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Journal of Bacteriology, December 2000, p. 6921-6926, Vol. 182, No. 24
Department of Microbiology, University of
Iowa, Iowa City, Iowa 52242
Received 19 May 2000/Accepted 27 September 2000
Acyl-homoserine lactones (acyl-HSLs) serve as dedicated
cell-to-cell signaling molecules in many species of the class
Proteobacteria. We have addressed the question of whether
these compounds can be degraded biologically. A motile, rod-shaped
bacterium was isolated from soil based upon its ability to utilize
N-(3-oxohexanoyl)-L-homoserine lactone as the sole source of energy and nitrogen. The bacterium was
classified as a strain of Variovorax paradoxus. The
V. paradoxus isolate was capable of growth on all of the
acyl-HSLs tested. The molar growth yields correlated with the length of
the acyl group. HSL, a product of acyl-HSL metabolism, was used as a
nitrogen source, but not as an energy source. Cleavage and partial
mineralization of the HSL ring were demonstrated by using radiolabeled
substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy
and nitrogen sources. This study provides clues about the metabolic
pathway of acyl-HSL degradation by V. paradoxus.
A diversity of bacterial species
self-regulate expression of specific sets of genes in response to their
own population density, a phenomenon that has become known as
quorum sensing (for reviews, see references 9, 27, and
33). Many members of the class Proteobacteria have
quorum-sensing systems that rely on acyl-homoserine lactone (acyl-HSL)
signals. The nature of the acyl side chain of the signal molecule
depends upon the quorum-sensing system. A diversity of acyl-HSL
structures have been elucidated. Signal specificity depends on
the length of and the substitutions in the acyl side chain (Fig.
1). Acyl-HSLs are dedicated signaling molecules with no other known function, and a specific enzyme is
required for their synthesis (13, 20, 22, 30). These signal
molecules reach concentrations on the order of 10 µM in laboratory
cultures of quorum-sensing bacteria (5, 24, 26).
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Metabolism of Acyl-Homoserine Lactone
Quorum-Sensing Signals by Variovorax paradoxus
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (7K):
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FIG. 1.
Generalized structure of acyl-HSLs produced by
quorum-sensing bacteria: R1, -H, -OH, or ==O;
R2, -CH3,
-(CH2)2-10CH3 or
-(CH2)3CH==CH(CH2)5CH3.
The available evidence is consistent with the idea that bacteria which synthesize acyl-HSLs do not degrade them, and acyl-HSLs are chemically stable at neutral or acidic pH in aqueous solutions (29). However, the HSL ring is subject to alkaline hydrolysis (32). The potential for biological decomposition of these signals is intriguing for several reasons. Other bacteria sharing the same local environment as quorum-sensing bacteria could conceivably gain a competitive advantage by degrading acyl-HSL signals. Enzymes that degrade acyl-HSLs might have commercial value as modulators of cell-to-cell signaling. Since acyl-HSLs are stable under slightly acidic conditions, biological degradation could play an important role in maintaining these signals at low environmental concentrations.
A recent report shows that acyl-HSL signaling molecules can be biologically inactivated by specific soil bacteria (4). A gene encoding this degradative ability was cloned from a Bacillus isolate. The purified gene product showed acyl-HSL-inactivating ability. It was not clear how the gene product served to inactivate acyl-HSLs or whether the Bacillus could use acyl-HSLs as nutrients for growth. To initiate our investigations into the biological degradation of acyl-HSL molecules, we have used enrichment and isolation techniques to obtain a pure culture of a bacterium capable of utilizing these signals as the sole source of energy and nitrogen. This is our initial description of that bacterium and its acyl-HSL-degrading capabilities.
