2010 NPR Quourum Sensing and Bacterial Biofilms PDF

Quorum sensing and bacterial biolms
Jeroen S. Dickschat
*
Received 29th October 2009
First published as an Advance Article on the web 3rd February 2010
DOI: 10.1039/b804469b
Covering: up to 2009
This review describes the chemistry of the bacterial biolms including the chemistry of their
constituents and signalling compounds that mediate or inhibit the formation of biolms. Systems are
described with special emphasis, in which quorum sensing molecules (autoinducers) trigger the
formation of biolms. In the rst instance, N-acyl-L-homoserine lactones (AHLs) are the focus of this
review, whereas the inter-species signal known as furanosyl borate diester and peptide autoinducers
used by Gram-positive bacteria are not discussed in detail. Since the rst discovery of an AHL
autoinducer from Vibrio scheri a large and further increasing number of different AHL structures
from Gram-negative bacteria have been identied. This review gives a summary of all known AHL
autoinducers and producing bacterial species. A few systems are discussed, where biolm formation is
suppressed by enzymatic degradation of AHL molecules or interference of secondary metabolites from
other species with the quorum sensing systems of communicating bacteria. Finally, the multi-channel
quorum sensing system, the intracellular downstream processing of the signal, and the resulting
response of whole populations including biolm formation are discussed for the Vibrio genus that has
been extensively investigated.
1 Introduction
2 Chemistry of biolms
3 Biosynthesis of N-acyl-L-homoserine lactones
4 Detection and identication of N-acyl-L-homoserine
lactones
5 The role of N-acyl-L-homoserine lactones in biolm
formation
6 Antagonists of N-acyl-L-homoserine lactones in nature
6.1 Enzymatic degradation of AHLs
6.2 Secondary metabolites
6.3 Biolm inhibition by azithromycin
7 A novel genus-specic autoinducer from Vibrio
8 Conclusions
9 Acknowledgements
10 References
1 Introduction
In the early days of microbiology bacteria were believed to
favour a planktonic and strictly unicellular way of life. This is
indeed true for one of the largest habitats, the worlds oceans,
and also for many culture asks in laboratories around the
world, where bacteria are grown in liquid media. Antonie van
Leeuwenhoek, who rst recognised the presence of animal-
cules in rain water and saliva, also encountered planktonic
bacteria. He was again the rst to nd a sessile formof bacteria in
dental plaque, where the microorganisms live in a biolm, but
van Leeuwenhoek didnt realise the difference between these two
forms of bacterial life with his simple microscope. Today it is
known that bacterial biolms are widespread in nature and are
formed by nearly all bacterial species. Biolms enable bacteria to
grow on surfaces in a self-produced polymeric matrix. These
biolms provide a mechanically stable and protective environ-
ment for the bacteria, resulting in a higher tolerance against
extreme conditions such as high or low pH or temperature.
Within the biolm matrix optimal conditions for cellcell inter-
actions and nutrient supply exist, and the bacteria are protected
against environmental stresses such as antibiotics. The formation
of bacterial biolms requires cellcell communication, and the
underlying chemistry of the bacterial language will be discussed
in this article.
Many Gram-negative bacteria use N-acyl-L-homoserine
lactones (AHLs) to communicate with each other. These bacte-
rial pheromones are often produced as mixtures of several AHLs
with one principle component that is species-specic as the
nature of its N-acyl chain varies from species to species. The rst
signal molecule of this type was identied from Vibrio scheri as
N-(3
0
-oxohexanoyl)-L-homoserine lactone (3-oxo-C
6
-HSL, 3)
that induces luminescence in this species.
1
The molecule is syn-
thesised by the LuxI protein, referred to as AHL synthase, in this
bacterium and excreted into the medium. The LuxR protein
encoded in the same lux operon is an AHL detector that upon
interaction with AHLs transcriptionally activates other genes in
order to cause their expression and phenotypic changes.
2
For
a signicant response of the bacteria a minimum AHL concen-
tration or, in other words, a threshold bacterial population
density is required. Due to these characteristics of the bacterial
Institute of Organic Chemistry, Technical University of Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany. E-mail: j.dickschat@
tu-bs.de
This journal is The Royal Society of Chemistry 2010 Nat. Prod. Rep., 2010, 27, 343369 | 343
REVIEW www.rsc.org/npr | Natural Product Reports
communicators, the AHLs are also called autoinducers or
quorum sensing molecules.
The autoinducers share the structural elements of a N-acyl
chain attached to a L-homoserine lactone ring, whereas species-
specic differences occur in the exact nature of the N-acyl chain
that can have different oxidation states in the 3-position such as
a 3-oxo function in 3, a methylene as in N-butyryl-L-homoserine
lactone (C
4
-HSL, 1) from Pseudomonas aeruginosa,
3
or
a 3-hydroxy group as in N-((3
0
R)-hydroxybutyryl)-L-homoserine
lactone ((R)-3-OH-C
4
-HSL, 5) from Vibrio harveyi.
4
Several
species developed more than one quorum sensing system to
tightly control different regulatory networks. To give some
examples, besides the already mentioned 3-oxo-C
6
-HSL system
in V. scheri a second AHL autoinduction cascade synthesises
and detects N-octanoyl-L-homoserine lactone (C
8
-HSL, 1).
5
Furthermore, N-(3
0
-oxododecanoyl)-L-homoserine lactone
(3-oxo-C
12
-HSL, 4) operates as autoinducer in addition to 1 in
P. aeruginosa.
6
The hereby used terminology for N-acyl-L-homoserine
lactones is consistently applied in the course of this review: the
abbreviation HSL denotes the L-homoserine lactone nature of
the signalling molecules and explicitly includes the L stereo-
chemistry, whereas the N-acyl moieties are dened by a termi-
nology as used for fatty acids. Generally, C
n
-HSL indicates
a saturated N-alkanoyl group and iso-C
n
is used as an identier
for an iso-branched, i.e. u-1 methyl branched N-alkanoyl group
with n carbons. Oxygen functionalities in the 3-position of the
attached N-alkanoyls are mentioned using the 3-oxo and 3-OH
prexes for N-3-oxoalkanoyl and N-3-hydroxyalkanoyl deriva-
tives, respectively. The usage of C
n:m
descriptors indicates the
presence of m double bonds in an N-alkanoyl chain with n
carbons, but saturated chains follow the C
n
and not the C
n:0
terminology unless the usage of C
n:0
is required for unambiguity.
The positions and E or Z congurations as well as the
R stereochemistry of 3-OH functions (S stereochemistry has
never been described so far) are denoted by the usual stereo-
chemical descriptors as prexes. To give an example, the AHL
molecule from Rhizobium leguminosarum, N-((3
0
R,7
0
Z)-3
0
-
hydroxytetradec-7
0
-enoyl)-L-homoserine lactone (6), also known
as small bacteriocin, is briey named (3R,7Z)-3-OH-C
14:1
-
HSL following this terminology.
Besides the mentioned luminescence in V. scheri and V.
harveyi,
1,4
several other phenotypic characteristics of Gram-
negative bacteria as different as carbapenem antibiotic produc-
tion in Pectobacterium carotovorum,
7
Ti plasmid conjugal
transfer in Agrobacterium tumefaciens,
8
synthesis of poly-
3-hydroxybutyrate in V. harveyi,
9
swarming in Serratia
liquefaciens,
10
root nodulation and growth inhibition in
R. leguminosarum,
11
exopolysaccharide production and virulence
in Pantoea stewartii,
12
virulence and biolm formation in
P. aeruginosa,
1315
and many more are expressed by quorum
sensing control. The focus of this review will, in the rst instance,
be on the mechanisms and chemical signals that are involved in
the formation of biolms.
Knowledge about the mechanisms of bacterial biolm
formation is particularly important, because biolms constitute
many persistent and chronic infections with inherent resistance
to antibiotics.
16
Biolm infections are frequently seen on
implantations such as pacemakers, in which case the effect of
antibiotic therapy is often restricted to revert the symptoms
caused by planktonic cells.
17
In many cases the human pathogen
P. aeruginosa permanently colonises the lungs of cystic brosis
patients and forms a biolm in the sputum.
13
These chronic
infections result in progressive lung damage and nally death by
respiratory failure for most cystic brosis patients, and due to the
biolmarchitecture of the P. aeruginosa colonies in these patients
even long-term antibiotic therapy does not eradicate the infective
agent.
18
Biolm formation in P. aeruginosa is mediated by
quorum sensing molecules.
13
The quorum sensing system of
Jeroen S: Dickschat
Dr Jeroen S. Dickschat studied
chemistry at the Technical
University of Braunschweig
(19972002), followed by
a PhD with Prof. Dr Stefan
Schulz as a fellow of the Fonds
der Chemischen Industrie. In
2005, he moved to the group of
Prof. Dr Rolf M uller at Saar-
land University, and from 2006
to 2008 he stayed in the lab of
Prof. Peter Leadlay as a fellow
of the Deutsche Akademie der
Naturforscher Leopoldina.
Recently he became a group
leader in the Emmy Noether Programme and within a Transre-
gional Collaborative Research Centre both founded by the Deut-
sche Forschungsgemeinschaft at the Technical University of
Braunschweig.
344 | Nat. Prod. Rep., 2010, 27, 343369 This journal is The Royal Society of Chemistry 2010
P. aeruginosa is operating through the two N-acylhomoserine
lactone autoinducers C
4
-HSL (1) and 3-oxo-C
12
-HSL (4).
3,6
These signal molecules are, as in any other known AHL-based
quorum sensing system, synthesised by LuxI-type AHL syn-
thases and detected by their cognate receptors, transcriptional
activator proteins of the LuxR-type. In the las system, the
autoinducer 3-oxo-C
12
-HSL (4) is produced by the signal syn-
thase LasI and detected by the receptor LasR,
19,20
whereas in the
second quorum sensing circuit, termed rhl system, RhlI catalyses
the synthesis of the signalling molecule C
4
-HSL (1) that binds to
the RhlR receptor.
