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. 2003 Sep;185(17):5029–5036. doi: 10.1128/JB.185.17.5029-5036.2003

Proteomic Analysis of Wild-Type Sinorhizobium meliloti Responses to N-Acyl Homoserine Lactone Quorum-Sensing Signals and the Transition to Stationary Phase†

Hancai Chen 1, Max Teplitski 2, Jayne B Robinson 2, Barry G Rolfe 1, Wolfgang D Bauer 2,*
PMCID: PMC180974  PMID: 12923075

Abstract

Proteome analysis revealed that two long-chain N-acyl homoserine lactones (AHLs) produced by Sinorhizobium meliloti 1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the wild type. Fifty-six of the corresponding proteins have been identified by peptide mass fingerprinting. The proteins affected by addition of these two AHLs had diverse functions in carbon and nitrogen metabolism, energy cycles, metabolite transport, DNA synthesis, and protein turnover. Two hours of exposure to 3-oxo-C16:1-homoserine lactone (3-oxo-C16:1-HL) affected the accumulation of 40 of the 56 identified proteins, whereas comparable exposure to C14-HL affected 13 of the 56 proteins. Levels of four proteins were affected by both AHLs. Exposure to 3-oxo-C16:1-HL for 8 h affected the accumulation of 17 proteins, 12 of which had reduced accumulation. Of the 80 proteins identified as differing in accumulation between early-log- and early-stationary-phase cultures, only 13 were affected by exposure to 3-oxo-C16:1-HL or C14-HL. These results provide a foundation for future studies of the functions regulated by AHL quorum sensing in S. meliloti and help to establish proteomic analysis as a powerful global approach to the identification of quorum-sensing regulatory patterns in wild-type bacteria.

Many bacteria are capable of responding to changes in population density and coordinating the behavior of individual cells in a local population through the exchange of extracellular signal molecules. This kind of regulation, called quorum sensing, affects a diversity of bacterial behaviors (33; reviewed in references 30 and 49). Quorum sensing appears to be particularly important in coordinating gene expression within a local bacterial population during its interaction with a eukaryotic host (49, 54). N-Acyl homoserine lactones (AHLs) are the most common of the signals used by gram-negative bacteria for quorum sensing regulation (49). With few exceptions (31), the proteins that serve as AHL receptors are transcriptional activators, homologs of the LuxR protein of Vibrio fischeri, and have both AHL- and DNA-binding domains (15, 41). Recent studies have indicated that AHLs can bind to the nascent receptor polypeptide, helping to ensure its proper folding into an active form and stabilizing the active form against proteolytic degradation (55, 56).

AHL quorum sensing can have global effects on bacterial physiology. Approximately thirty proteins were differentially accumulated or modified in AHL synthase-deficient mutants of Yersinia enterocolitica and Serratia liquefaciens in response to added AHLs (19, 45). In Pseudomonas aeruginosa, the addition of AHLs to a mutant deficient in AHL production was found to activate more than 250 random transcriptional fusions (50), and recent microarray studies have revealed that expression of about 6% of the genes in this species are affected by AHL-mediated quorum sensing (40, 46, 47). Other recent studies have shown that expression of subsets of AHL-regulated genes requires the presence of additional regulators (RpoS, MvfR, RsmA, and MvaT) in addition to high AHL concentration (5, 11, 36, 38, 51).

We have been interested in contributing to the systematic characterization of quorum sensing regulation in the nitrogen-fixing bacterial symbiont of legumes, Sinorhizobium meliloti. The genomic sequences of the circular chromosome (SmC) and the two megaplasmids (SmA and SmB) from strain 1021 have been recently published (16). Proteomic reference maps have been developed for both free-living S. meliloti 1021 (21, 22, 48) and differentiated nitrogen-fixing bacteroids (32). Its symbiotic partner, Medicago truncatula, a model legume closely related to alfalfa (9), was shown recently to produce compounds that can mimic AHL signals and disrupt AHL-mediated quorum sensing in various bacteria (2, 17, 43). In addition, M. truncatula was shown recently to respond extensively and specifically to AHLs from S. meliloti and other bacteria (29).

