aurora, Colorado 80045,Department of Chemistry, University of Colorado at Denver, 1200 Larimer Street, Denver, Colorado 80217,Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523,Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, P.O. Box 2087, Fort Collins, Colorado 80522

    ABSTRACT

    The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-L-homoserine lactone and N-3-oxo-hexanoyl-L-homoserine lactone.

    TEXT

    In 2001, an effort to understand the fundamental characteristics of plague virulence was initiated with the genome sequence of Yersinia pestis (14). This project led to the identification of two genes, yspI and ypeI, encoding putative LuxI family acyl-homoserine lactone (AHL) synthases (1). The phenomenon of bacterial cell-to-cell signaling, known as quorum sensing, coordinates the transition from individual cell growth to collective virulence in many bacterial species (reviewed in references 4, 7, and 13). In Y. pestis, it is not yet known how quorum sensing may be involved in the organism's lifestyle, but the hierarchical regulation within the quorum-sensing system of Pseudomonas aeruginosa (16) and Yersinia pseudotuberculosis (1) suggests that a similar structure may exist within the ysp and ype quorum-sensing systems of Y. pestis (Fig. 1A). Studies of Y. pseudotuberculosis suggest a role for the ypsI/ytbI systems in clumping and motility and in production of N-3-oxo-hexanoyl-homoserine lactones (HSLs) (1). Identification of the principal AHLs employed by the ysp quorum-sensing system may be valuable in the effort to characterize gene regulation and virulence in Y. pestis.

    Nomenclature. An acyl chain of a specific length is indicated by "Cn" for the number of carbons in the chain (i.e., hexanoyl is C6). The substitution type and position are designated 3-oxo or 3-hydroxy, and HSL refers to D/L-homoserine lactone (e.g., 3-oxo-C6-HSL). AHL refers to N-acyl-HSL, with any chain length or degree of substitution.

    Recombinant YspI in Escherichia coli produces a similar AHL profile to Y. pestis. The YspI protein of Y. pestis is identical in amino acid sequence to the YtbI protein of Y. pseudotuberculosis (Fig. 1B). Identification of the AHLs obtained from culture supernatant extracts of Y. pseudotuberculosis and mutants with mutation in the ytbR/I system suggests that the profile of AHL signals produced depends on the pH and growth temperature (1, 12, 20). Therefore, the YspR/I system of Y. pestis is expected to produce the same profile of AHLs as the YtbI enzyme. In order to determine the AHLs produced by YspI, the gene was expressed in E. coli and the AHL signals released into the surrounding media were examined using the CV026 thin-layer chromatography (TLC) overlay (12, 19) and lasR lasB'-lacZ reporter strain liquid culture (15, 18) bioassays.

    Y. pestis strain KIM6+ (biovar Medievalis) was obtained from Robert Perry (University of Kentucky) and used in this study. This strain lacks the virulence plasmid pCD1 and is, therefore, avirulent, exempt from select agent guidelines, and can be grown under biohazard safety level 2 conditions. Standard methods were used to extract KIM6+ DNA (17). PCR was used for amplification of the yspI gene from strain KIM6+ DNA using primers YSPI-U (5'-AAAGCATATGTTAGAAATTTTCGATGT), which introduced an NdeI site (underlined) at the yspI start codon, and YSPI-D (5'-TGGATCCTATTAAGCCGATTCTGG), which introduced a BamHI site (underlined) after the stop codon. The 666-bp PCR fragment was cloned into pCR2.1 (Invitrogen) to form pYSPI. A 722-bp NdeI and HindIII digestion product of pYSPI was then subcloned into the similarly digested expression plasmid pET-28a (Novagen) and transformed into E. coli DH5 cells. The sequence of the pET-28a-yspI plasmid was confirmed before transformation into E. coli strain BL21(DE3) to give E. coli BL21(DE3)/pET-28a-yspI; E. coli BL21(DE3)/pET-28a was used as a control. Ten-milliliter bacterial cultures grown in Luria-Bertani (LB) medium with 50 μg/ml kanamycin were inoculated and incubated at 37°C with shaking at 225 rpm. After 12 to 18 h, 10-ml volumes of fresh LB medium with 50 μg/ml kanamycin were inoculated with 200 μl of each culture and incubated to an optical density at 600 nm (OD600) of 0.6. IPTG (isopropyl--D-thiogalactopyranoside; 0.2 mM) induced protein expression during an additional hour of incubation at 37°C. The cultures were then centrifuged, and the supernatants were immediately decanted and passed through a 0.22-μm-pore-size nitrocellulose syringe filter, extracted twice with 10 ml of ethyl acetate acidified with 0.01% acetic acid, dried, and stored at –20°C. Prior to use, extracts were reconstituted in 100 μl methanol. Similar approaches were used to grow Y. pestis and to extract the AHL signals.