21,22
Despite the high similarities in all quorum
sensing systems that are using the same type of AHL signalling
molecules, the sequence homologies between the known LuxI-
type AHL synthases and also between the known LuxR-type
receptors are fairly low. Importantly, the possibility to correlate
the sequences of the LuxI (or LuxR) proteins to the structures of
their synthesised (or detected) AHLs has been questioned.
23
The luxI-type genes are often transcriptionally activated in the
presence of the LuxR-type protein and the cognate AHL mole-
cule, a phenomenon referred to as positive feedback loop that is
known from several AHL quorum sensing systems such as the
lux operon from V. scheri, the las operon from P. aeruginosa, or
the tra operon in A. tumefaciens.
2428
In this review a brief summary of the accumulated knowledge
about the chemistry of biolms will be given (chapter 2), followed
by considerations about the biosynthesis of AHLs (chapter 3).
Biosynthetic aspects are considerably important, because only
a detailed understanding of the underlying pathways can give
explanations for the structural variants including stereochemical
details of the AHL molecules produced by bacteria, and, vice
versa, a complete structure elucidation of AHLs including their
absolute congurations can point to or exclude the participation
of well-known primary metabolic pathways. Therefore, the
biosynthesis chapter is closely connected to chapter 4 that deals
with methods for the identication of AHLs. At the end of this
chapter a tabulated summary of all known AHL structures and
AHL producing species is given. The following chapters will
cover the role of AHLs for the formation of biolms (chapter 5),
and mechanisms of AHL antagonism in nature (chapter 6).
Finally, one of the most important and best investigated systems
involving quorum sensing signalling from the Vibrio genus will
be discussed in detail (chapter 7).
Several reviews on AHLs and biolm formation covering
different aspects of bacterial communication systems have
appeared in the recent past. Uroz et al. summarised the accu-
mulated knowledge about quorum quenching mechanisms
that interrupt AHL-mediated cellcell communication,
29
Stein-
dler and Venturi gave an overview about the detection of AHLs
using bacterial bioreporters,
30
and Richards and Melander dis-
cussed how biolms can be controlled.
31
Specic aspects of the
biolmformation in P. aeruginosa and the Vibrio genus have also
been covered by recent articles.
32,33
In contrast to AHL signalling
used by Gram-negative bacteria, microorganisms of the Gram-
positive Staphylococcus genus use small post-translationally
modied peptides as autoinduction signals that are involved in
the formation of biolms. This type of autoinduction system will
not be discussed here, but has been described in previous
reviews.
34,35
2 Chemistry of biolms
Bacterial biolms are sessile communities of microorganisms
that coexist as highly differentiated associates in an extracellular
matrix produced by their constituent cells. The formation of
these complex multicellular structures requires cell-to-cell
communication via extracellular messenger molecules or direct
cell-to-cell contact, in which also small molecules may conduct
the signal, similar to concerted processes within single-species
communities such as the fruiting body formation of Stigmatella
aurantiaca and sporulation of Streptomyces coelicolor that are
controlled by stigmolone and the A-factor, respectively.
3638
The biolm matrix typically consists of extracellular poly-
saccharides (also termed exopolysaccharides, EPS), and can also
comprise proteins and DNA, but the precise structure and
composition of biolms strongly vary with the resident species
and environmental conditions. Before going into the details
about the structure and mode of action of signalling molecules
that initiate biolm formation, this chapter provides a brief
introduction to the chemistry of biolms.
As outlined above, EPSs usually belong to the major compo-
nents of bacterial biolms. These EPSs can be composed of only
one type of monosaccharide or can be formed from various
monosaccharides that are usually O- or N-acylated, respectively.
A widespread EPS is poly-b-1,6-N-acetylglucosamine (7) that is
excreted by Actinobacillus pleuropneumoniae,
39
Staphylococcus
aureus,
40
and Escherichia coli.
41
In some cases only partial
N-acetylation is found,
39,40
but N-acetylation can also be
complete,
41
and, in addition, O-succinyl groups can be attached
to the carbohydrate backbone.
40
An example for an EPS formed
from different monosaccharides is 8 from Erwinia persicina. The
polysaccharide is composed of an iterated pentameric branched
subunit containing two a-D-galactose, one b-D-galactose, and
two b-D-glucose monomers that are irregularly O-acylated. The
deacetylated structure has been elucidated by
1
H and
13
C NMR
spectroscopy.
42
Cellulose (9) is the most abundant organic
molecule in nature, and is also an important component of the
biolm matrix of bacteria such as Salmonella and E. coli.
43,44
The
mucoid phenotype of P. aeruginosa that causes chronic respira-
tory infections in cystic brosis patients is characterised by an
overproduction of alginate (10),
45,46
a polysaccharide that is
composed of a-L-guluronic and a-L-mannuronic acid. For many
years 10 was generally believed to be a matrix component of
P. aeruginosa biolms, but recent investigations revealed that
10 is indeed not a signicant component of non-mucoid strains
such as P. aeruginosa PAO1 and PA14.
47
Extracellular DNA is required for the formation of bioms in
P. aeruginosa PAO1, and young biolms can be dissolved by
DNAse treatment, whereas mature biolms are not affected by
DNAse, suggesting that the stability of the matrix in young
biolms highly depends on extracellular DNA, whereas other
components are holding the cells together in aged biolms.
48
In
contrast, mature biolms of four independent clinical P. aeru-
ginosa isolates could be degraded by DNAse treatment, sug-
gesting that DNA is considerably important for the cell-to-cell
interconnection in these strains.
49
Although extracellular DNA is
a major component of P. aeruginosa PAO1 biolms, gene inac-
tivation experiments in a putative EPS biosynthetic gene cluster
(psl, polysaccharide synthesis locus) revealed the critical role of
an unidentied EPS for the biolm matrix.
50,51
Similar investi-
gations identied two loci in P. aeruginosa PA14 and ZK2870, psl
and pel that are involved in the production of a mannose-rich
and a glucose-rich matrix material, respectively. Maturation of
P. aeruginosa biolms requires at least one of these EPSs, and
either of the two matrix components is sufcient for the forma-
tion of a mature biolm.
52,53
Recent investigations revealed that
extracellular DNA in P. aeruginosa PAO1 biolms is highly
similar to chromosomal DNA and is mainly excreted during the
late-log phase in a process that depends on acyl homoserine
lactone signalling, suggesting that extracellular DNA is gener-
ated by quorum sensing-regulated cell lysis of a subpopulation of
the bacteria.
54
3 Biosynthesis of N-acyl-L-homoserine lactones
Early feeding experiments with
15
N-labelled methionine sug-
gested S-adenosyl-L-methionine (SAM) as a precursor for AHL
biosynthesis.
55
Winans and coworkers showed by an incubation
experiment with the puried recombinant AHL synthase TraI
from A. tumefaciens that bacteria synthesise the AHL molecules
from acyl carrier protein (ACP) bound fatty acyl derivatives, but
not fatty acyl-CoAderivatives, and SAM(Scheme 1).
56
The same
observations have been made independently and in the same year
by Greenberg and coworkers on the puried LuxI protein from
V. scheri.
57
Generally, the LuxI homologues acylate the amino
group of SAM followed by an intramolecular nucleophilic
substitution and loss of methylthioadenosine to generate the
homoserine lactone.
58
In consequence, the stereochemistry of the
homoserine lactone ring is generally believed to be S (i.e. L)
congured, although the absolute conguration has only been
experimentally established in a few cases: by circular dichroism
for (S)-3-oxo-C
6
-HSL ((S)-3) from Pectobacterium car-
otovorum,
7
by chiral GC for (S)-C
6
-HSL from Erwinia psidii as
well as (S)-C
6
-HSL and (S)-C
7
-HSL from Pantoea ananatis,
5961
and, using both methods, to elucidate the S conguration of C
4
-
HSL (1) produced by another strain of Pantoea sp.
62
The
mechanism of AHL biosynthesis is particularly interesting,
because the two substrates used adopt roles that drastically
deviate from their usual cellular functions, in which SAM nor-
mally acts as a biological methyl donor and the acyl-ACPs are
prone to yield fatty acids.
The ACP-bound fatty acyl derivative to be transferred to the
amino group of SAM can be of varying chain length, saturated
or unsaturated in different positions with E or Z double bond
geometry, oxidised in the 3-position (3-OHor 3-oxo), and methyl
branched, depending on the substrate specicity of the LuxI-type
protein. In summary, the variety of the N-acyl chains represents
a broad range of modications that are generated in fatty acid
biosynthesis. The LuxI homologue often shows a preference for
one specic fatty acyl-ACP derivative leading to the formation of
one major AHL product, but closely related AHLs with similar
chain lengths and/or oxidation states in the 3-position frequently
occur in the same bacterial species. To give an example, the
highly virulent human pathogen Yersinia pestis produces mainly
3-oxo-C
8
-HSL in combination with the two carbons
shorter 3-oxo-C
6
-HSL (3) and the two carbons longer 3-oxo-
C
10
-HSL. In addition, C
8
-HSL (2) and traces of C
6
-HSL with
Scheme 1 LuxI-catalysed synthesis of N-acyl-L-homoserine lactones from fatty acyl-ACP derivatives and S-adenosyl-L-methionine (SAM).
a 3-methylene moiety are found. All these AHL derivatives are
synthesised by the YspI protein as demonstrated by the heter-
ologous expression of the recombinant protein in E. coli.
63
The
main AHL product of one AHL synthase reects its preferred
substrate, but especially in the case of AHL synthases with
a broad substrate specicity, the relative abundance of the
different AHL products may also feature the available fatty
acyl-ACP pool.
Random mutagenesis of the AHL synthases LuxI from
V. scheri and RhlI from P. aeruginosa identied critical regions
for enzyme activity.
64,65
A cysteine or serine residue within the
active site of AHL synthases was suggested to take up the fatty
acyl moiety from the fatty acyl-ACP derivative during AHL
biosynthesis.