Examination of the S. meliloti 1021 genomic sequence indicates the presence of a LuxI AHL synthase homolog (sinI = Smc00168), an HdtS-like AHL synthase (24) homolog (Smc00714), and perhaps five potential LuxR AHL receptor homologs (16, 37), suggesting that AHL-mediated quorum sensing is both important in S. meliloti and likely to be complex. Recent studies by Marketon et al. (26, 27) have provided evidence that sinI is required for the synthesis of several novel AHLs, with long acyl side chains, including C16:1-homoserine lactone (C16:1-HL), 3-oxo-C16:1-HL, and C18-HL, plus two previously known long-chain AHLs, 3-oxo-C14-HL (6) and C12-HL, but is not required for synthesis of short-chain AHLs. AHLs with short acyl side chains were not detected by Marketon et al. in cultures grown in defined medium (27). Disruption of the adjacent luxR homolog, sinR (= Smc00170), resulted in diminished synthesis of the long-chain AHLs, suggesting that SinR is an active AHL receptor and is involved in amplification of SinI-mediated signal synthesis (27). Recent studies have also provided evidence that ExpR (= Smc03896) functions as an AHL receptor in S. meliloti. The expR gene is interrupted by a native insertion sequence in strain 1021, but spontaneous excision of this insertion sequence element results in enhanced production of exopolysaccharide II in response to C16:1-HL and 3-oxo-C16:1-HL (28, 37). Functional expR is present in other native S. meliloti isolates (28, 37).

We conducted a proteomic analysis of quorum sensing in S. meliloti 1021 in order to obtain a global overview of the number and nature of AHL-regulated functions and to learn which functions might be affected by specific AHLs in a wild-type strain. In this initial study, we treated low-population-density cultures of the wild-type S. meliloti with purified AHLs to identify responses at the level of protein accumulation. Any study of quorum-sensing responses in a wild-type background, rather than in an AHL synthase mutant background, requires that the level of endogenous AHLs be minimized so that the effects of the added AHLs can be reliably detected. Our approach was to repeatedly wash and regrow low-density cultures of the bacterium to reduce the levels of endogenous AHLs and then expose the cultures to levels of AHLs that they would encounter normally only at late log or early stationary phase. Over 100 proteins accumulated to significantly different levels in response to the added AHLs. The two AHLs tested affected the levels of two quite different sets of proteins.

MATERIALS AND METHODS

Bacterial strains, media, and growth conditions.

S. meliloti 1021 was grown in TA (YTB) medium (7) for proteome analysis so that results could be compared to earlier proteomic studies of this bacterium (7, 8, 21, 22, 32, 48) and in a defined NM medium (39) supplemented with 3g of glucose/liter, 0.505 g of KNO3/liter, and Gotz vitamins for AHL extraction and purification in order to minimize contamination of AHL preparations with organic components from rich media. The AHL reporter strains, Escherichia coli pSB401 (LuxRI′::luxCDABE) and pSB1075 (LasRI′::luxCDABE) (53) were grown in Luria-Bertani (LB) medium (18). For bioassays to detect AHLs, the E. coli reporter strains were first grown overnight in LB broth with appropriate antibiotics, then diluted 100-fold in fresh LB with antibiotics twice at 2-h intervals, and then allowed to grow to an optical density at 600 nm (OD600) of 0.2. The culture was then centrifuged, and the pellet was resuspended in 10 volumes of fresh LB, vigorously vortexed, and used for the bioassays as described below.

AHL extraction and purification.

To obtain purified AHLs for proteomic studies, 1.5-liter cultures of S. meliloti were grown in 2.8-liter wide-bottom flasks to early stationary phase (OD600 = 1.4 to 1.8). Cell-free culture supernatants (pH 6.4 to 7.1) were extracted twice with 0.5 volume of ethyl acetate acidified with 0.1 ml of glacial acetic acid/liter and then dried over anhydrous sodium sulfate, filtered, and rotary evaporated over a 40°C water bath.

For purification of AHLs by reverse phase high-performance liquid chromatography (HPLC), the extracts from 6 liters of the cell culture supernatants were dissolved in 1 ml of acetonitrile and injected onto a semipreparatory C18 column (Whatman Partisil 10, ODS-3), fitted with a guard column and equilibrated with 10:90 acetonitrile-water. The column was eluted at 2 ml/min with a linear water-acetonitrile gradient to reach 100% acetonitrile after 65 min, followed by an additional 20 min in 100% acetonitrile. Synthetic AHLs were chromatographed on the same gradient to establish their retention times. Synthetic 3-oxo-C12-L-HL, C6-L-HL, 3-oxo-C6-L-HL, and C14-L-HL were purchased from Quorum Sciences (Coralville, Iowa). Other AHL standards were synthesized as described previously (12, 13).

To detect AHLs, samples of the HPLC fractions were aliquoted into 96-well black microtiter plates, dried in a laminar-flow hood, and then mixed with 80 μl of an E. coli LasRI′::CDABE or LuxRI′::CDABE AHL reporter (53) in LB. After approximately 3 to 5 h of incubation at 37°C, the luminescence of the E. coli reporters was measured with a microtiter plate reader (Wallac Victor-2; Perkin-Elmer Inc, Gaithersburg, Md.) as described previously (17).