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MATERIALS AND METHODS |
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Materials and Methods
Results
Discussion
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Bacterial strains, media, and culture conditions. The bacterial strains used were Variovorax paradoxus VAI-C (isolation described below), V. paradoxus ATCC 17713, and, for biological production of radioactive N-butanoyl-HSL (C4-HSL), Escherichia coli XL1-Blue containing pRHL1 (21). For growth of E. coli, we used Luria-Bertani (28) broth or agar amended as indicated. For enrichment, isolation, and growth experiments with V. paradoxus, we used a defined medium. The composition of the medium (per liter) was 1 g of NaCl, 0.5 g of KCl, 0.4 g of MgCl2 · 6H2O, 0.3 g of NH4Cl (unless otherwise specified), 0.1 g of CaCl2 · 2H2O, 0.2 g of KH2PO4, 0.15 g of Na2SO2, and 1 g of 2-(N-morpholino)-ethanesulfonic acid (MES). Trace elements and a selenate-tungstate mixture (18) were added, and the pH was adjusted to 5.5 with 1 M NaOH. This basal medium was autoclaved, and, after cooling, vitamins were added unless otherwise noted. The final vitamin composition per liter of medium was 100 µg of riboflavin and 1 mg of L-ascorbic acid, biotin, DL-calcium-panthothenate, folic acid, niacinamide, nicotinic acid, p-aminobenzoic acid, pyridoxal-HCl, thiamine-HCl, lipoic acid, and cyanocobalamin. For cultivation on solid media, agarose (Gibco Ultrapure) was incorporated at a final concentration of 1.0%. Growth substrates were added to the autoclaved, vitamin-amended medium as indicated.
For routine maintenance of V. paradoxus we used the medium described above supplemented with 5 g (wt/vol) of Difco yeast extract · liter
1 as an energy and nitrogen source.
Stock solutions of acyl-HSLs were used at 5 mg · ml1 (except for
N-3-oxohexanoyl-L-HSL [3OC6-HSL], which was
used at 50 mg · ml1) in ethyl acetate acidified
with glacial acetic acid (0.1% [vol/vol]). The stock solutions were
stored at 
20°C. For liquid media, the solutions of acyl-HSLs were
dispensed into sterile tubes, the ethyl acetate was removed by
evaporation under a stream of nitrogen gas, and sterile medium was
added to the remaining acyl-HSL. The acyl-HSL-containing media were
used immediately after preparation. Acyl-HSLs with carbon chain lengths
of >8 did not fully dissolve in the medium at concentrations of >100
µM. Cells were grown in 3 or 5 ml of medium in 13- or 18-mm-diameter
tubes, respectively, with shaking at 30°C unless otherwise noted. For
agarose plates, the ethyl acetate solutions were spread on the surface
of the agarose medium, and the plates were used shortly after the ethyl acetate evaporated. Acyl-HSL molecules are stable for weeks under the
conditions of low pH in our defined medium. At higher pH values (ca.
8.0), the half-life of an acyl-HSL can be <3 h (29; A. Eberhard, personal communication).
Enrichment and isolation procedures.
Turf soil was collected
in September 1998 at the University of Iowa. The soil was disrupted
with a metal spatula until all particles were finely dispersed, and the
remaining large particles were removed. One hundred milligrams of the
soil preparation was added to 3 ml of the basal medium containing
3OC6-HSL as the sole source of nitrogen and energy (500 µg · ml1). Vitamins were not added to the enrichment medium.
After 48 h, a 5% (vol/vol) transfer was made to fresh enrichment
medium, and after an additional 48 h, a second transfer was made.
After a further 48-h incubation, cells in the third transfer tube were streaked on a plate of 3OC6-HSL-containing agarose medium.
Growth studies. We constructed a 3OC6-HSL consumption curve by analyzing duplicate 10-µl samples of culture fluid collected during growth. Duplicate 1-ml samples were collected after the culture had entered the stationary phase. The analysis was by means of a 3OC6-HSL bioassay as described elsewhere (23).