56
However, this suggested enzyme mechanism was
contradicted by site-specic mutagenesis of several cysteine and
serine residues within the critical regions of LuxI and RhlI that
did not result in the loss of catalytic activity.
64,65
Further proof
against covalent binding of the acyl intermediate to the AHL
synthase was given by resolving the crystal structure of EsaI from
Pantoea stewartii.
66
The active site of EsaI was identied by
substrate modelling into a hydrophobic cavity spanned by
several conserved residues. Site-directed mutagenesis of an active
site tyrosine residue to alanine (T140A) altered the enzymes
substrate specicity from 3-oxo-C
6
-HSL (3) to C
6
-HSL.
According to the model the OH-function of the tyrosine residue
is crucial for the selection of the 3-oxo-C
6
-ACP by the formation
of a hydrogen bond to the 3-oxo group of this substrate. The
crystal structure of LasI from P. aeruginosa revealed an enlarged
hydrophobic binding pocket for the uptake of the 3-oxo-C
12
-
ACP substrate, whereas it is unknown by which mechanism the
enzyme excludes shorter acyl-ACP substrates that are prevalent
in P. aeruginosa.
23
Mutagenesis of two enzyme residues in the
active site of the ExpI synthase from P. carotovorum changed the
substrate specicity froma 3-oxo-C
6
-HSL (3) to a 3-oxo-C
8
-HSL
producer.
67
The fatty acyl-ACP pool available to AHL production is fed
from intermediates of the fatty acid biosynthesis that is catalysed
by an assembly of separate enzymes in bacteria termed type II
fatty acid synthase system.
68
The pathway starts from acetyl-
CoAthat is selected and transferred to the ACP by the (malonyl-)-
acyltransferase (MAT, Scheme 2). Chain elongation proceeds
via the incorporation of malonyl-CoA units that are loaded onto
the ACP by the MAT. The ketosynthase (KS) catalyses the
Claisen condensation of the starter unit and the malonyl-ACP
with concomitant decarboxylation to yield a 3-oxoacyl-ACP
intermediate. The subsequent reductive loop includes ketor-
eduction by the ketoreductase (KR) to the (R)-3-hydroxyacyl-
ACP, elimination of water by the dehydratase (DH) to the
(2E)-enoyl-ACP, and C]C double bond hydrogenation by the
enoylreductase (ER) to an acyl-ACP intermediate that is sub-
jected to the next round of chain elongation. Iteration of this
two-carbon elongation process results in fatty acyl derivatives
with an even number of carbons. The ACP-bound intermediates
obtained in the different stages of the reductive loop react to
form the even-numbered N-acyl-L-homoserine lactones of the
C
n
-HSL, 3-oxo-C
n
-HSL, (R)-3-OH-C
n
-HSL, and (2E)-C
n:1
-
HSL types. AHL molecules of the (2E)-C
n:1
-HSL type
are relatively rare, possibly because the reactivity of the
Scheme 2 Fatty acid biosynthesis and biosynthesis of N-acyl-L-homoserine lactones.
a,b-unsaturated enoyl-ACP substrates is different from the
reactivity of the saturated acyl-ACP substrates. Therefore, the
generation of (2E)-C
n:1
-HSLs from 3-OH-C
n
-HSLs by an
enzyme-catalysed (DH) or chemical reaction e. g. during the
analytical procedure cannot be ruled out. Although the biosyn-
thesis of AHLs with an unsaturated N-acyl chain is not investi-
gated in detail, double bonds may be introduced by the usual
processes during the bacterial biosynthesis of unsaturated fatty
acids. Two clearly different pathways, i.e. anaerobic and aerobic
biosynthesis of unsaturated fatty acids, can be distinguished. The
anaerobic pathway of the bacterial type II system involves
introduction of a double bond into the growing acyl-ACP chain
at the stage of the b-hydroxydecanoyl-ACP intermediate by the
b-hydroxydecanoyl-ACP dehydratase FabA that is also able to
isomerise the dehydration product (2E)-decenoyl-ACP to
(3Z)-decenoyl-ACP.
69
The ongoing chain elongation yields
a series of unsaturated fatty acyl-ACP intermediates composed
of (3Z)-C
10:1
-ACP, (5Z)-C
12:1
-ACP, (7Z)-C
14:1
-ACP, (9Z)-C
16:1
-
ACP, and (11Z)-C
18:1
-ACP. The aerobic pathway proceeds rst
via generation of the saturated carbon chain and subsequent
dehydration usually by a D
9
-desaturase yielding (9Z)-C
n:1
-ACP
derivatives.
69
Alternatively, the action of a desaturase on a satu-
rated AHL derivative might be a plausible mechanism.
The fatty acyl-ACP pool used by the AHL synthases is only
fed by the described anabolic pathway, whereas fatty acid
catabolism of long chain fatty acids by b-oxidation proceeds via
fatty acyl-CoA and not via ACP-bound intermediates. The fatty
acid b-oxidation is a repetitive chain shortening process that
degrades fatty acids to produce acetyl-CoA. The degradative
pathway involves similar acyl intermediates as the fatty acid
biosynthesis, albeit in a reverse order. A major difference occurs
in the absolute conguration of the 3-hydroxyacyl-CoA inter-
mediate that is S congured in the catabolic pathway, in contrast
to (R)-3-hydroxyacyl-ACP intermediates in fatty acid biosyn-
thesis.
68,70
Therefore, the absolute congurations of 3-OH-C
n
-
HSLs point to their origin from fatty acid biosynthesis or
b-oxidation, respectively. The stereochemistry of the autoinducer
fromV. harveyi was determined by a chiral NMRshift reagent as
(R)-3-OH-C
4
-HSL (5).
71
Inhibition of HSL production in this
species by the antibiotic cerulenin that is known to block fatty
acid biosynthesis, further corroborated an anabolic rather than
a catabolic origin of the N-acyl moieties in AHLs. Similarly, the
absolute conguration of small bacteriocin from R. legumi-
nosarum was elucidated using a chiral NMR shift reagent as
(3R,7Z)-3-OH-C
14:1
-HSL (6).
72
Recently, the autoinducers
3-OH-C
8
-HSL (11) from Aeromonas culicicola and 3-OH-C
10
-
HSL (12) fromPhaeobacter gallaeciensis have been established to
be enantiomerically pure and R congured by methanolysis and
separation of the obtained methyl 3-hydroxyalkanoates on
a chiral GC phase.
73
A mutational disruption of the fatty acid
b-oxidation pathway in E. coli heterologously expressing TraI
did not affect the formation of 3-oxo-C
8
-HSL, whereas specic
inhibitors of fatty acid biosynthesis abolished AHL
production.
74
All these data equally support the biosynthesis of
AHLs via fatty acid biosynthesis and not the b-oxidation
pathway.
Acetyl-CoA is the most frequently used starter unit in fatty
acid biosynthesis. In consequence, the majority of known AHL
signalling molecules carries an even-numbered N-acyl moiety.
Fatty acids with an odd number of carbons arise from the pro-
pionyl-CoA starter unit that is used less often, and consequently,
AHL molecules with an odd-numbered N-acyl chain are
comparably rare. Methyl branched AHLs are likely produced in
a similar manner to methyl branched fatty acids from the amino
acid-derived starter units isobutyryl-CoA (valine), isovaleryl-
CoA (leucine), or 2-methylbutyryl-CoA (isoleucine) by trans-
amination and oxidative decarboxylation. The isobutyryl-CoA
starter may be used in the biosynthesis of iso-even N-acyl-L-
homoserine lactones, whereas isovaleryl-CoA can lead to iso-odd
AHL molecules, and 2-methylbutyryl-CoA would be the
precursor for anteiso-odd AHL derivatives. The putative occur-
rence of anteiso-even AHLs is only explainable by a-oxidation of
an isoleucine-derived anteiso-odd fatty acyl-CoA intermediate.
Surprisingly, although iso- and anteiso-fatty acids are quite
widespread in bacteria, and this in combination with the known
AHL biosynthesis from fatty acyl-ACP intermediates suggests
that methyl branched AHLs should also frequently occur in
bacteria, there is only one recent report about iso-branched
AHLs from A. culicicola.
73
It seems possible that further C
n
-
HSLs identied so far are indeed methyl branched, especially if
compound identication was only based on low resolution
techniques such as thin layer chromatography (TLC).
4 Detection and identication of N-acyl-L-
homoserine lactones
Several different bioassays, spectroscopic, and chromatographic
methods have been used for the detection and identication of
AHLs in culture extracts from bacteria. In Tables 24 an effort
was made to summarise all AHLs that have been identied until
today, including information about the producing species, the
main component in complex AHL mixtures, and the methods
that have been applied for compound identication. Curiously,
there is one report about an AHL from a cyanobacterium in the
literature, whereas all other AHLs identied until today are from
alpha-, beta-, or gammaproteobacteria.
75
One of the most sensitive methods to sense AHLs in bacterial
samples includes a radioactive assay that has been developed for
the detection of AHLs in P. aeruginosa biolms in the lungs of
cystic brosis (CF) patients.
13,76
The assay is based on the uptake
of radiolabelled [1-
14
C]-L-methionine that is incorporated via
SAM into the AHL molecules. This method allows the detection
of all different types of AHLs without any bias for a particular
side chain length or oxidation state in the 3-position. Because the
assay is unbiased it can be used for the quantication of the
relative amounts of different AHLs produced by one organism.
77
Several bacterial species have been screened for the production of
AHLs using this fast radiotracer assay.
