Journal of Bacteriology, December 1998, p. 6384-6388, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
N-Acyl-L-Homoserine Lactone
Autoinducers Control Production of an Extracellular Lipopeptide
Biosurfactant Required for Swarming Motility of Serratia
liquefaciens MG1
P. W.
Lindum,1
U.
Anthoni,2
C.
Christophersen,2
L.
Eberl,3
S.
Molin,1 and
M.
Givskov1,*
Department of Microbiology, Technical
University of Denmark, 2800 Lyngby,1 and
Marine Chemistry Section, H. C. Ørsted Institute,
University of Copenhagen, 2100 Copenhagen,2
Denmark, and
Lehrstuhl für Mikrobiologie, Technische
Universität München, D-80290 München,
Germany3
Received 15 May 1998/Accepted 23 September 1998
 |
ABSTRACT |
A nonswarming Serratia liquefaciens mutant deficient in
serrawettin W2 production was constructed by transposon mutagenesis. Sequence homology indicated that insertion had occurred in gene swrA, which encodes a putative peptide synthetase.
Expression of swrA is controlled by quorum sensing.
| |
TEXT |
Several bacterial species are able
to form expanding colonies on the surface of semisolid growth media by
means of swarming motility (1, 7, 8, 24). Serratia
liquefaciens MG1 swarms on the surface of AB minimal medium
(3) solidified with 0.5 to 1.0% agar supplemented with
0.5% Casamino Acids and 0.5% glucose (6, 7). Development
of a swarming colony requires the sensing and integration of a variety
of environmental, as well as intracellular, signals involving surface
contact and local high population density. According to our current
working hypothesis for the development of a swarming colony (11,
12), exposure of the cells to surfaces with a certain viscosity
is recognized by an unknown sensor, and signal transduction then
progresses via the flhDC master operon. Stimulation of this
operon initiates swarm cell differentiation that involves
development of characteristic traits such as cell elongation,
multinucleation, and hyperflagellation (6). The population density is recognized by a homoserine lactone-dependent quorum-sensing system constituted by the swrI and
swrR genes (7, 11, 12). On defined growth medium,
expansion of the swarm colony is strictly dependent on a functional
swrI gene, a member of the luxI family of
autoinducer synthetase genes (7). The swrI gene
product catalyzes the formation of
N-butanoyl-L-homoserine lactone (BHL) and
N-hexanoyl-L-homoserine lactone in a molar ratio of 10:1 (7). The flagellar master and the quorum-sensing
system are global regulators which control two separated regulons
(12). We are now in the process of identifying target genes
of the quorum-sensing system, as well as macromolecules and compounds
that are synthesized to facilitate swarming motility.
MG1 produces an extracellular biosurfactant.
The ability to
synthesize compounds with surface tension-reducing properties is
widespread among bacteria, and the significance of surface-active
compounds or surfactants in interaction with interfaces has been
reviewed (27). The application of a drop-collapsing test has
been used to identify bacteria that produce surfactants (14). A 10-µl drop of an outgrown bacterial culture or of
a suspended bacterial colony is placed on a hydrophobic surface such as
the lid of a petri dish. The drop either remains globular or collapses
rapidly while increasing the wet area on the surface. The latter
situation indicates the presence of a surfactant capable of lowering
the surface tension of the water (Fig.
1). This simple test indicates that
S. liquefaciens MG1 produces a biosurfactant. The
swrI mutant MG44, which is defective in BHL and
N-hexanoyl-L-homoserine lactone synthesis
(7), is impaired in surfactant production (Fig. 1). However,
addition of exogenous BHL to the growth medium restores surfactant
production (Fig. 1). This strongly suggests that the quorum-sensing
mechanism controls the production of a biosurfactant. The presence of
an inactivating mutation at the flhD locus does not affect
surfactant production (Fig. 1). After removal of the cells from the
cell suspensions depicted in Fig. 1, the drop-collapsing test result
was reproduced with the cell-free supernatants (data not shown). This
indicated that the surface tension-reducing activity is caused by the
presence of an extracellular compound(s).
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FIG. 1.
Drop-collapsing test. Ten-microliter volumes of the
following bacterial cultures were placed on the lid of a petri dish.
(A) Side view. (B) Top view. 0, 0.9% NaCl; wt, S. liquefaciens MG1; swrI, S. liquefaciens
MG44; swrA, S. liquefaciens PL10;
flhD, S. liquefaciens MG3; +BHL, strain grown in
the presence of 200 nM BHL.
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Quorum sensing regulates expression of a gene necessary for
surfactant production.