Treatment of bacteria for proteomic analysis.

To minimize the levels of endogenous AHLs in wild-type S. meliloti 1021 cells, a starter culture was grown to an OD600 of 1.7, and then 1 ml was pelleted, resuspended, and inoculated into 1 liter of TA medium in a 2.8-liter wide-bottom glass flask and cultured overnight at 28°C to and OD600 of 0.2 on a gyratory shaker at 200 rpm. A 100-ml portion was then centrifuged, the pellet was resuspended in 1 liter of TA medium, and the suspension was incubated on a shaker for 5 h to an OD600 of 0.05. Three-hundred-milliliter portions of this low-density cell culture were washed again, and the resuspended pellets were inoculated into flasks containing 1.5 liter of TA supplemented with one of the two purified AHLs. The concentrations of the two purified AHLs added to the washed, low-density cultures were adjusted to be approximately equal to those present in an early-stationary-phase NM culture of the bacterium. The treated cultures were incubated for 2 h at 28°C and 200 rpm to a final OD600 of 0.03 to 0.04, yielding 0.1 g of dry cells per 1.5 liters of culture. Duplicate control cultures with no added AHLs were grown under the same conditions. All centrifugations were conducted at room temperature to minimize stress. Viability and growth of the cells were monitored throughout the induction experiment by dilution plating. In addition, another low-density culture of wild-type 1021 was exposed for 8 h to one of the AHLs. The cell density of this culture reached a final OD600 of 0.08. Additional control cultures were grown to early stationary phase (24 h old; OD600 = 1.8) under the same conditions. All treatments and controls were duplicated.

Protein extraction and separation.

Proteins were extracted from freeze-dried cells and the samples were subjected to proteomic analysis as described previously (7, 8). Isoelectric focusing was performed with linear immobilized pH 4 to 7 IPG strips (Amersham Pharmacia Biotechnology, Uppsala, Sweden). Aliquots containing 1 mg of solubilized cellular protein were cup-loaded onto the acidic end of each IPG strip and run for 200 kV · h. Chromatography in the second dimension was performed on precast polyacrylamide ExcelGel XL SDS 12 to 14 gel from (Amersham Pharmacia Biotechnology, Uppsala, Sweden) according to the manufacturer's instructions.

Image processing and protein identification.

Preparative gels were stained with Coomassie brilliant blue in a stepwise colloidal staining procedure as described elsewhere (8, 34). Gels were scanned at 600 dpi on a UMAX 2400 Astra scanner, and relative protein abundance was quantified with the help of Melanie 3.05 software (Swiss Institute of Bioinformatics, Geneva, Switzerland) on triplicate gels for each treatment. Positions of the spots were compared to the 10 landmark proteins, and matched against a specialized proteomic database for S. meliloti 1021 (48). The OD of each spot over its area (volume) as a percentage of the relative OD of the gel image was used to quantify each spot, as described by Natera et al. (32). Analysis of variance (using a P of <0.05) was performed with Genstat software (version 5.0; Lawes Agricultural Trust) to test for statistical significance of differences in protein accumulation between the different treatments and controls.

Proteins were identified by tryptic digestion of the polypeptides isolated from the Coomassie-stained control gels followed by peptide mass fingerprinting with MALDI-TOF mass spectrometry performed on a Micromass TofSpec 2E Time of Flight Mass Spectrometer (Waters Corporation, Milford, Mass.) at the Australian Proteome Analysis Facility (Macquarie University, Sydney). Peptide mass fingerprints (PMF) were identified by comparison against the 1021 proteomic database using MassLynx software (Micromass; Waters Corporation) as described previously (48). Scoring of PMF matches was done based on the following criteria: (i) a minimum of four peptides matched within 100 ppm to the theoretical mass of the polypeptide without any protein modification, (ii) matched peptides collectively comprise >30% of the entire protein, (iii) there is a good agreement between the actual and predicted molecular mass and pI, and (iv) no other polypeptide matches. For smaller proteins (<15 kDa), three precisely matching nonmodified peptides were allowed, but a higher coverage (>40%) was considered. For proteins larger then 60 kDa, more then six peptides were required, but the coverage requirement was relaxed to 20%. A confidence rating of 3 was assigned if all criteria were met; a score of 2 was assigned if one criterion was not met. A confidence score of 1 means that at least one of the criteria was met.