78,79
Another sensitive bioassay for the specic detection of AHLs
includes the usage of reporter strains. The method does not
require a complex experimental instrumentation and has been
applied to detect AHLs from a large number of different
bacterial species. Until today, several AHL reporter strains have
been constructed (a selection is summarised in Table 1). The
principle for most AHL reporter strains is as follows: a promoter
or gene that is transcriptionally activated by the presence of
AHLs is fused to a reporter gene, and the fusion construct is
introduced into a reporter strain. It is of course crucial that the
reporter strain itself is a natural non-producer of the AHLs to be
detected by the reporter construct, or does not produce such
AHLs due to an inactivation of the respective AHL synthase
gene. One extensively applied system was constructed after this
principle involving a traG::lacZ fusion and the AHL receptor
TraR from Agrobacterium tumefaciens that transcriptionally
activates traG (Fig. 1). The co-transcribed lacZ gene expresses
b-galactosidase activity and enables the detection of AHLs by the
formation of blue-coloured cultures on agar plates containing
X-Gal.
80,81
A major drawback of this methodology is the strongly varying
sensitivity of the reporter strains to different AHLs depending on
the length and functional group in the 3-position of the N-acyl
chain. The traG::lacZ fusion reporter in A. tumefaciens responds
to C
n
-HSLs with a chain length from n 612 and to 3-oxo-C
n
-
HSLs (n 412). A similar Ensifer meliloti Rm41 reporter strain
complements this pattern and is capable of specically detecting
long chain AHLs (C
n
-HSL: n 1216, 3-oxo-C
n
-HSL: n 14).
82
This reporter system is particularly interesting, because it
contains a sinI::lacZ fusion derived from the sinI gene including
the sinI promoter region that encodes the AHL synthase for long
chain AHLs. As a side effect of this transcriptional fusion the
production of sinI-encoded AHLs is impaired, whereas the sinI
gene is at the same time transcriptionally activated in the pres-
ence of a SinR-AHL complex due to a positive feedback loop. In
consequence, the sinI::lacZ fusion suppresses the natural AHL
backgroud in E. meliloti and results in b-galactosidase activity in
the presence of long chain AHLs.
Several bioluminescent AHL reporter plasmids have been
constructed. To cover a broad range of structural variations of
the AHL autoinducers, different quorum sensing response
regulator genes (luxR: LuxR detects 3-oxo-C
6
-HSL (3) in
V. scheri, rhlR: RhlR is the detector protein for C
4
-HSL (1)
in P. aeruginosa, and lasR: LasR responds to 3-oxo-C
12
-HSL (4)
in P. aeruginosa) and the promoter regions of the respective luxI
homologues (P
luxI
, P
rhlI
, and P
lasI
) have been fused to the lux-
CDABE operon from Photobacterium luminescens that responds
with bioluminescence upon transcriptional activation.
83
In these
constructs the luxCDABE operon is under control of the
promoters of luxI (or homologues) that are addressed by auto-
inducer detection of LuxR (or homologues). The plasmids have
been transformed into E. coli JM109 that does not produce any
AHL signals. The reporter strains respond to AHLs with
different chain lengths and oxidation states at C-3 of the N-acyl
chain (C
n
-HSL: n 412, 3-oxo-C
n
-HSL: n 414), with the
highest sensitivities observed for the cognate activator molecules,
followed by structurally closely related AHLs.
Very similar plasmids have been constructed based on the
green uorescent protein (GFP). Plasmid pJBA132 contains
a luxR-P
luxI
::gfp(ASV) fusion construct that was introduced into
E. coli MT102.
84
Although the range of AHL molecules detect-
able by the resulting reporter strain has not been systematically
investigated, several AHLs including C
6
-HSL, C
8
-HSL (2),
3-oxo-C
6
-HSL (3), and 3-oxo-C
12
-HSL (4) can be detected with
high sensitivity. The related plasmids pKR-C12 and pAS-
C8 contain P
lac
::lasR and P
lasB
::gfp(ASV) or P
lac
::cepR and
P
cepI
::gfp(ASV) fusions, respectively, using genes from the
Fig. 1 The A. tumefaciens NT1 (pDCI41E33) traG::lacZ fusion reporter
strain.
Table 1 AHL reporter strains
Reporter strain Genotype Phenotype C
n
-HSL 3-oxo-C
n
-HSL Ref.
A. tumefaciens NT1 (pDCI41E33) traG::lacZ b-galactosidase activity n 612 n 412 80,81
E. meliloti Rm41 (pVIKSinIsub) sinI::lacZ b-galactosidase activity n 1216 n 14 82
E. coli JM109 (pSB401) luxR-P
luxI
::luxCDABE bioluminescence n 412 n 414 83
E. coli JM109 (pSB406) rhlR-P
rhlI
E. coli JM109 (pSB1075) lasR-P
lasI
E. coli MT102 (pJBA132) luxR-P
luxI
::gfp(ASV) green uorescence n 68 n 612 84
P. aeruginosa PAO-JP2 (pKR-C12) P
lac
::lasR green uorescence not sensitive n 1012 85,86
P
lasB
::gfp(ASV)
B. cepacia H111-I (pKR-C12) P
lac
::lasR green uorescence n 612 n 612 85,86
P
lasB
::gfp(ASV)
P. aeruginosa PAO-JP2 (pAS-C8) P
lac
::cepR green uorescence n 612 n 1012 85,86
P
cepI
::gfp(ASV)
B. cepacia H111-I (pAS-C8) P
lac
::cepR green uorescence n 612 n 612 85,86
P
cepI
::gfp(ASV)
C. violaceum CV026 cviI::mini-Tn5 AHL production impaired n 48 (n 1014) n 48 (n 1014) 87
P. aeruginosa las system and the luxR and luxI homologues cepR
and cepI fromBurkholderia cepacia (the major product of CepI is
C
8
-HSL(2)).
85,86
Both plasmids were introduced into the lasI rhlI
double mutant P. aeruginosa PAO1-JP2 and the cepI mutant
B. cepacia H111-I that do not produce any AHLs. Highest
sensitivities of the reporter strains are found for the original
signal molecules of the used receptors, but structurally similar
molecules can also be detected (summarised in Table 1). Inter-
estingly, some differences in the detectable range of AHL
molecules occur, if one plasmid is transformed into different
reporter strains, e. g. the P. aeruginosa PAO1-JP2 reporter con-
taining plasmid pKR-C12 does not detect any C
n
-HSLs, whereas
the same plasmid in the B. cepacia H111-I reporter enables
sensitivity against C
n
-HSLs (n 612).
Another simple, but very efcient and extensively used
reporter strain is Chromobacterium violaceum CV026.
87
The
wildtype of C. violaceumATCC 31532 is a producer of the purple
pigment violacein that is tightly controlled by AHLs. The strain
CV026 was obtained by transposon insertion mutagenesis and
carries an insertion in the luxI homologue cviI, resulting in
a white phenotype. The mutant can be used as a reporter strain to
detect AHLs (C
n
-HSL: n 48, 3-oxo-C
n
-HSL: n 48) by
a restored pigment production. C. violaceum CV026 can also be
used for the detection of long chain AHLs (C
n
-HSL: n 1014,
3-oxo-C
n
-HSL: n 1014), because the presence of these AHLs
blocks the detection of the short chain signals.
Several reporter strains have been applied for the rapid
detection of AHLs in culture extracts by a method as simple as
TLC.
80
The procedure is fast and cheap, but a disadvantage of
TLC like any other chromatographic method is that synthetic
standards are required for unambiguous compound identica-
tion. Nevertheless, TLC is extensively used, since today several
C
n
-HSLs (n 4, 6, 7, 8, 10, 12, 14) and 3-OH-C
n
-HSLs (n 6, 8,
10, 12, 14) are commercially available. For the detection of
AHLs after the chromatographic procedure the TLC plates are
overlaid with agar and inoculated with a reporter strain to
visualise the presence of AHLs. Major drawbacks of this method
are that the reporter strains used for AHL detection are naturally
biased towards one or a few autoinducer molecules due to the
selectivity of the LuxR homologue. Furthermore, minor struc-
tural differences between the detected AHL molecules and the
synthetic standards such as methyl branches or C]C double
bonds in the N-acyl chain may not always result in a clearly
different chromatographic behaviour, and, in consequence, the
method bears the risk of wrong compound identications.
Gas chromatography in combination with mass spectrometry
(GC-MS) has been applied for the identication of several AHLs
from a large number of alphaproteobacteria.
73,88
Characteristic
fragment ions can be used to trace back the different classes of
AHLs in culture extracts. The mass spectra of 3-OH-C
n
-HSLs
such as 11 are dominated by fragment ions at m/z 102 and m/z
172 representing the ion C
4
H
8
NO
2
+
originating from the
homoserine lactone moiety and the ion C
7
H
10
NO
4
+
formed by
a-cleavage, respectively, whereas the fragment ions at m/z
102 and m/z 143 (C
6
H
9
NO
3
+
, McLafferty rearrangement) are
typical for C
n
-HSLs such as C
8
-HSL (2). For the quantication
of 3-oxo-C
n
-HSLs in complex culture extracts a useful derivati-
sation method has been developed.
89
The 3-oxo-C
n
-HSLs are
transformed into their pentauorobenzyloxime (PFBO)
derivatives that exist as E and Z diastereomers, e. g. (E)- and (Z)-
13, and detected with high sensitivity in electron capture-negative
ionisation mode. Their mass spectra are dominated by charac-
teristic fragment ions at m/z 181 (C
7
H
2
F
5
) representing the
pentauorobenzyl group and at m/z M-181.
The presence of C]C double bonds in the N-acyl chain of
AHL autoinducers is easily deduced from their molecular ions,
however, the determination of double bond positions or cong-
urations from mass spectra is not possible. A straightforward
method for the localisation of double bonds in AHLs is the
iodine-catalysed addition of dimethyl disulde (DMDS) result-
ing in bis(thiomethyl) derivatives such as 14. The preferential
a-cleavage between the methylthio groups allows conclusions
about the double bond position in the original C
n:1
-HSL.
Notably, a,b-unsaturated AHLs fail to react with DMDS.