The swrI mutant MG44 was
randomly mutagenized with a Tn5-derived transposon carrying
a promoterless luxAB reporter as described by Kristensen et
al. (18). The resulting double mutants were screened for
BHL-induced bioluminescence on agar plates supplemented with 0.2 µM
BHL. This gave rise to the isolation of 19 mutants in which expression
of the luxAB reporter was BHL dependent. One mutant,
designated PL10, was unable to swarm in the presence of externally
added BHL. The drop-collapsing test indicated that the PL10 mutant is
completely defective in surfactant production, even in the presence of
BHL (Fig. 1). From this we concluded that the luxAB
transposon had most likely been integrated into a gene, designated
swrA, which is essential for surfactant production. A
Southern blot analysis targeting luxAB in PL10 and the
parent strain MG44 was performed. Chromosomal DNA was digested with
five different restriction enzymes that had no recognition sites in the
luxAB sequence. The Southern blot confirmed the presence of a single transposon insert in PL10 and no insertion in MG44 (data not shown).
PL10 cells were grown as colonies, washed off, and suspended in fresh
growth medium. When expression of bioluminescence per unit of optical
density at 450 nm was determined, cells that previously had been grown
in the presence of 0.2, 2.0, and 20 µM BHL showed 50-fold higher
light emission than cells from a colony grown in the absence of BHL
(data not shown). We also monitored a liquid culture of PL10 and
determined the expression of bioluminescence per unit of optical
density at 450 nm throughout the growth cycle in the absence or
presence of 0.2, 2.0, and 20 µM BHL. The presence of BHL in the
medium caused a maximum fivefold stimulation of bioluminescence
(data not shown).
The product of the gene swrA is a putative peptide
synthetase.
Chromosomal DNA of PL10 was prepared as described by
Givskov et al. (10), restricted with either PmlI
or BamHI, and ligated into the SmaI or
BamHI site of pUC18Not (32),
respectively. Ligation mixtures were electroporated into
Escherichia coli MT102 [araD139 (ara-leu)7697 
lac
thi hsdR]). Two hybrid plasmids, pPL1051, carrying 3.5 kbp of
PL10 DNA upstream of the transposon insert, and pPL1052, carrying 3 kbp downstream of the transposon insert, were isolated (Fig.
2). From plasmid 1051, approximately 1.5 kb was sequenced in one direction upward from
the transposon. This DNA sequence exhibited no homology to known
genes. The insert of pPL1052 was completely sequenced in both
directions. A BLASTX homology search for the DNA sequence was done via
the worldwide web at the BCM Nucleic Acid Sequence Searches of the
Human Genome Center, Baylor College of Medicine
(http://dot.imgen.bcm.tmc:9331/seq-search/nucleic_acid-search.html). Results of the BLASTX homology search (Fig. 2) indicate that
swrA encodes a peptide synthetase with high homology to,
e.g., the surfactin synthetase SrfA of Bacillus subtilis
(5) and the tyrocidine synthetase TycABC of B. brevis (25), both of which are members of a superfamily
of peptide synthetases all encoded by large operons (31).
Several characteristic conserved amino acid motifs or core sequences of
this superfamily have been defined (13) within the larger
conserved modular domains. Two such motifs with homology to the
consensus sequences LKAGGA and HHILDGV are found in the predicted SwrA
open reading frame (ORF) (Fig. 2). The stretch of weak homology in the
putative SwrA (Fig. 2, open region) separating the areas of high
homology (Fig. 2, hatched and squared regions) seems to be larger than
in most petide synthetases. This region might house a modification
module, e.g., an epimerase, but this is purely speculative. The
biosynthesis of lipodepsipeptides is often accomplished by
nonribosomal, multifunctional peptide synthetases (16, 17,
19). The surfactin biosynthesis in B. subtilis is one
of the best characterized of these systems. Surfactin is a small,
cyclic peptide consisting of seven amino acids and a
3-hydroxy-13-methyltetradecanoic fatty acid side chain (2)
which is synthesized by the Srf enzyme complex. The Srf complex
contains seven highly homologous amino acid binding domains present in
four subunits, Srf1-4, encoded by four ORFs. These domains determine
the seven specific amino acids and their order in the final surfactin
molecule; thus, the synthetase acts as a protein template during
peptide formation. Also, the complex contains two thioesterase active
site-like sequences, and finally, the third and sixth amino acid
binding domains are followed immediately by modifying domains which
epimerize the bound amino acid to the D form present in
surfactin (5).
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FIG. 2.
DNA region of S. liquefaciens PL10 (including
size markers) containing the transposon insert. The extent of cloned
and sequenced DNA on plasmids pPL1051 and pPL1052 is indicated by
arrows. The predicted SwrA ORF of 911 amino acids (aa) is shown as a
thick arrow. Homology between the putative SwrA and the SrfA surfactin
synthetase subunits Srf1 and Srf2 of B. subtilis
(7) is as follows: hatched regions of 450 aa are 28%
identical and 42% similar and squared regions of 75 aa are 54%
identical and 70% similar. The positions of motifs characteristic of
peptide synthetases are indicated by black symbols and given by amino
acid number. The motif sequences, as well as the specific amino acids
(Glu, Leu, etc.) that the corresponding domains bind are shown in the
box. Race denotes a domain which catalyzes the L- to
D-form epimerization of the adjacent bound Leu.