RESULTS

Purification of AHL quorum-sensing signals.

The AHLs present in ethyl acetate extracts of S. meliloti 1021 culture filtrates were fractionated by C18 reverse-phase HPLC. Bioassays revealed a broad peak of substances in the hydrophobic region of the elution profile (fractions 49 to 75) that stimulated responses in the LasR- and LuxR-based AHL reporters (Fig. 1A) The putative AHLs detected by these reporters eluted after the retention time of 3-oxo-C12-HL (Fig. 1A). Refractionation of the substances in this broad activity peak resulted in the separation of two peaks of material capable of activating the reporters (Fig. 1B). The strong LasR-LuxR activity in fractions 46 to 51 (Fig. 1B) was refractionated (Fig. 1C). The purified AHL present in fractions 47 to 49 (designated peak A) was used in proteomic experiments. The second major AHL peak in Fig. 1B (fractions 60 to 63) was resolved into two by further HPLC fractionation (Fig. 1D). The AHL present in an earlier-eluting peak, fractions 32 to 35 (designated peak B), was also used for proteomic experiments. Using the electrospray ionization mass spectrometry (MS)-MS and gas chromagtography-MS methods described by Marketon et al. (27), the AHL present in peak A was identified as 3-oxo-C16:1-HL, and the AHL present in peak B was identified as C14-HL (A. Eberhard, M. R. Gronquist, M. Teplitski, and W. D. Bauer, unpublished data).

FIG. 1.

FIG. 1.

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HPLC fractionation of S. meliloti 1021 AHLs. Culture filtrate extracts were fractionated by HPLC and bioassayed for AHLs as described in Materials and Methods. Dark and light vertical bars indicate the relative activities of compounds in HPLC fractions capable of activating the LuxR and LasR AHL reporters, respectively. (A) The active fractions (fractions 49 to 63), indicated by inward-facing arrows, were pooled and rechromatographed (B) with a shallower water-acetonitrile step gradient (thick dark line). (B) The substances present in the two peaks indicated by arrows (fractions 47 to 52 [C] and 60 to 63 [D]) were subsequently rechromatographed. Numbered arrows indicate elution times for synthetic AHL standards as follow: 1, C4-HL; 2, 3-oxo-C6-HL; 3, C6-HL; 4, 3-oxo-C12-HL; 5, C12-HL; 6, C14-HL. Pooled fractions 48 and 49 (C) and pooled fractions 32 to 34 and 43 to 45 (D) were analyzed by electrospray ionization MS-MS. Substances in fractions indicated by dark bars, fractions 48 and 49 of (C) (peak A) and fractions 32 to 34 of (D) (peak B) were identified as 3-oxo-C16:1-HL and C14-HL, respectively, and used to treat 1021 cells for proteomic analysis.

Effects of added AHLs on protein accumulation in wild-type S. meliloti 1021.

Treatment of early-log-phase cultures of S. meliloti 1021 with 3-oxo-C16:1-HL (peak A) or C14-HL (peak B), at concentrations adjusted to equal those in the early-stationary-phase cultures from which they were purified, was found to reproducibly and significantly affect the accumulation of over 100 polypeptides (data not shown). As indicated in Table 1, 56 of these polypeptides correspond to proteins previously identified in S. meliloti 1021 (21, 22, 48). Identification of the other AHL-responsive polypeptides is currently in progress.

TABLE 1.

Proteins differentially accumulated in early log phase cultures of wildtype S. meliloti treated with 3-oxo-C16:1-HL or C14-HL