Following the localisation of double bond(s) in AHLs the
elucidation of the double bond conguration(s) is possible by an
E/Z-selective synthesis. Using this approach the structures of
(7Z)-C
14:1
-HSL (15) and (2E,9Z)-C
16:2
-HSL (16) have been fully
established.
73,88
The mass spectra of methyl-branched AHLs are very similar to
the mass spectra of their unbranched counterparts, but methyl
branched AHLs elute with signicantly shorter retention times
from the GC column compared to unbranched ones making
GC-MS analysis a useful tool for the identication of methyl
branched AHLs.
73
The position of the methyl branching in the
N-acyl chain can be determined using a retention index-based
empirical model that has previously successfully been applied to
the structure elucidation of methyl branched lipids from several
other compound classes.
9093
The empirical model has been used
to suggest the structures of the rst methyl branched AHLs
iso-C
9
-HSL (17) and 3-OH-iso-C
9
-HSL (18) from A. culicicola.
73
Another extensively used chromatographic method for the
identication of AHLs is HPLC in combination with different
detection methods such as radiodetectors (after feeding of radi-
olabelled methionine), fraction screening with reporter strains, or
mass spectrometry. The usage of capillary electrophoresis is in
contrast rather exotic. Details about the application of these
methods will not be further discussed here, but are summarised in
Tables 24.
Among the spectroscopic methods NMR spectroscopy is the
most powerful technique and has been used for the structure
elucidation of several AHL molecules. The rst structurally
characterised AHL autoinducers have been identied as 3-oxo-
C
6
-HSL (3) from V. scheri,
1
followed by the planar structure of
(R)-3-OH-C
4
-HSL (5) from the related bacterium V. harveyi and
C
8
-HSL (2) as the second messenger of V. scheri.
4,5
After these
initial isolations and characterisations of AHLs from the genus
Vibrio, several other AHL derivatives have been identied by
purication and NMR spectroscopy from a broad range of
Gram-negative bacteria including C
4
-HSL (1) from P. aerugi-
nosa, S. liquefaciens, and Pantoea sp.,
3,10,62,94
3-oxo-C
6
-HSL (3)
from P. carotovorum,
7
3-oxo-C
8
-HSL (19) from A. tumefaciens,
8
and 3-oxo-C
12
-HSL (4) from P. aeruginosa.
6
NMR spectroscopy
is also a very powerful method for the localisation and deter-
mination of the conguration of C]C double bonds in the
N-acyl chain, highlighted by the structure elucidations of
(5Z)-C
12:1
-HSL (20) and (5Z)-3-oxo-C
12:1
-HSL (21) from Meso-
rhizobium sp.,
95
(7Z)-C
14:1
-HSL (15) from Rhodobacter sphaer-
oides,
96
and of (3R,7Z)-3-OH-C
14:1
-HSL (6) from R.
leguminosarum,
11,72
that is also termed small bacteriocin. The
structures of two unsaturated AHLs from Methylobacterium
extorquens have only been solved in part as (Z)-C
14:1
-HSL (22)
and (2E,Z)-C
14:2
-HSL (23) with unidentied localisation of the Z
double bonds.
97
In a few cases the NMR-spectroscopic structure
elucidation of 3-oxo-C
n
-HSLs has been assisted by the applica-
tion of IR spectroscopy.
1,7,8
5 The role of N-acyl-L-homoserine lactones in
biolm formation
First hints for the involvement of AHLs in the formation of
biolms have been obtained by their detection in aquatic
biolms.
187
As outlined in chapter 2, biolms are aggregates of
microorganisms that adhere to a solid surface in a matrix
composed of extracellular biopolymers. Biolms can have
enormous impact on the virulence of pathogens. For example,
Pantoea stewartii that is the etiological agent of Stewarts wilt
in sweet corn, produces the autoinducer 3-oxo-C
6
-HSL (3).
167
This AHL negatively regulates the biosynthesis of the complex
extracellular polysaccharide stewartan composed of a repeating
heptameric subunit.
12,188,189
A gene cluster for the EPS
biosynthesis has been identied that is functionally involved in
the virulence by several mechanisms.
190
The EPS capsules,
efciently protecting the pathogen against plant host defence
factors,
191,192
are required for the formation of lesions by
water-soaking,
193
and cause wilting by a blockage of the free
ow of water in the plant vascular system.
192
Another plant
pathogen with AHL-dependent EPS production is Pseudo-
monas syringae, the causal agent of brown spot disease in
beans.
194
Several human-pathogenic bacteria are known to produce
AHL-dependent biolms. These pathogens are particularly
problematic among cystic brosis (CF) patients. CF is caused
by a genetic defect
195
that affects a chloride ion channel regu-
lating the transport of chloride ions and, as a consequence, of
uids across epithelial cells.
196
The CF gene defect results in
mucoid secretions that form the matrix for chronic bacterial
infections in the lungs. The most important human pathogen in
this context is P. aeruginosa that forms a biolm in the lungs of
CF patients in an AHL-regulated process.
14,15
Another impor-
tant opportunistic pathogen in CF lung infections is Bur-
kholderia cepacia that forms biolms under control of AHL
autoinducers.
78,197
The opportunistic pathogen Serratia marescens, particularly
important in ocular infections and infections of immunocom-
promised patients, is another AHL-dependent biolm-
producer.
198
The formation of biolms has also been shown to be
AHL dependent in the sh pathogen Aeromonas hydrophila,
199
the soil bacterium Ensifer meliloti,
200
and the plant associate
Methylbacterium extorquens.
201
T
a
b
l
e
2
C
n
:
m
-
H
S
L
S
p
e
c
i
e
s
T
a
x
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n
a
4
:
0
5
:
0
6
:
0
7
:
0
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s
o
-
7
:
0
i
s
o
-
7
:
1
8
:
0
9
:
0
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s
o
-
9
:
0
1
0
:
0
1
2
:
0
1
2
:
1
1
4
:
0
1
4
:
1
1
4
:
2
1
6
:
0
1
6
:
1
1
6
:
2
1
8
:
0
1
8
:
1
1
8
:
2
A
c
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6 Antagonists of N-acyl-L-homoserine lactones in
nature
6.1 Enzymatic degradation of AHLs
AHLs can be enzymatically degraded by several species of
bacteria of phylogenetically diverse origin. This potential seems
to be quite widespread in bacteria, since 25 different AHL-
degrading bacterial species were isolated from the rhizosphere of
Nicotiana tabacum. Six strains that efciently metabolised AHLs
were identied as Pseudomonas, Variovorax, Camomonas, and
Rhodococcus spp., respectively, and investigated in detail for
their substrate specicity unravelling a variable potential for
AHL metabolism.
202
This and other studies demonstrated that
some bacteria are catalysing the transformation only of AHLs
with a certain chain length,
202,203
whereas others degrade a broad
range of substrates with different chain length and oxidation
states at C-3 of the N-acyl group.
202,204208
Sometimes only the
natural L form and in other cases both the L and the unnatural
D form can be metabolised.
203,206,209
Recently a few classes of
enzymes have been identied that catalyse the degradation of
quorumsensing messengers, termed quorumquenching enzymes.
The different mechanisms for the transformation of AHLs
include opening of the lactone ring by AHL lactonases, cleavage
of the amide bond by AHL aminoacylases, and transformations
by oxidoreductases.
First hints for a bacterial enzyme that degrades N-acylhomo-
serine lactones were obtained by cloning of the aiiA gene from
Bacillus sp. 240B1 and its heterologous expression in P. car-
otovorum that strongly diminished the virulence of this plant
pathogen.
210
A broad range of taxonomically diverse plants are
attacked by Pectobacterium spp. that are the etiologic agents of
soft-rotting diseases. Different subspecies of P. carotovorum
possess quorum sensing systems that are composed of the AHL
synthases AhlI and usually two receptors ExpR1 and ExpR2.
The synthase of each subspecies produces varying amounts of the
signalling molecules 3-oxo-C
6
-HSL and 3-oxo-C
8
-HSL. ExpR1
is sensitive to 3-oxo-C
8
-HSL, whereas ExpR2 detects both 3-oxo-
C
6
-HSL and 3-oxo-C
8
-HSL.
211
The AHL lactonase fromBacillus
sp. 240B1 transforms both signalling molecules into N-(3-oxo-
hexanoyl)-L-homoserine and N-(3-oxooctanoyl)-L-homoserine,
and thereby the quorum sensing signalling pathway is disrupted.
The potential of N-acylhomoserine lactonases to protect plants
against plant pathogens that control the expression of virulence
genes using N-acylhomoserine lactone-dependent quorum
sensing systems has impressively been demonstrated by the
expression of the AHL lactonase AiiA in N. tabacum and
Solanum tuberosum leading to a resistance against P. car-
otovorum.
204
Besides the P. carotovorum quorum sensing messengers, the
AHL lactonase fromBacillus sp. 240B1 inactivates a broad range
of other AHLs with different chain lengths (412 carbons) and
oxidation states at C-3 of the acyl chain by hydrolysis of the
lactone moiety, e. g. C
4
-HSL and 3-oxo-C
12
-HSL from P. aeru-
ginosa.
205
The substrate specicity of AHL lactonases was further
tested with the respective enzyme from Bacillus thuringiensis
against the two enantiomers of C
6
-HSL. This lactonase hydro-
lyses the lactone moiety of C
6
-L-HSL, whereas the enantiomeric
C
6
-D-HSL is not cleaved.
206
T
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h
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.
AHL lactonases contain a highly conserved
104
HXHXDH
109
H
169
zinc binding motif.
210
Although it was rst
suggested that AHL lactonases are not metalloproteins,
205
it was
shown that the conserved histidine-rich motif indeed binds two
equivalents of zinc that are required for catalytic activity and
form a dinuclear site.
206
The AHL lactonase from Bacillus thur-
ingiensis subsp. kurstaki HD263 has been crystallised indepen-
dently by two different groups,
207,212
and recently the crystal
structure of another AHL lactonase (AiiB) from Agrobacterium
tumefaciens has been obtained.