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Isolation and identification of the biosurfactant serrawettin
W2.
Growth conditions were found to affect surfactant production.
When cultured in liquid, MG1 gave rise to only small amounts compared
to the large quantities produced when the strain was cultured on an
agar surface (data not shown), an observation consistent with the
observed light emission pattern of PL10. The combined contents of 15 petri dishes that had been completely colonized by swarming colonies of
S. liquefaciens MG1 over 48 h were extracted three
times with equal amounts of 1% formic acid in ethyl acetate for
24 h. The combined extracts were evaporated to dryness, suspended in water, lyophilized, extracted with methanol, and then filtered, and
the filtrate was evaporated to dryness leaving a total of 358 mg of
crude extract. A 300-mg sample of the extract was subjected to
high-performance liquid chromatography (HPLC) separation on an RP18C
column (Merck) isocratically with acetonitrile-water (80:20) containing
0.05% trifluoroacetic acid as the eluent and monitored at 215 nm. The
chromatogram showed five fractions, the major one (56 mg) being pure
serrawettin W2. Serrawettin W2 is a colorless solid. Fast atom
bombardment (FAB)-mass spectrometry (MS): m/z 732.5 (M+H)+, 754.5 (M+Na)+, 1,463.9 (2M+H)+, 467.2 (M+H-C10H17COLeu)+,
266.3 (C10H17COLeu+H)+. In glycerol
matrix: m/z 732.3 (M+H)+. Electron impact MS
with direct inlet at 202°C: m/z (% relative intensity)
732 (M+, 8), 714 (9), 696 (100), 688 (25);
compatible with the composition C28H61N5O9. FAB-MS data
were determined in an m-nitrobenzylic alcohol-glycerol-thioglycerol 1:1:2 matrix containing 1%
trifluoroacetic acid. The FAB-MS exhibited a cluster ion at
m/z 1,463.9 representing the (2M+H)+
ion. Fragmentation of an acylated linear peptide formed by ring opening between the protonated C-terminal L-Ile and the
-hydroxydecanoyl moiety form the protonated linear
N-decenoyl peptide ion which, in turn, loses the
N-decenoylleucine moiety, forming m/z 467. The
N-decenoylleucine pseudomolecular ion appears at
m/z 266. This finding is analogous to the recorded
fragmentation of massetolides (9).
Optical rotation and UV spectra were determined as
[
]D20 
10.3° (c 0.0975, ethanol
[EtOH]). UV (EtOH) 
max nm (log 
): 251 (2.51), 258 (2.51), 264 (2.44), 268sh (2.37). The circular dichroism (CD) spectra
measured were CD (1.33 × 10
3 mol/liter, EtOH) (): 270 (0.15), 263 (0.10), 255 (0.10), 246 (0.15), 223 (3.45).
Nuclear magnetic resonance (NMR) spectra were recorded at 400.0 and
100.6 MHz for 1H- and 13C-NMR, respectively, on
samples in dimethyl sulfoxide-d6. The 1H- and 13C-NMR data are compatible with this
structure (Table 1). The partial
assignments presented in Table 1 are supported by pulse gradient,
multiquantum-filtered, phase-sensitive correlation spectroscopy and
two-dimensional total correlation spectroscopy experiments, establishing the scalar couplings between neighboring protons. The
carbon resonance assignments resulted from pulse gradient, reverse-detected heteronuclear multiple quantum coherence (HMQC) experiments optimized for a JC,H of 140 Hz and
the connectivities from pulse gradient, reverse-detected heteronuclear
multiple bond coherence (HMBC) experiments optimized for a
JC,H of 7 Hz.
The sequence of the amino acid units and the points of attachment of
the hydroxyacyl group were revealed by a rotating frame nuclear
Overhauser and exchange spectroscopy (ROESY) experiment using a mixing
time of 300 ms. Cross peaks were present between Leu-NH to the protons of the decanoyl moiety and to Ser-NH, thus placing the Leu
moiety at the acylated N-terminal position followed by Ser. The Ser protons are close to Thr-NH which, in turn, magnetically relaxes with
Phe-NH. The C terminus is occupied by Ile, since Ile-NH interacts with
the Phe proton. The same experiment served to verify the
assignments of the proton resonances of the various constituting units.
We conclude that the surface-active compound is identical to
serrawettin W2, a compound that was originally isolated from S. marcescens NS 25 (21) with the chemical structure
proposed in Fig. 3. Based on automated
amino acid analysis, MS, and NMR (1H and nuclear Overhauser
enhancement spectroscopy) data on a sample containing at least 20%
impurity, the structure of W2 was proposed by Matsuyama and coworkers
(21). The sterochemistry of the amino acid units was
determined, as well as the absolute configuration of the
3-D-hydroxydecanoic moiety (23). The
spectroscopic investigations described here confirm the proposed
structure.