Proteina Genomic accession no.a,b Ratingc Accumulation response (%)d
3-oxo-C16: 1-HLe 2 h 3-oxo-C16: 1-HLe 8 h C14-HLf 2 h
Hypothetical SMc00040 3 200
Peroxiredoxin SMc00072 2 290
Phosphoglycolate phosphatase SMc00151 3 240
Signal peptide hypothetical/global homology SMc00242 3 150
Fatty-acid-CoA ligase SMc00261 1 320
Orotidine 5′-phosphate decarboxylase SMc00412 3 40
Hypothetical protein SMc00496 3 150
Conserved hypothetical protein SMc00538 3 230
Hypothetical SMc00651 2 60
IMP dehydrogenase SMc00815 3 270
Glutamine synthetase I SMc00948 3 New New
Acetyltransferase SMc01017 1 40
Polypeptide deformylase SMc01101 1 150
NADP-dependent malic enzyme SMc01126 3 200
Nitrogen regulatory IIa protein SMc01141 2 40
Elongation factor G SMc01312 3 130
Biotin carboxyl carrier protein of acetyl-CoA carboxylase SMc01344 2 70
Ribose 5-phosphate isomerase B (isoform 1) SMc01613 3 290
Ribose 5-phosphate isomerase B (isoform 2) SMc01613 3 240
Ribose 5-phosphate isomerase B (isoform 3) SMc01613 3 230
2,3,4,5-Tetrahydropyridine-2-carboxylate N-succinyltransferase SMc01732 3 50
Glycine dehydrogenase decarboxylating protein SMc02049 3 770
30S ribosomal protein S2 SMc02101 3 280
Conserved hypothetical protein SMc02111 3 280
Ferredoxin-NADP reductase SMc02122 3 200
Orotate phosphoribosyltransferase SMc02165 3 410
ABC transporter periplasmic binding protein SMc02171 3 230
O-Succinylhomoserine sulfhydrylase SMc02217 3 310 260
Peptide chain release factor 1 SMc02436 3 50
Transaldolase-like protein (isoform 1) SMc02495 3 1,100
Phosphoenolpyruvate carboxykinase SMc02562 3 120 40 240
CLP protease proteolytic subunit (isoform 1) SMc02720 3 300 150 360
CLP protease proteolytic subunit (isoform 2) SMc02720 3 160
ABC transporter glycine betaine-binding protein SMc02737 3 170
Phosphoglycerate mutase 1 SMc02838 3 220
Conserved hypothetical protein SMc02852 3 65 40
ABC transporter periplasmic binding protein SMc02873 3 160
Flagellar hook protein SMc03047 3 270
Precorrin-2 C20-methyltransferase SMc03191 2 370 45
Precorrin-8x methylmutase SMc03192 3 260
Transmembrane hypothetical SMc03233 3 310 200
ABC transporter peptide-binding periplasmic protein SMc03269 3 120
Aconitate hydratase SMc03846 2 190 50
ABC transporter amino acid-binding periplasmic protein SMc03891 3 190
Transketolase SMc03978 3 50 230
Conserved hypothetical protein SMc04266 1 270
Putative d-threonine dehydrogenase SMa0237 2 510
Conserved hypothetical protein SMa0241 3 50 290
Hypothetical protein SMa0247 3 150
Conserved hypothetical protein SMa1326 2 130
Probable ABC transporter, periplasmic solute-binding protein SMa1375 3 170
Probable ARCD2 arginine/ornithine antiporter SMa1668 1 230 40
Conserved hypothetical protein SMa2245 1 710
Immunogenic protein SMb20292 1 40
Putative methylcrotonoyl-CoA carboxylase biotinylated subunit SMb21124 3 490 20
Putative heat shock protein groEL SMb21566 3 240
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a

Based on S. meliloti 1021 genomic database (http://sequence.toulouse.inra.fr/meliloti.html) (16).

b

Smc, Sma, and Smb refer to products of ORFs located on the chromosome and symbiotic plasmids A and B, respectively.

c

Confidence ratings were assigned as described in Materials and Methods.

d

Differential accumulation was determined as described in Materials and Methods, and is based on statistical analysis of at least three gels per treatment, and two cultures per treatment. Numbers represent average percentage differences in spot volumes between the AHL-treated and untreated cultures, and each value is significant at the P = 0.05 level. Underlining and boldface type represent enhanced and reduced accumulation, respectively. “Missing” and “New” refer to polypeptides not detectable in the treated and untreated gels.

e

Purified AHL from peak A, Fig. 1C, identified as 3-oxo-C16:1-HL.

f

Purified AHL from peak B, Fig. 1D, identified as C14-HL.

Of the 56 proteins identified, 42 showed changes of at least 2- to 10-fold in accumulation after exposure to one or both of the AHLs (Table 1). Two hours of exposure to C14-HL affected the accumulation of 13 proteins. In contrast, 2 h of exposure to 3-oxo-C16:1-HL led to the differential accumulation of 40 of the 56 proteins. Only 4 of the 56 proteins were affected by both 3-oxo-C16:1-HL and C14-HL after 2 h.

Responses to 3-oxo-C16:1-HL changed quite substantially between 2 and 8 h. Only 15% of the 40 proteins affected by 3-oxo-C16:1-HL after 2 h still showed significantly altered accumulation after 8 h exposure. Interestingly, 12 of the 17 proteins that were differentially accumulated after 8 h exposure to 3-oxo-C16:1-HL accumulated to levels lower than controls.