213
The crystal structures exhibit
the organisation of the catalytically active dinuclear Zn
2+
centre,
in which the rst Zn
2+
ion is coordinated by His-104, His-106,
and His-169, and the second Zn
2+
ion is surrounded by the His-
109, His-235, and Asp-108 ligands. Furthermore, Asp-191
coordinates both zincs in a bridging fashion, and Tyr-194 points
towards the catalytically active centre.
207,212
As shown by
comparison of the
1
H NMR spectra of the dizinc and the apo
AHL lactonase, the presence of two Zn
2+
ions is required for
proper enzyme folding.
214
Site-directed mutagenesis of either of
these highly conserved metal-binding histidine or aspartate
residues and of Tyr-194 in the AiiA enzyme demonstrated their
requirement for catalytic activity (Table 5). All mutated amino
acid residues are critical for the catalytic activity of the enzyme as
shown by a strong reduction by the exchange of the histidine,
aspartate, or tyrosine residues against alanine or leucine,
respectively. However, exchange of histidine against serine or
aspartate against glutamate can result in a residual enzyme
activity (H104S, H106S, D108E, H169S), likely because the
substituting amino acid residues can also function as zinc
ligands. Furthermore, the Y194F and, surprisingly, the H109A
mutants also show some residual activity.
207,210,215
A catalytic
mechanism for the ring-opening reaction of AHL lactonases has
been proposed based on the crystal structures of the enzyme and
its complex with homoserine lactone (Fig. 2).
207
However, an
alternative substrate orientation relative to the two zinc ions
in the tetrahedral transition state was proposed.
213
Incubation
of the AHL substrate C
6
-HSL with the AHL
lactonase from B. thuringiensis in H
2
18
O and D
2
O established the
Table 4 3
0
-OHC
n:m
-HSL
Species Taxon
a
4:0 6:0 7:0 iso-7:0 8:0 iso-9:0 10:0 12:0 14:0 14:1 15:0 16:0
Acidithiobacillus ferrooxidans ATCC1927
98
g +
c,f
+
c,f
+
c,f
+
c,f
+
c,f
98
g +
c,f
+
c,f
+
c,f
+
c,f
+
c,f
99
g +
c,f
+
c,f
+
c,f
+
c,f
+
c,f
98
g +
c,f
+
c,f
+
c,f
+
c,f
+
c,f
Acidithiobacillus ferrooxidans DSMZ583
98
g +
c,f
+
c,f
+
c,f
+
c,f
+
c,f
Acidithiobacillus ferrooxidans DSMZ9464
98
g +
c,f
+
c,f
+
c,f
+
c,f
+
c,f
Acidovorax sp. N35
177
b +
c,f
Acinetobacter baumannii M2
178
g +
b,c,f
Aeromonas culicicola MTCC3249
73
g +
d,f
+
d,f
+
d,f
+
d,f
Azospirillum lipoferum B518
103
a +
b,c,f
Azospirillum lipoferum TVV3
103
a +
b,c,f
Burkholderia mallei ATCC23344
114,115
b +
c
Burkholderia phytormans PsJN
179
b +
b
Burkholderia pseudomallei DD503
119
b +
c,f
Burkholderia pseudomallei PP844
121
b +
c,f
+
c,f
+
c,f
Chromobacterium violaceum ATCC12472
180
b +
b,f
Ensifer meliloti Rm41 (AK631)
129
a +
f
+
f
Erwinia amylovora OMP-BO1077/7
181
g +
b
Herbaspirillum frisingense DSM13130
135
b +
f
Listonella anguillarum 90-11-287
164
g +
b,c,f
+
c,f
+
c,f
Listonella anguillarum NB10
136,164
g +
b,c,f
+
c,f
+
c,f
Mesorhizobium sp. R8-Ret-T53-13d
95
a +
c,f
+
c,f
+
c,f
Pectobacterium carotovorum SCC3193
67
g +
c,f
Phaeobacter gallaeciensis T5
73,88
a +
d,f
Photobacterium phosphoreum P100
182
g +
b,c,f
Pseudomonas chororaphis 3084
142
g +
b,c,f
+
b,c,f
+
b,c,f
+
b,c,f
Pseudomonas chororaphis PCL1391
142
g +
b
+
b
+
b
+
b
Pseudomonas uorescens 279
80
g +
f
+
f
+
f
Pseudomonas uorescens 5064
183
g +
c,f
Pseudomonas uorescens F113
147
g +
c,f
Ralstonia solanacearum AW1
130
b +
b
Ralstonia solanacearum K60
80,130
b +
b
Rhizobium leguminosarum bv. viciae 248
72
a +
g
Rhizobium leguminosarum bv. viciae 8401
11,148
a +
c,f,g
Serratia liquefaciens ATCC27592
152
g +
c,f
Serratia plymuthica IC1270
184
g +
b
+
b
Vibrio harveyi MAV
4
g +
b,f,g
Vibrio scophthalmi A102
185
g +
b,c,f
Yersinia pseudotuberculosis YpIII
159
g +
c,f
+
c,f
Yersinia ruckeri NCIMB1316
161
g +
c,f
Xenorhabdus nematophilus 19061
186
g +
b
a
Taxon: a alphaproteobacteria, b betaproteobacteria, g gammaproteobacteria.
b
Identication by TLC/biosensor assay.
c
Identication by
HPLC.
d
Identication by GC.
e
Identication by CE.
f
Identication by MS.
g
Identication by NMR spectroscopy.
h
IR spectroscopy.
addition-elimination mechanism with attack of water at the
carbonyl group and the alcohol as a leaving group.
214
Kinetic
investigations on the B. thuringiensis AHL lactonase with
different metal substitutions (Zn
2+
, Mn
2+
, Co
2+
, Cd
2+
) demon-
strated a direct involvement of the metal ion in the turnover of
AHL substrates by their binding.
214
To date several AHL lac-
tonases have been identied from Arthrobacter sp.,
216
A. tumefa-
ciens,
217,218
andespeciallydiverseBacillusspp.
206,207,210,212,215,219222
A. tumefaciens produces the autoinducer 3-oxo-C
8
-HSL to
control the conjugal transfer efciency of the Ti plasmid during
the exponential growth phase.
223
The expression of the AHL
lactonase Attm of A. tumefaciens is upregulated when the
bacterial cells enter the stationary phase and Attm catalyses
the degradation of 3-oxo-C
8
-HSL that accumulates during the
exponential growth phase. This mechanism allows the bacterial
cells to exit the quorum sensing phase, terminates the Ti conjugal
plasmid transfer, and establishes an AHL autoregulatory
circuit.
217
Interestingly, three AiiA homologs are encoded by the
genome of this species that are all located on plasmids. One AHL
lactonase gene (aiiB) is located together with the AHL synthase
gene traI, the traR gene for its cognate receptor, and virulence
genes on the Ti plasmid, suggesting that the virulence genes along
with the genes for their regulation can be transferred to another
host.
218
Under alkaline conditions AHLs undergo the same ring-
opening reaction of the lactone moiety as catalysed by AHL
lactonases. The N-acylhomoserine lactones become unstable
within a narrow physiological pH range (pH 78) and rapidly
undergo lactonolysis at higher pH.
224,225
This characteristic of
AHLs is used by eukaryotic organisms that can combat patho-
genic bacteria by the non-enzymatic degradation of their
N-acylhomoserine lactones. Interestingly, plants that have been
infected with Pectobacterium immediately respond by increasing
the pHof the apoplastic uid around the source of infection from
pH < 6.4 to pH > 8.2 that is accomplished by a rapid proton
inux into the cells.
226
A second class of AHL-degrading enzymes was identied as
AHL aminoacylases that cleave the amide bond of AHLs. A
pure culture of the betaproteobacterium Variovorax paradoxus
was isolated from soil and is able to grow on a broad range of
AHLs with different acyl chain lengths (412 carbons) as the
sole carbon and nitrogen source. The AHLs are degraded to
L-homoserine lactone and a fatty acid derivative that is further
metabolised as an energy source, but the encoding gene as well
as the enzyme that catalyses the cleavage reaction remains to be
identied from this species.
208,227
However, such genes and
enzymes have subsequently been reported from other bacteria.
The gene aiiD was cloned from the biolm isolate Ralstonia
eutropha XJ12A and XJ12B (betaproteobacteria) and
sequenced. The puried gene product AiiD catalyses the rapid
conversion of 3-oxo-C
8
-HSL, 3-oxo-C
10
-HSL, and 3-oxo-C
12
-
HSL into L-homoserine lactone and the respective 3-oxo fatty
acid, whereas degradation of short-chain AHLs is slow. AiiD
shares several highly conserved amino acid residues with other
acylases. Site-directed mutagenesis of Gly-232 and Ser-233
Table 5 Analysis of functional residues in AHL lactonases by site-
specic mutagenesis
Enzyme Activity
d
References
WT 1.00
H104S,
a
H104A
b
1.00, 0.01 207,210
H106S,
a
H106A
b
0.61, 0.02 207,210
D108E,
a
D108S,
a
D108A
b
0.91, 0.00, 0.02 207,210
H109S,
a
H109A
b
0.00, 0.16 207,210
H169S,
a
H169A
b
0.61, 0.01 207,210
D191A,
b
D191L
b
0.02, 0.01 207
Y194F,
b
Y194A
c
0.33, strong reduction 207,215
H235A
b
0.15 207
a
Experiment carried out with the AHL lactonase AiiA from Bacillus sp.
240B1.
b
AiiA from Bacillus thuringiensis subsp. kurstaki HD263.
c
AiiA
from Bacillus cereus Y2.
d
Relative activity compared to the respective
wildtype enzyme.
Fig. 2 Catalytic mechanismfor the hydrolysis of the homoserine lactone ring of AHLs by AHL lactonases (A),
207
and alternative tetrahedral transition
state with reversed substrate orientation relative to each zinc (B).