Molar growth yields with acyl-HSLs as energy sources were determined in NH4Cl-replete medium containing the indicated acyl-HSL at a final concentration of 0.5, 0.75, 1.0, or 1.5 mM. Growth yields with nitrogen sources other than NH4Cl were determined in a medium containing 20 mM sodium succinate as the energy source. The nitrogen sources used in place of NH4Cl were HSL (at concentrations of 0 to 10 mM), homoserine (at concentrations of 0 to 10 mM), or 3OC6-HSL (at concentrations of 0 to 1 mM). A factor for converting optical density to cell dry mass was constructed by using cells grown in a medium containing succinate as the energy source and NH4Cl as the nitrogen source, washed with 50 mM ammonium acetate buffer (pH 5.5), and then dried to a constant weight. Experiments were done at least twice.Metabolism of radiolabeled C4-HSL.
We prepared
C4-L-[1-14C]HSL for radiotracer experiments
by modification of a previously described procedure (12).
E. coli XL1-Blue cells containing the C4-HSL synthase
expression vector pRHLI were grown in 50 ml of Luria-Bertani broth
containing ampicillin (100 µg · ml1).
Isopropyl-
-thiogalactoside (1 mM) was added after 2 h at
37°C. Cells were harvested by centrifugation when the culture reached an optical density of 0.7 at 600 nm. The cells were suspended in 2 ml
of phosphate-buffered saline (28) containing 10 mM glucose in a 15-ml conical tube. After 10 min at 37°C with shaking, we added
10 µCi of L-[1-14C]methionine (55 mCi
· mmol1; American Radiolabeled Chemicals, Inc., St.
Louis, Mo.) and incubated the cell suspension for an additional 4 h. The cells were then removed by centrifugation, and the C4-HSL was
extracted from the remaining culture fluid with 2 equal volumes of
acidified ethyl acetate. The ethyl acetate evaporated, and the residue
was dissolved in 200 µl of 20% methanol in water. The C4-HSL in the
methanol-water was purified by reversed-phase high-performance liquid
chromatography (29). The purified, radioactive C4-HSL was
dried and stored at 20°C. Radioactivity was measured with a liquid
scintillation counter and was quench corrected by using an internal standard.
1)
was added to each reaction vessel (as described above). Immediately after inoculation, the amount of radioactivity in the culture broth was
measured. After cultures had reached the stationary phase, the
headspace above each culture was flushed with N2 for 60 min
into two CO2 traps connected in tandem (1). The
radioactivity in the second vial ranged from 0.1 to 1.0% of that
trapped in the first. Culture fluid was then removed, and after
centrifugation, the clarified culture fluid was extracted with 8 equal
volumes of acidified ethyl acetate. Radioactivity in both the extract and the culture fluid after extraction was measured. The cell pellet
was washed three times with 10 mM MES (pH 5.5). The radioactivity present in the washed cells was determined after boiling for 2 min in
100 µl of 1 M NaOH.
Nucleotide sequence analysis of the 16S rDNA. The nucleotide sequence of a PCR-amplified fragment of the 16S ribosomal DNA (rDNA) of our bacterial isolate was determined by previously described procedures (18). Genomic DNA was isolated with a QIAamp tissue kit (QIAGEN, Inc., Valencia, Calif.). We used the Expand Long Template PCR system (Boehringer-Mannheim) to amplify 16S rDNA with about 50 ng of bacterial DNA as the template with previously described 27-forward and 1494-reverse primers (17). The PCR product was purified and ligated into pCRII by using a TOPO TA cloning kit (Invitrogen, San Diego, Calif.). DNA sequencing was by routine automated methods at the University of Iowa DNA Core Facility. The sequencing primers were the standard M13 forward and M13 reverse primers and primers previously designed to target internal regions of the 16S rRNA genes of most bacteria (17). For sequence analysis, we used ARB software (www.mikro.biologie.tu-muenchen.de/pub/ARB/linux/).
Other analyses. Phase-contrast microscopy and epifluorescence microscopy were performed with an Olympus BH2 microscope. Acridine orange staining and confocal-epifluorescence microscopy were performed as described elsewhere (2, 6). To demonstrate that HSL was released as a product of acyl-HSL degradation during culture growth, growth supernatants were analyzed at the University of Iowa College of Medicine Molecular Analysis Facility with a Beckman 6300 high-performance ion-exchange analyzer operated according to the manufacturer's specifications. The bioassays used to screen for production of 3OC6-HSL, C4-HSL, and related molecules by V. paradoxus have been described previously (23, 25).