Differences in protein accumulation during the maturation of early log phase cultures to early stationary phase were also examined. As shown in Table 2 it was possible to identify 80 proteins that accumulated to significantly different levels in S. meliloti 1021 cells taken from early-log-phase cultures and early-stationary-phase cultures. The levels of only 13 of these 80 proteins were affected by addition of C14-HL or 3-oxo-C16:1-HL to the early-log-phase cultures (Table 2).

TABLE 2.

Proteins differentially accumulated in stationary phase cultures of S. meliloti 1021 and those also affected by addition of AHLs

Proteina Genomic accession no.a,b Ratingc Accumulation response (%)d
Stationary phase 3-oxo-C16: 1-HLe 2 h 3-oxo-C16: 1-HLe 8 h C14-HLf 2 h
Hypothetical SMc00040 3 1,600 200
Conserved hypothetical protein SMc00048 1 670
Ribose 5-phosphate isomerase SMc00152 2 300
Periplasmic binding protein SMc00265 3 340
Polyribonucleotide nucleotidyltransferase SMc00324 1 40
30S ribosomal protein S1 (isoform 1) SMc00335 3 30
30S ribosomal protein S1 (isoform 2) SMc00335 3 40
30S ribosomal protein S1 (isoform 3) SMc00335 3 40
Regulator of nucleoside diphosphate kinase SMc00347 3 370
Elongation factor P SMc00357 3 50
Phosphoribosylformyl glycinamidine synthetase II (isoform 1) SMc00488 2 40
Phosphoribosylformyl glycinamidine synthetase II (isoform 2) SMc00488 2 40
Hypothetical SMc00530 3 20
50S ribosomal protein L9 SMc00565 3 40
Nucleoside diphosphate kinase SMc00595 3 40
TRP repressor binding protein homologue SMc00943 2 720
Conserved hypothetical protein SMc01002 1 New
Acetyltransferase SMc01017 1 Missing 40
Dihydrolipoamide S-acetyltransferase SMc01032 3 40
Polypeptide deformylase SMc01101 1 30 150
Carbamoyl-phosphate synthase large chain SMc01215 3 10
Elongation factor G SMc01312 3 40
50S ribosomal protein L7/L12 SMc01318 3 50
Transcription antitermination protein SMc01322 3 40
Biotin carboxyl carrier protein of acetyl-CoA carboxylase SMc01344 2 800 80
Glutamyl-tRNA amidotransferase subunit B SMc01350 3 50
Signal peptide hypothetical/global homol SMc01418 2 290
creA protein SMc01465 3 390
Conserved hypothetical protein SMc01587 3 New
Urease β subunit SMc01939 2 New
30S ribosomal protein S2 SMc02101 3 60 280
Citrate synthase SMc02087 3 New
Elongation factor TS (ET-TS) protein SMc02100 3 50
Conserved hypothetical protein SMc02111 3 550 280
ABC transporter l-amino acid-binding periplasmic protein SMc02118 3 200
Orotate phosphoribosyltransferase SMc02165 3 200 410
ABC transporter periplasmic binding protein Smc02171 3 190 230
O-Succinylhomoserine sulfhydrylase SMc02217 3 1,300 300 260
DNA-directed RNA polymerase ω chain SMc02408 3 40
Transaldolase-like protein SMc02495 3 330 1,100
ATP synthase alpha chain SMc02499 3 50
ABC transporter iron transport ATP-binding protein SMc02508 3 1,100
ABC transporter iron-binding periplasmic protein SMc02509 3 470
Ferric uptake regulation protein SMc02510 3 440
Transmembrane hypothetical protein SMc02634 3 30
50S ribosomal protein L25 SMc02692 3 50
ABC transporter periplasmic binding protein SMc02774 3 650
ABC transporter periplasmic binding protein SMc02873 3 400 160
Heat shock protein 70 (hsp70) chaperone (isoform 1) SMc02857 3 50
Heat shock protein 70 (hsp70) chaperone (isoform 2) SMc02857 3 50
Heat shock protein 70 (hsp70) chaperone (isoform 3) SMc02857 3 30
ABC transporter alpha-glucoside-binding periplasmic protein SMc03061 3 520
Hypothetical protein SMa0312 3 330
Transmembrane outer membrane lipoprotein SMc03157 3 250
Transmembrane hypothetical SMc03233 3 Missing 310 200
Bacterioferritin (cytochrome B-1) SMc03786 3 660
Acetoacetyl-CoA reductase SMc03878 2 630
Acetyl-CoA acetyltransferase SMc03879 3 250
Hypothetical SMc03880 1 650
Histidinol-phosphate aminotransferase SMc03885 3 670
Fructose-bisphosphate aldolase class I (isoform 1) SMc03983 3 1,600
Fructose-bisphosphate aldolase class I (isoform 2) SMc03983 3 740
Bifunctional protein SMc04088 3 35
Hypothetical/global homology SMc04240 3 2,800
Signal peptide mannitol-binding periplasmic protein SMc04251 3 440 /PICK>
Conserved hypothetical protein SMa0241 3 720 50 300
Hypothetical protein SMa0947 1 330
Hypothetical protein SMa1169 1 New
Putative ABC transporter, periplasmic solute-binding protein SMa1438 2 510
Probable thiolase (isoform 1) SMa1450 3 360
Probable thiolase (isoform 2) SMa1450 3 820
Dihydrolipoamide succinyltransferase SMb20019 2 1,200
Transporter periplasmic binding protein SMb20325 3 450
Periplasmic solute-binding protein SMb20442 3 250
Putative rhizopine uptake ABC transporter SMb20712 3 660
Probable sugar uptake ABC transporter periplasmic SMb20895 2 190
d-β-Hydroxybutyrate dehydrogenase SMb21010 3 230
Putative glutaryl-CoA dehydrogenase SMb21181 3 1,300
Signal peptide hypothetical/partial homology SMb21221 3 300
Probable lactose uptake ABC transporter periplasmic solute-binding protein precursor SMb21652 3 New
Open in a new tab
a