    AHL signals were detected by the lasR lasB'-lacZ reporter gene system, which responds to AHLs but does not indicate which specific AHLs are present (Fig. 2A). E. coli strain MG4/pKDT17 contains a lasB'-lacZ detection system and a lasR gene under control of the lac operon promoter (15, 18). The lasB and lasR genes are from P. aeruginosa and encode the quorum-sensing-regulated elastase and the LuxR-type regulator LasR, respectively. This reporter strain produces -galactosidase in response to a wide range of AHLs, including 3-hydroxy-, 3-oxo-, and unsubstituted AHLs with side chain lengths of 8 to 14 carbons (17). For detection of AHLs, a 10-ml culture of MG4/pKDT17 grown in LB medium with 100 μg/ml ampicillin was incubated with shaking for 12 to 18 h at 30°C. The culture was diluted to an OD600 of 0.1 with A medium (17). Concentrated AHL samples, including negative controls, were prepared in triplicate by adding 5 μl of undiluted, methanol-reconstituted extract to a tube and evaporating the samples to dryness. A 100-μl aliquot of diluted overnight culture and 900 μl of Z buffer (12) were added to each dried sample. Samples were incubated at 30°C with shaking for 5.5 h, and their OD600 was measured. Cells were permeabilized with sodium dodecyl sulfate-chloroform, -galactosidase activities were measured, and Miller units were calculated as previously described (12). Extracts from BL21(DE3)/pET-28a cultures produced considerably less activity than extracts from BL21(DE3)/pET-28a-yspI cultures, as shown in Fig. 2A. The pET-28a-yspI construct obviously produces a protein that is required for synthesis of AHLs. To identify the AHL signals produced by YspI in E. coli, TLC overlay assays were performed with the Chromobacterium violaceum mutant strain CV026 for comparison with previous studies (12, 19). CV026 cannot synthesize AHL signals to activate violacein production but produces the pigment with added AHLs. The reporter strain responds well to AHLs with acyl chain lengths of 4 to 8 carbons but weakly to 3-oxo-substituted AHLs and not at all to AHLs with acyl chain lengths of 10 to 14 carbons (12). To perform the assay, a reverse-phase TLC plate (Whatman KC18F) was prerun, dried, loaded with AHLs and culture extracts, and developed with 60% methanol-40% water. After drying, the plate was overlaid with the CV026-containing agar. This overlay was prepared by suspending CV026 cells from a 50-ml LB medium-grown overnight culture in 250 ml autoclaved 1.5% LB agar with 50 μg/ml streptomycin and 50 μg/ml kanamycin. The plate was allowed to incubate for 12 to 18 h at 37°C for the violacein pigment to develop. The TLC plate in Fig. 2B shows that when this detection technique was utilized, the recombinant YspI expressed in E. coli produced an AHL profile similar to that of the wild-type Y. pestis strain KIM6+. Several AHL species, including 3-oxo-C6 and AHLs with longer acyl chains, were observed.