213
demonstrated their importance for catalytic activity. Heterolo-
gous expression of AiiD in P. aeruginosa disrupts quorum
sensing by this bacterium.
228
Surprisingly, AHL acylases are
also produced by P. aeruginosa itself. P. aeruginosa PAO1
degrades long-chain AHLs (814 carbons), but AHLs with
a shorter acyl chain are not used. Both the L and the D forms are
transformed into L-homoserine lactone and the fatty acid
derivative via the acylase mechanism. In contrast to R. eutropha
the initial product L-homoserine lactone is hydrolysed to
L-homoserine after prolonged incubations, suggesting the
enzymatic activity of a HSL lactonase in Pseudomonas. Two
AHL acylases are encoded by the genes pvdQ and quiP in
Pseudomonas.
203,229
The AHL acylase PvdQ has been puried,
and this enzyme degrades only AHLs with a long-chain N-acyl
group (1114 carbons) regardless of the oxidation state in the
3-position.
230
The heterologous expression of PvdQ or QuiP in
E. coli enables the degradation of long chain AHLs by this
species. Furthermore, the constitutive expression of either of
these enzymes in P. aeruginosa prevents the formation of its
native messenger 3-oxo-C
12
-HSL, but not of C
4
-HSL.
203,229
Both L-homoserine lactone messengers are produced by the
wildtype, and therefore the expression of these AHL acylases in
the wildtype seems to be tightly regulated to allow the
production of 3-oxo-C
12
-HSL in early growth phases or up to
certain levels. Both AHL acylases might be used to regulate the
exit from quorum sensing processes in P. aeruginosa similar to
the autoregulation of AHL levels in A. tumefaciens by the AHL
lactonase Attm.
203,229,230
In contrast to the AHL acylases from
P. aeruginosa the AHL acylase AiiC from Anabaena sp.
PCC7120 has a broad substrate specicity and is able to cleave
AHLs with different chain lengths (414 carbons) and oxidation
states at C-3, but degradation of long-chain AHLs is faster.
231
Streptomyces sp. M664 secretes the AHL acylase AhlM into the
culture medium that efciently degrades long-chain AHLs,
whereas C
6
-HSL and 3-oxo-C
6
-HSL are only slowly metab-
olised, and C
4
-HSL is not cleaved at all.
232
Rhodococcus eryth-
ropolis W2 that has been isolated from the rhizosphere of
Nicotiana tabacum also shows an AHL acylase activity, and, in
addition, an oxidoreductase activity that transforms the L- and
D-enantiomers of 3-oxo-C
n
-HSLs into the respective 3-OH-C
n
-
HSLs.
209
A combined mechanism for the degradation of N-acylhomo-
serine lactones was described to occur in the gut of lepidopteran
larvae that accommodate a broad diversity of AHL-producing
bacteria such as Acinetobacter spp., E. coli, Pseudomonas spp.,
Enterobacter spp., Ochrobactrum spp., and Erwinia spp.
233,234
Due to the basic pH in the foregut and the midgut of the cater-
pillars a rapid ring opening of the bacterial AHLs to N-acylho-
moserines takes place.
235
Large amounts of N-acylglutamines
with structural similarity to N-acylhomoserines are present in the
gut of lepidopteran larvae that induce the emission of plant
volatiles from damaged leaves. These volatiles in turn attract
parasitic wasps that are the natural enemies of the insect
larvae.
236,237
An N-acylamino acid hydrolase (AAH) from the
insect gut bacterium Microbacterium arborescens was cloned,
puried, and functionally characterised; the enzyme cleaves
N-acylglutamines into the respective fatty acids and L-gluta-
mine,
238
and also N-acylhomoserines into fatty acids and
L-serine, but not AHLs.
235
6.2 Secondary metabolites
Besides AHL-antagonistic enzymes some species produce small
molecules that block the effect of AHLs. An extensively inves-
tigated class of compounds includes brominated furanones that
have been isolated from the macroalga Delisea pulchra. Among
these, compounds 24 and 25 reduce the AHL-dependent
swarming motility in Serratia liquefaciens and luminescence in
V. scheri by a blockage of the receptor site of the LuxR
homologue,
239
and 24 inhibits AHL-regulated luminescence and
toxin production in a virulent strain of V. harveyi.
240
The related
compound 26 inhibits the biosynthesis of the carbapenem anti-
biotic 1-carbapen-2-em-3-carboxylic acid and the production of
virulence factors in P. carotovorum.
241
Although E. coli is not
able to synthesise AHL molecules due to a lack of a luxI gene, it
possesses the LuxRhomologue SdiAand responds to AHLs such
as C
6
-HSL and 3-oxo-C
6
-HSL.
242
As in V. harveyi, in E. coli
another quorum sensing system using the furanosyl borate
diester 27 (AI-2) is fully functional (chapter 7).
243
This auto-
inducer is used by a wide range of bacteria and serves as a sig-
nalling compound in inter-species communication. The
brominated furanone 24 interrupts biolm formation in E. coli
and the expression of virulence in V. harveyi by interference with
the AI-2 system, making compound 24 a non-specic auto-
induction antagonist.
244246
Indole (28) that is secreted into the
medium by E. coli in the stationary phase, decreases biolm
formation mediated by its detection by the SdiA receptor.
247
Its
derivatives 5-hydroxyindole (29) and 7-hydroxyindole (30) also
strongly suppress the formation of biolms in E. coli, whereas
isatin (31) induces biolm formation.
248
The two brominated
tryptophane-derived alkaloids 32 and 33 have been isolated from
the North Sea bryozoan Flustra foliaceae and reduce signal
intensities in different AHL reporter strains.
249
The fungus Fusarium oxysporum produces the picolinic acid
derivative fusaric acid (34). This compound suppresses the
production of AHLs in Pseudomonas chlororaphis, and in
consequence the AHL-dependent biosynthesis of the antifungal
metabolite phenazine-1-carboxamide (35).
250
Finally, Medicago
sativa produces L-canavanine (36) that interferes with the
quorum sensing system of its nitrogen-xing symbiont Ensifer
meliloti and inhibits the expression of genes for the production of
exopolysaccharides in the bacterium.
251
Three AHL inhibitors, oridoside (37), betonicine (38), and
isethionic acid (39), have been isolated from the red alga Ahn-
feltiopsis abelliformis in a bioactivity-guided fractionation
approach using an AHL reporter strain.
252
The two structurally related acetogenins 40 and 41 have been
isolated from Annona cherimolia. Curiously, 40 promotes the
formation of biolms in P. aeruginosa, whereas 41 is inhibitory.
The sesquiterpene lactones 4247 from Acanthospermum hispi-
dum all inhibit the production of biolms by P. aeruginosa.
253
The diterpenoid salvipisone (48) isolated from roots of Salvia
sclarea inhibits the biolm formation in the Gram-positive
bacteria Staphylococcus aureus and S. epidermis.
254
These
bacteria like other Gram-positives do not use AHLs for cell-cell
communication, and the mode of action of 48 is unknown. The
plant metabolite salicylic acid (49) that has an important role in
plant defense mechanisms during bacterial infection, down-
regulates the virulence factor production and biolm formation
of the plant pathogen P. aeruginosa.
255
6.3 Biolm inhibition by azithromycin
Antibiotic treatment of bacterial infections is complicated by the
formation of mature biolms in terms of the resistance of
bacterial cells within the biolm matrix. Therefore it is of high
importance to prevent the formation of biolms during infec-
tions to enable an effective antibiotic therapy. A promising
possibility to treat biolm infections is the suppression of biolm
formation by the interruption of the cell-cell communication
systems of pathogenic bacteria, while bacterial growth is not
inhibited and the evolvement of bacterial resistance might be of
lowrisk. Several compounds presented in chapter 6.2 are efcient
quorum sensing blockers, but their usage as drugs may be pre-
vented by their high toxicity to humans. In contrast, azi-
thromycin (50) is a macrolide antibiotic that is approved for
clinical use and marketed by Pzer. Macrolide antibiotics are
active against Gram-positive bacteria and inhibit the protein
biosynthesis in binding to the bacterial ribosomes.
256
In contrast,
these antibiotics do not have a signicant effect against Gram-
negative bacteria such as P. aeruginosa, e. g. the minimum
inhibitory concentration (MIC) of 50 is above 1 mg mL
1
.
However, at sub-inhibitory concentrations azithromycin has
a benecial effect on cystic brosis patients.
257,258
At these levels
the antibiotic has been shown to retard the production of bio-
lms, to reduce virulence factor production, and to interrupt
quorum sensing.
259262
In spite of this promising perspective
further investigations will be needed for the development of
drugs against biolm infections.
7 A novel genus-specic autoinducer from Vibrio
The Gram-negative, luminescent marine bacterium V. harveyi
exists in a free-living form and as an associated pathogen in
marine animals.
263
Similarly, the life cycle of the human pathogen
V. cholerae includes a free-living and a host-associated phase, in
which it causes serious epidemic infections of the human intestine
resulting in the diarrhoeal disease cholera. The interepidemic
free-living period in the aquatic environment requires the
formation of biolms on surfaces of the ora and fauna. The
disease is acquired by oral ingestion of contaminated water or
food sources. The virulence of V. cholerae depends on two critical
factors. Colonisation of the human intestine by V. cholerae is
mediated by the toxin-coregulated pilus that is primarily
responsible for the adhesion of bacteria to the intestine
epithelium. Enterotoxicity is caused by cholera enterotoxin,
a potent exotoxin that activates the production of cyclic AMP
(cAMP) from ATP. The increased intracellular cAMP levels
result in an increased Cl
secretion into the lumen causing an

osmotic gradient. In consequence, massive volumes of water ow
into the lumen and exceed its volumetric capacity, leading to the
clinical disease pattern of an intense watery diarrhoea that can
quickly result in severe dehydration and death.
264
Three autoinduction systems have been reported from
V. harveyi, two of which also operate in V. cholerae (Fig. 3).
Moreover, genetic information manifests the presence of all three
signaling cascades in V. parahaemolyticus.