Based on S. meliloti 1021 genomic database (http://sequence.toulouse.inra.fr/meliloti.html) (16).

b

Smc, Sma, and Smb refer to products of ORFs located on the chromosome and symbiotic plasmids A and B, respectively.

c

Confidence ratings were assigned as described in Materials and Methods.

d

Differential accumulation was determined as described in Materials and Methods, and is based on statistical analysis of at least three gels per treatment, and two cultures per treatment. Numbers represent average percentage differences in spot volumes between the AHL-treated and untreated cultures, and each value is significant at the P = 0.05 level. Underlining and boldface type represent up-regulation and down-regulation, respectively. “Missing” and “New” refer to polypeptides not detectable in the treated and untreated gels, respectively.

e

Purified AHL from peak A, Fig. 1C, identified as 3-oxo-C16:1-HL.

f

Purified AHL from peak B, Fig. 1D, identified as C14-HL.

DISCUSSION

This initial proteomic study of quorum sensing in S. meliloti was conducted in a wild-type background because the double (SinI, HtdS-like) AHL synthase knockout mutant was not yet available. There appear to be no reports of proteome-level responses to AHLs in wild-type bacteria. If quorum-sensing-regulated functions could be identified effectively in a wild-type background, many comparisons would be facilitated and the potentially invisible accumulation of second site mutations in AHL synthase mutants and downstream polar effects of mutant constructs in AHL synthase genes (3) could be avoided. The extensive and AHL-specific changes in protein levels seen in this study indicate that treating washed, early-log cultures of wild-type bacteria with purified AHLs can indeed be an effective approach to the analysis of quorum sensing regulation. This may encourage future studies of AHL-induced responses with wild-type bacteria in situ (e.g., in eukaryotic hosts or natural environments) where AHL synthase mutants cannot establish normal starting populations. Additional proteomic studies in an AHL synthase mutant background are now needed for comparison and in order to test uncertainties regarding second site mutations and potential construct problems.

Roughly 5% of the total polypeptides resolved on the gels were significantly affected by the addition of purified 3-oxo-C16:1-HL and C14-HL to the S. meliloti wild type. This percentage is comparable to the proportion of the AHL-responsive genes identified in an AHL synthase minus mutants of P. aeruginosa (40, 46, 47, 50). Our proteomic analysis in S. meliloti indicates that the two AHLs directly or indirectly affected the levels of proteins involved in carbon and nitrogen metabolism, bacterial energy cycles, protein processing, and nucleotide synthesis as well as in secondary metabolism (Table 1). Several of the proteins identified in Table 1 as AHL-responsive (e.g., nitrogen regulatory protein II, elongation factor G, GroEL5, and phosphoenoylpyruvate carboxykinase) are differentially regulated in bacteroids compared to free-living cells (32, 35). This invites further tests to determine whether AHL-mediated quorum sensing is involved in bacteroid formation or function.