    YspI in E. coli produces predominantly 3-oxo-C8-HSLs and 3-oxo-C6-HSLs. A physical/chemical method, based on liquid chromatography-tandem mass spectrometry (8), was used to structurally identify the AHL molecular species produced by YspI and Y. pestis, independent of the selectivity of CV026 reporter strain (6). The ethyl acetate extracts of the E. coli BL21(DE3)/pET-28a and BL21/(DE3)pET-28a-yspI cultures were further purified by solid-phase extraction prior to liquid chromatography-tandem mass spectrometry. YspI AHL extract reconstituted with 100 μl of methanol was loaded onto prewashed silica Sep Pak (Waters) with 5 ml isooctane-ether. The Sep Pak was washed two times with 5 ml of isooctane-ether and eluted with 7.5 ml acidified ethyl acetate. The eluate was dried to 500 μl, transferred to an autosampler vial, evaporated to dryness, and finally reconstituted with 50 μl methanol. The samples were analyzed with a PE Sciex API-3000 triple-quadrupole tandem mass spectrometer (Perkin-Elmer Life Sciences, Thornhill, Ontario, Canada). Chromatographic separation on a C18 column was achieved using a gradient of 20% solvent B to 95% solvent B over 30 min (mobile phase A, water, 0.1% acetic acid; mobile phase B, methanol, 0.1% acetic acid) at a 200-μl/min flow rate. The tandem mass spectrometer was operated first in precursor ion-scanning mode to determine which AHLs were present and then in the multiple reaction monitoring mode, where ion transitions, based on previously studied collisionally induced dissociation behavior of the [M + H]+ ion for several synthetic AHLs, were detected. Specific product ions corresponding to the fatty acyl and lactone moieties were measured for each AHL as previously reported (9, 10).

    The high-performance liquid chromatography separation chromatogram (Fig. 3A) revealed four distinct homoserine lactone-containing components in the YspI extract from E. coli. The detection of each AHL was obtained by multiple reaction monitoring, specifically 3-oxo-C6-HSL (m/z 214102), 3-oxo-C8-HSL (m/z 242102), C8-HSL (m/z 228102), and 3-oxo-C10-HSL (m/z 270102). 3-Oxo-C8-HSL and 3-oxo-C6-HSL were the major signals more than twofold over the next signals, which were C6-HSL and C8-HSL. This semiquantitative approach permitted measurement of C6-HSL amounts due to the presence of the deuterated D3-C6-HSL internal standard and comparison of relative amounts of the other AHLs due to inherent differences in extraction and purification efficiencies. A comparison of the relative amounts of AHLs made by the Y. pestis strain (Fig. 3B) compared to the recombinant YspI in E. coli (Fig. 3A) shows similar profiles.

    The abundance of N-3-oxo-HSLs detected in E. coli BL21(DE3)/pET-28a-yspI and Y. pestis culture supernatant extracts in this study differs from the AHL production reported for YtbI (1, 20). Specifically, in previous studies, E. coli JM109-ytbI clones yielded culture supernatant extracts containing predominantly C6-HSL and C8-HSL, as detected by CV026 TLC overlay assay. The mass spectrometry studies (Fig. 3) did not detect any C6-HSL, although C8-HSL was present in small but detectable amounts. The TLC spot migrating between C6-HSL and C8-HSL that was previously unidentified, and which was a major AHL produced by Y. pestis, appeared to be 3-oxo-C8-HSL. The environment in which AHLs are produced could affect AHL synthase specificity, because there may be different respective acyl-acyl carrier protein (acyl-ACP) pools. However, although the differences between E. coli strains JM109 and BL21(DE3) with respect to the production of acyl-ACPs are not known, they are not expected to be large enough to affect the AHL profiles of YtbI or YspI under comparison here. Furthermore, even differences that could be present in the acyl-ACP pools of diverse expression hosts such as E. coli and Pseudomonas aeruginosa do not alter AHL production of a particular AHL synthase in the same way (8).

    Conclusions. N-(3-Oxooctanoyl)-L-homoserine lactone and N-(3-oxohexanoyl)-L-homoserine lactone were the most abundant AHLs produced by recombinant YspI in E. coli and by nonrecombinant wild-type Y. pestis. Therefore, 3-oxo-C6-ACP and 3-oxo-C8-ACP are the primary substrates for YspI. Further study is warranted to determine which genes are activated in Y. pestis once the AHL signals are sensed. Because the AHL profiles of E. coli strains expressing YspI and Y. pestis were very similar, the role of the second AHL synthase YpeI in AHL production and Y. pestis quorum sensing remains puzzling but warrants further studies.

    ACKNOWLEDGMENTS

    We thank K.-H. Choi for assistance with analysis of Y. pestis AHLs.

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