Nucleotide sequence accession number. The 16S rDNA sequence has been assigned GenBank accession no. AF250030. All other rDNA sequences were from the ARB database or from GenBank.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Enrichment and isolation of acyl-HSL-degrading bacteria. Enrichment was in vitamin-free basal medium containing 3OC6-HSL. Enrichment tubes were inoculated with soil, and growth was evident as turbidity within 48 h. No obvious turbidity was observed in the absence of 3OC6-HSL. After two transfers in the 3OC6-HSL-containing medium, a complex microbial community including a variety of bacterial and eukaryotic microbes was observed by phase-contrast microscopy. There was an obvious biofilm near the air-liquid interface. The biofilm was disrupted by vortexing the culture for 30 s, and then a sample of the culture was streaked on a plate of vitamin-free agarose medium containing 3OC6-HSL. Three colony types arose over a period of a week. Pure cultures of each colony type were obtained by repeated streaking. The three isolates obtained were designated VAI-A (a dimorphic rod-coccus), VAI-B (a highly motile spirillum), and VAI-C (a weakly motile rod). These isolates were screened for growth on 3OC6-HSL in the liquid medium used for the enrichments. The pure VAI-A and VAI-C cultures grew on 3OC6-HSL, and VAI-C was chosen for further study.
Phylogenetic analysis of VAI-C.
A nearly complete sequence for
the 16S rDNA was obtained. The sequence corresponds to E. coli 16S rRNA nucleotide positions 28 to 1489. Web-based
similarity searches against the GenBank and Ribosomal Database
Project databases suggested that VAI-C belonged to the subclass
-Proteobacteria, clustering with the family
Comamonadaceae. The 16S rDNA of VAI-C shared 99.3 to
99.8% sequence identity with the 16S rDNA of three strains of
V. (formerly Alcaligenes)
paradoxus. A further phylogenetic analysis supported the
conclusion that VAI-C is a strain of V. paradoxus. By
any of the FastDNAML maximum-likelihood, maximum-parsimony, and Desoete distance treeing algorithms, VAI-C clustered tightly with the other
V. paradoxus strains (Fig.
2). Thus, we consider our isolate to be a
strain of V. paradoxus. An additional confirmation of this assignment was obtained by demonstrating that the type strain of
V. paradoxus (ATCC 17713) was capable of growth in
3OC6-HSL broth. Growth of this strain was not as rapid as growth of
VAI-C in the test medium, irrespective of the substrate tested (data not shown).
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General properties of V. paradoxus VAI-C.
Growth of V. paradoxus VAI-C on 3OC6-HSL occurred
at 30°C, but not at 37°C. The isolate formed spreading, yellow
colonies on plates of 3OC6-HSL defined agarose medium and raised,
yellow colonies with an occasional spreading edge on yeast extract
agarose plates (Fig. 3A). Cells from
late-logarithmic-phase 3OC6-HSL broth cultures were 1.1 by 0.7 µm in
dimension (Fig. 3C). Despite the spreading of colonies on agarose
plates, cells in broth were sporadically motile. A putrid aroma was
produced during growth, especially on yeast extract agarose plates. As
is the case for other strains of V. paradoxus, our
isolate was capable of growth with pantothenic acid or folic acid as
the sole source of energy and nitrogen (10, 11, 19).
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Acyl-HSLs as energy sources for growth.
Pure cultures of
V. paradoxus VAI-C grew slowly (24- to 48-h doubling
times) on 3OC6-HSL in the absence of vitamins. In vitamin-supplemented medium, the doubling time improved to 18 h, with a molar growth yield of 94 g (dry weight) of cell · mol of
3OC6-HSL1. With NH4Cl added to the medium,
the doubling time improved to 3.5 h (Fig.