Addition of AHLs to early-log-phase cultures resulted, either initially or after 8 h, in the diminished accumulation of approximately one-third of the AHL-responsive proteins (Table 1). It is not yet clear how this should be interpreted. Added AHLs have been reported to reduce the accumulation of polypeptides in two other bacteria (19, 45), so the phenomenon is not unique to S. meliloti. AHLs are normally activators of gene expression through interaction with and stabilization of their cognate receptors (4, 41, 56). The immediate consequence of AHL addition should thus be enhanced transcription and increased accumulation of the corresponding protein. However, the addition of AHLs was found to directly repress the transcription of certain genes in Erwinia carotovora (23) Brucella melitensis (42), and P. aeruginosa (40, 47). In addition, C8-HL in V. fischeri was found to antagonize transcriptional activation via LuxR by its other cognate AHL, 3-oxo-C6-HL, thus reducing the accumulation of certain polypeptides (4). Givskov et al. (19) observed that AHL addition diminished the accumulation of certain proteins in S. liquefaciens within just 5 min, which may also reflect reduced transcription. Thus, the addition of 3-oxo-C16:1-HL and C14-HL may have resulted in reduced transcription of genes corresponding to at least some of the proteins listed in Table 1 rather than reducing their levels via enhanced proteolysis or through other downstream regulatory effects. The repression of transcription and protein levels by AHLs may prove to be a biologically important yet under appreciated aspect of quorum sensing regulation.

The addition of 3-oxo-C16:1-HL and C14-HL to 1021 cells affected the accumulation of two quite distinct sets of proteins (Table 1), suggesting that responses to these AHLs are mediated by different receptors. The two sets of proteins help to define the functional quorum-sensing regulons for each signal. Future studies with synthetic 3-oxo-C16:1-HL and C14-HL and various AHL receptor mutants will be needed to confirm and extend the definition of these regulons. Addition of 3-oxo-C16:1-HL affected the levels of a larger number of polypeptides after 2 h (40) than did C14-HL (13), and the changes in level of accumulation were generally larger after 3-oxo-C16:1-HL addition than after C14-HL addition. These differences in response to the two AHLs are consistent with the possibility that 3-oxo-C16:1-HL may be dominant in a regulatory hierarchy over C14-HL, as seen in P. aeruginosa (49) and R. leguminosarum (25).

The map of polypeptides that differentially accumulated in late log phase cultures of strain 1021 (26) was quite similar to the 2D polypeptide map obtained during our comparison of early-log- versus early-stationary-phase polypeptides (data not shown). The 80 differentially accumulated proteins characteristic of stationary phase cultures shown in Table 2 were quite diverse in their functions, and included 14 transporters (all upregulated), five regulatory proteins (up- and down-regulated), and three elongation factors (all down-regulated). The gene corresponding to d-β-hydroxybutyrate dehydrogenase was reported to be upregulated in stationary phase cultures of S. meliloti (1), consistent with the differences we found in polypeptide accumulation.

It is not yet clear why 3-oxo-C16:1-HL and C14-HL affected the accumulation of only 13 of the 80 “stationary-phase” proteins (Table 2). It is certainly possible that more of the stationary-phase proteins would be affected by AHL addition if different AHLs or higher AHL concentrations or longer exposure times were used. Alternatively, it may be that early-log-phase cultures of S. meliloti are not yet ready to respond fully to these AHLs. Work elsewhere has shown that even though treatment with AHLs can stimulate various physiological changes characteristic of a stationary-phase culture (e.g., see references 10, 20, 44, and 52), additional regulators and stationary-phase sigma factors are required for full activation of certain AHL-regulated genes (5, 11, 14, 23, 51). Further studies are required to determine whether AHL responsiveness in S. meliloti is similarly conditioned by such global, growth phase dependent regulators. The S. meliloti 1021 genomic database (16) does not appear to contain any sequences similar to those of the regulators described in P. aeruginosa (MvaT, RsmA, RsaL, and RpoS).

Acknowledgments

We are grateful to Michael Djordjevic, Uli Mathesius, and Jeremy Weinman for their help in interpreting and analyzing the proteomic data. We are indebted to James Metzger and Brian Ahmer for allowing us access to their equipment and guiding us though its use. Synthetic AHLs were generously provided by Anatol Eberhard. We thank Matt Gronquist and A. Eberhard for mass spectral identification of AHLs.

Work presented in this paper was supported by USDA grant 2002-3531911559 to W.D.B., J.B.R., and B.R.; an Ohio Plant Biotechnology Consortium grant to W.D.B. and J.B.R.; Ohio State University (OSU) Office of International Education travel grants to W.D.B. and M.T.; and a Presidential Fellowship, an OARDC Research Enhancement grant, and an OSU Extension Sustainable Agriculture grant to M.T. Partial salary support was also provided to W.D.B. by the Ohio Agricultural Research and Development Center.

Footnotes

†

Contribution 02-22 from the Ohio Agricultural Research and Development Center.

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