An inherent complication of the identification of this type of
structure is the lack of a single physical parameter allowing its
identity to be established unambiguously. A realistic method must
present a unique fingerprint in order to be generally reliable. The
13C-NMR spectrum, recorded under carefully controlled
conditions, seems to be ideally suited for this purpose, since it
displays, in this case, 28 distinct signals distributed on a scale
covering around 160 ppm (Table 1). CD data have not been recorded much for this type of compound either. In order to establish a reference for
future discussions concerning the stereochemistry and conformation of
this type of cyclodepsipeptides, these data have been included in this report.
Surfactants promote swarming motility of S. liquefaciens.
The following complementation experiments completely
restored the swarming motility of the PL10 and MG44 mutants. The
strains were inoculated on 0.6% agar plates supplemented with three
purified biosurfactants: either 1-µg/ml surfactin (Sigma) produced by
B. subtilis (2) (data not shown), 10-µg/ml
serrawettin W1 produced by S. marcescens (20)
(data not shown), or HPLC-purified serrawettin W2 (Fig.
4A). The presence of W2 above the
critical concentration of 1 µg/ml restores the swarming motility of
PL10 to the level of the wild-type strain (Fig. 4A). When W2 is
dissolved in water at 1 µg/ml and above, rapid drop collapse follows,
as demonstrated in Fig. 4B. This critical concentration of W2 could
reflect a physical property of the compound itself, e.g., a critical
micelle-forming concentration.
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FIG. 4.
(A) Swarming motility of S. liquefaciens PL10
on medium supplemented with HPLC-purified serrawettin W2 at the
concentrations indicated. (B) Drop-collapsing test of water
supplemented with HPLC-purified serrawettin W2 at the concentrations
indicated.
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Conclusion.
The present study is in agreement with previously
published data demonstrating that biosurfactant production is crucial
for the swarming motility of Serratia spp. (22).
B. subtilis also exhibits swarming motility on surfaces, and
mutants unable to produce the biosurfactant are defective in swarming
motility (24). Swarming motility appears to be an
intrinsically linked surface and cell density phenomenon. It is
therefore not surprising to find that S. liquefaciens
employs a quorum-sensing regulatory circuit in the process of surface
conditioning. Since the swarming motility of the swrI and
swrI swrA mutants could be restored simply by supplementing
the medium with pure surfactant, the only quorum-sensing-controlled gene involved in swarming motility may be swrA. The fact
that external addition of BHL leads to 10-fold higher induction of the
swrA-luxAB fusion on the surface of agar plates compared to liquid is in accordance with the finding that only small surfactant amounts are produced in a liquid culture. These observations are intriguing; we speculate that either the surface itself, i.e., a form
of surface recognition, or dense growth within a colony, i.e., intimate
cell-cell contact, accounts for the observed differences. In this
context, it is interesting that the recently identified quorum-sensing
system in Rhodobacter sphaeroides controls the switch
from aggregated to dispersed growth (29). A screening of our
Serratia strain collection revealed a correlation between swarming motility and surfactant production. Many produced detectable amounts of acylated homoserine lactone (our unpublished observations), and this indicates that surfactant production in Serratia in
general is responsive to cell density by quorum-sensing mechanisms that may control the expression of biosurfactant synthetases. Recently it
was shown that B. subtilis regulates surfactin production by a cell density-responsive mechanism not based on homoserine lactone but
utilizing a peptide pheromone, ComX (4), as is usually the
case in gram-positive bacteria (15). From an evolutionary point of view, the ubiquitous peptide synthetases are highly
interesting, being present in both gram-negative and gram-positive
bacteria, as well as in certain filamentous fungi (30). The
different products of the peptide synthetases display a range of
powerful biological properties, such as antibiotic, hemolytic,
antitumor, and surface-conditioning activities (16, 26, 28).
Nucleotide sequence accession number.
The nucleotide sequence
of swrA has been assigned GenBank accession no. AF039572.
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ACKNOWLEDGMENTS |
We are indebted to S. E. Harnung for determination of the CD data.
The CD data were measured on a modified JASCO 710 instrument financed
by the Danish Natural Science Research Council (grant 0373-1). We thank
Linda Stabell for excellent technical assistance and Tohey Matsuyama
for kindly providing a purified sample of serrawettin W1.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Bldg. 301, Technical University of Denmark, 2800 Lyngby, Denmark. Phone: 45 45252769. Fax: 45 45932809. E-mail:
mg{at}im.dtu.dk.
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Journal of Bacteriology, December 1998, p. 6384-6388, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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