4); the molar growth yield with
NH4Cl was about the same as without it (as was the final pH
in the culture medium [5.7 to 6.4]). The concentration of 3OC6-HSL
decreased during logarithmic growth, and it was below 100 nM just after
the onset of the stationary phase (Fig. 4).
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) and without (
) 3OC6-HSL is shown, as is the concentration of
3OC6-HSL in the 3OC6-HSL-containing culture (
).
8 carbons did not completely dissolve in
the medium at the concentrations we used (>100 µM), so accurate
growth rates on these substrates were not obtained. However, upon
incubation, the insoluble acyl-HSL particles disappeared, and final
culture densities could be measured. Epifluorescence microscopy
of acridine orange-stained cells during early growth on C12-HSL
showed acyl-HSL particles covered with V. paradoxus
cells (Fig. 3B). Few cells were found unattached in the growth medium
until the visible C12-HSL particles had disappeared.
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-butyrolactone, DL-homocysteine thiolactone, or
N-acetyl-L-homocysteine thiolactone. Growth on
acyl-HSLs was comparable to that with many other energy sources in
terms of rate and yield on a per carbon basis.
The molar growth yield studies (Fig. 5) indicate that the HSL ring
moiety of an acyl-HSL does not serve as an energy source. Nevertheless,
the ring may be degraded. To gain insights into the fate of the HSL
moiety, we grew cultures with C4-HSL radiolabeled in ring carbon 1. With C4-HSL as the sole source of energy and NH4Cl as a
source of nitrogen, nearly half of the radiolabel was recovered as
14CO2. The production of
14CO2 demonstrates the cleavage of a
significant fraction of the homoserine lactone ring. Of the radioactive
label that was not recovered as 14CO2, most was
accounted for in the culture fluid, even after ethyl acetate
extraction. We suspected that this might be HSL. To test this
hypothesis, we grew a culture on unlabeled C4-HSL. HSL was the only
compound detected by quantitative amino acid analysis (Fig.
6). When taken together with the
radiolabling experiments, this indicates that about 25% of the HSL in
the C4-HSL was recovered as HSL in the culture fluid. These experiments
indicate that HSL is an intermediate in the degradation of C4-HSL.
Because HSL accumulates in the culture medium, we suggest that
acyl-peptide bond cleavage occurs outside of cells or that there is an
export system for intracellular HSL.
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Acyl-HSLs as nitrogen sources for growth.
V.
paradoxus VAI-C was capable of growth with acyl-HSLs as the
sole source of organic carbon, energy, and combined nitrogen. The
acyl group serves as an energy source, and HSL, which is a product of
acyl-HSL metabolism, can serve as a sole source of combined nitrogen
for growth. Furthermore, the HSL ring is metabolized even though it
does not appear to be utilized as an energy source (Table
2). Thus, we wanted to study growth of
the isolate with succinate as an energy source and acyl-HSLs as the
only source of nitrogen. Both C4-HSL and 3OC6-HSL served as a nitrogen
source. Homoserine and HSL also served as nitrogen sources, but only
the acyl-HSLs and homoserine would serve as the sole energy and
nitrogen substrate for growth. The doubling times with homoserine, HSL, or an acyl-HSL as the nitrogen source were slow compared to doubling times with NH4Cl (about 30 h compared to 4 h).
However, final cell yields with excess succinate were
comparable to the cell yield with NH4Cl (data
not shown). When 14C-labeled C4-HSL was used as a
nitrogen source, nearly all of the radioactivity was recovered as
CO2 (Table 2). We do not know whether CO2
release is an early or late step in the ring cleavage process, but we
conclude that ring cleavage and CO2 release occur prior to
utilization of the nitrogen. Whether any HSL carbon, or only its amino
group, is incorporated into cellular proteins remains unanswered.
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DISCUSSION |
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Introduction
Materials and Methods
Results
Discussion
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We have isolated a bacterium capable of degrading and growing on a
number of different acyl-HSLs. This organism appears to be a strain of
V. paradoxus, and it was able to utilize acyl-HSLs as
both energy and nitrogen sources. Apparently only the acyl group is
used in energy generation (Table 1 and Fig. 5). In fact, HSL is
produced by cultures growing on C4-HSL (Fig. 6). Thus, we believe that
the first step in the metabolism of acyl-HSLs involves a
carboxypeptidase or aminoacylase that releases the fatty acid from HSL
(Fig. 7). By using
C4-L-[1-14C]HSL, we showed that V. paradoxus cleaves the HSL ring (Table 2). Because the other
carbons in the C4-HSL were not labeled, we do not know their metabolic
fate.
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Although further studies are required to elucidate the pathway of
acyl-HSL metabolism, we can speculate that after hydrolysis of the
acyl-amide bond, the next step in the degradative pathway might
involve the action of a novel 
, -eliminating deaminase-lyase yielding -ketobutyrate and NH4+ (Fig. 7).
Alternatively, a lactone hydrolase (14, 16) could convert
HSL to homoserine. However, the latter hypothesis seems inconsistent
with the observation that V. paradoxus VAI-C utilized homoserine, but not HSL (added exogenously or derived from acyl-HSLs), as an energy source.
The acyl-HSL-degrading activity of V. paradoxus VAI-C expands the list of diverse metabolic traits exhibited by members of this bacterial species. Many strains of the species can grow via H2 + CO2 + O2 chemolithoautotrophy (3). Other capabilities of strains of V. paradoxus include degradation of bioplastics (31), involvement in the dechlorination of xenobiotic compounds (7), and the ability to accumulate the rare metal yttrium (15). Of particular interest, strain VAI-C shares with other Variovorax isolates the ability to grow on acyl-amide bond-containing vitamins such as pantothenate (pantoyl-N-3-alanine) and folate (pteroyl-N-glutamate). The degradation of these vitamins requires two different amino acid-specific carboxypeptidases (10, 11). We believe it is likely that V. paradoxus contains an additional acyl-HSL-specific carboxypeptidase.
Acyl-HSLs have received considerable attention as quorum-sensing signals and key regulators of the community behavior of a number of genera of Proteobacteria (9, 27, 33). Many acyl-HSLs are quite stable in mildly acidic or neutral pH environments. However, there is no evidence that they accumulate in such environments. If they accumulated over long periods of time, their function as quorum-sensing signals would be disarmed. The signal concentration would not reflect cell number after fluctuations in population density. An intriguing question arises from the identification of bacteria with acyl-HSL-degrading capabilities. Can acyl-HSL-degrading bacteria influence the gene expression of quorum-sensing bacteria when both groups intermingle with or reside in close proximity to each other? This report, along with the report by Dong et al. (4) on a Bacillus isolate exhibiting acyl-HSL-inactivating activity, should open up investigations of the metabolic pathway for acyl-HSL degradation, the genetics of acyl-HSL degradation, and the ecological significance of acyl-HSL degradation. As pointed out by Dong et al.(4), the application of acyl-HSL degradation could have value in control of specific plant and animal diseases that are caused by bacteria that employ these quorum-sensing signals to control virulence or biofilm formation. Conversely, there has been interest in exploiting beneficial quorum-regulated activities such as the inhibition of pathogenic fungi around the roots of plants (8, 34). It may be useful to consider biological signal degradation as one factor decreasing the potential effectiveness of acyl-HSL-mediated biocontrol regimes.
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ACKNOWLEDGMENTS |
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This research was supported by grants from the National Institutes of Health (GM59026), the National Science Foundation (MCB 9808308), and the Cystic Fibrosis Foundation. J.R.L. was supported in part by a traineeship from the National Institutes of Health (T32-AI07343) and in part by a National Science Foundation Postdoctoral Fellowship in the Biosciences Related to the Environment (DBI-9804278).
We thank M. Parsek and A. Schaefer for many intellectual and technical discussions and E. Rus for performing quantitative amino acid chromatography.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7990. Fax: (319) 335-7949. E-mail: epgreen{at}blue.weeg.uiowa.edu.
Present address: California Institute of Technology, Pasadena, CA 91125.
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Discussion
References
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