265
The rst quorum
sensing molecule is the species-specic signal Harveyi auto-
inducer 1 (HAI-1, (R)-3-OH-C
4
-HSL, 5),
4,71
that is produced by
LuxM and detected by the two-component hybrid sensor kinase
LuxN.
266
The autoinducer 2 (AI-2), furanosyl borate diester
27,
267
is produced and detected by various bacteria and believed
to be involved in interspecies communication.
243,268
The unbo-
rated AI-2 precursor is made by LuxS,
243,269
from which AI-2 is
formed spontaneously, and response to AI-2 is dependent on the
LuxPQ sensor.
270
LuxP is a member of a large family of peri-
plasmic receptor proteins for the binding of diverse ligands,
271
and the crystal structure of AI-2 bound to LuxP from V. harveyi
has been solved,
267
whereas LuxQ is a two-component hybrid
sensor kinase similar to LuxN.
270
The V. cholerae and V. para-
haemolyticus genome sequences revealed that the AI-2 system is
also used by this species, whereas the genes representing the
V. harveyi HAI-1 system are only present in V. para-
haemolyticus.
265,272
A second quorum sensing cascade in
V. cholerae uses the Cholerae autoinducer 1 (CAI-1) that is
Fig. 3 Quorum sensing systems in V. cholerae, V. harveyi, and V. parahaemolyticus. A: signaling cascade at low cell densities, B: signaling cascade at
high cell densities.
synthesised by CqsA and detected by the CqsS receptor.
273
The
crystal structure of the CqsA protein was obtained and led to
a suggested biosynthetic pathway of CAI-1 from decanoyl-CoA
and an amino acid such as L-2-aminobutyric acid or L-threo-
nine.
274
A V. cholerae reporter strain carrying the luxCDABE
luciferase operon was used to detect CAI-1 activity in several
other Vibrio spp. In particular, this experiment revealed that the
CqsA/CqsS system is also present in V. harveyi and V. para-
haemolyticus establishing the CAI-1 system as a third quorum
sensing circuit in these species.
275
This nding was corroborated
by the genetic information of V. parahaemolyticus.
265
Recently,
the structure of the underlying signalling molecule has been
determined as (S)-3-hydroxytridecan-4-one (51).
276
This novel
type of autoinducer is not only structurally unique, but is also the
rst example of a genus-specic signaling molecule.
The signals of all three autoinducers are relayed from their
sensor kinases LuxN, LuxQ, and CqsS, respectively, by a phos-
phate transfer mechanism via the histidine phosphotransferase
LuxU to the negative response regulator LuxO (Fig. 3).
277279
At
low cell densities in the absence of autoinducers the autophos-
phorylating sensor kinases transduce information to LuxU by its
phosphorylation that in turn passes on phosphate to LuxO.
279
LuxO-phosphate is an activator protein that together with the
sigma factor s
54
encoded by rpoN initiates the expression of
genes to yield four small regulatory RNAs (sRNAs) in V. chol-
erae termed Qrr14 (quorum regulatory RNAs).
280,281
The
sRNAs in cooperation with the sRNA chaperone Hfq control
the expression of the transcriptional regulators LuxR (V. harveyi
and V. parahaemolyticus, this protein is different from LuxR in
V. sheri) or HapR (V. cholerae), respectively, by destabilisation
of their mRNAs.
282,283
In contrast, at high cell densities the
sensors LuxN, LuxQ, and CqsS switch from kinase to phos-
phatase activity inverting the direction of phosphate ux.
Dephosphorylated LuxO is inactive, the qrr genes are no longer
transcribed, and LuxR or Hap, respectively, are produced. A
detailed biochemical study showed that the HAI-1 sensor LuxN
autophosphorylates with high ATP turnover rates, and LuxN-
phosphate quickly phosphorylates LuxU, whereas phosphatase
activity of LuxN against LuxU-phosphate is slow. HAI-1
strongly decreases the kinase, but not the phosphatase activity of
LuxN, suggesting that the autoinducer-mediated repression of
the LuxN kinase activity characterises the key regulatory
mechanism in the HAI-1 quorum sensing system of V. harveyi.
284
But how are the quorum sensing cascades linked to pathoge-
nicity and biolm formation in these species? In fact, the tran-
scriptional regulators LuxR and HapR, respectively, control the
expression of several genes involved in biolm formation and the
production of virulence factors in Vibrio. Both traits are
controlled by closely linked regulatory cascades, and a common
discussion of these phenomena allows some insights into inter-
esting ecological aspects of these pathogens. V. parahaemolyticus
and V. harveyi express type III secretion systems (TTSS) that are
also used by several other pathogenic bacteria to inject bacterial
virulence factors via a needle-like structure into the cytoplasm of
target host cells. The respective gene clusters encoding for TTSSs
have been identied in both Vibrio spp. Their expression is in
contrast to TTSSs in other species negatively regulated by
quorum sensing, meaning that in growth phases with a low cell
density, during which the formation of LuxR is suppressed,
TTSS-related virulence occurs, while at high cell densities the
non-virulent form evolves.
285
In V. cholerae a TTSS is missing,
but instead the strong pathogenicity of V. cholerae is caused by
the synthesis of cholera enterotoxin and of the toxin-coregulated
pilus that are under control of the quorum sensing systems
(Fig. 4).
286
Virulence gene expression depends on a complex
regulatory cascade termed the ToxR virulence regulon.
287
Its
Fig. 4 Regulatory cascade for the expression of genes for virulence factors and biolm formation in V. cholerae.
central transcriptional regulators are AphA and AphB, and at
high cell densities HapR binds to aphA and prevents its expres-
sion.
288
At low cell densities the AphA promoter is generated and
activates in direct contact with AphB the tcpPH operon that
encodes the regulatory protein TcpP.
289,290
The activity of TcpP is
enhanced by ToxR that is encoded by the toxRS operon, and
their concerted action promotes the expression of ToxT, the
major regulator of virulence gene transcription in V. cholerae.
291
ToxT binds to AT-rich regions directly upstream of the ctxAB
genes that encode for cholera enterotoxin biosynthesis, or tcpA,
the rst gene of a large operon (tcpAF) for the toxin-coregulated
pilus, and activates the expression of these genes.
292294
Another parallel regulatory cascade controls the expression of
virulence genes and genes for the formation of biolms in
V. cholerae via the quorum sensing-dependent transcriptional
regulator HapR and the bacterial second messenger
bis(3
0
,5
0
)-cyclic diguanylic acid (53, c-di-GMP). One branch of
this pathway functions also via the regulator pair AphA/AphB
by the AphA-dependent repression of the acgA and acgB tran-
scriptions. The respective gene products AcgB and AcgA contain
the conserved motifs GGDEF and EAL and modulate the
intracellular c-di-GMP levels by activating its synthesis from
guanosine triphosphate (52, GTP, activated by GGDEF) or its
degradation to 5
0
-O-phosphonoguanylyl-(3
0
/5
0
)-guanosine
(54, pGpG, activated by EAL, Scheme 3).
295,296
A third newly
discovered class of proteins involved in the modulation of
c-di-GMP concentration exhibits HD-GYP motifs and degrades
c-di-GMP to guanosine monophosphate (55, GMP).
297
Other
diverse genes in V. cholerae encode proteins that belong to either
of the three types that catalyse c-di-GMP synthesis or degrada-
tion to modulate the intracellular c-di-GMP level.
298
The tran-
scription of several of these genes is controlled by the quorum
sensing regulator HapR.
299
The net result of this altered tran-
scription levels are a depletion of the c-di-GMP concentration at
high cell density, whereas at low cell density an increase of the
c-di-GMP pool is found. The EAL domain enzyme VieA is part
of the incompletely characterised VieSAB signaling system that
seems not to be controlled by HapR, but possibly by environ-
mental signals.
300,301
The expression of ToxT is negatively regulated by c-di-GMP,
and therefore high levels of c-di-GMP prevent the transcription
of virulence genes, whereas low concentrations promote viru-
lence.
300
Biolmformation in V. cholerae is also controlled by the
second messenger c-di-GMP.
302
The V. cholerae biolms result
from secretion of a matrix-forming exopolysaccharide (EPS). A
biosynthetic gene cluster for EPS with two operons (vpsAK and
vpsLQ) and two regulators that each promote the expression of
both operons (vpsR, vpsT) has been identied.
303306
Direct
evidence for the regulation of the vps genes by c-di-GMP was
given by a transcriptome analysis at an increased c-di-GMP level,
in which V. cholerae responds with an increased transcription of
the vps genes.
307
Before knowledge about the bacterial second
messenger c-di-GMP accumulated, investigations on the
V. cholerae quorum sensing regulatory system revealed that the
expression of the vps biosynthetic gene cluster is negatively
controlled by the central transcriptional regulator HapR, i.e.
biolm formation is favoured at low cell density and suppressed
at high cell densities.
308,309
The recent unravelling of the signaling
Scheme 3 Synthesis and degradation of c-di-GMP (53) by GGDEF, EAL, and HD-GYP proteins.
pathways via the second messenger c-di-GMP provides an
explanation for this nding, although not all participating
mechanisms in the regulatory cascades are understood to date.
In summary, low cell densities coincide with low concentra-
tions of the three V. cholerae autoinducers CAI-1, HAI-1, and
AI-2. Their sensors function as kinases and relay the signal by
phosphate transfer to LuxO allowing the transcription of
sRNAs. This blocks the expression of HapR, and the tran-
scription levels of different GGDEF and EAL domain proteins
are altered, resulting in a net increase of the intracellular
c-di-GMP pool. This nally causes the expression of vps genes
and the formation of biolms. Contrarily, a high cell density
results in a strong autoinduction signal, and the sensor proteins
switch to phosphatase activity. LuxO-phosphate is dephos-
phorylated and the sRNAs are not transcribed resulting in HapR
expression. This has a reverse effect on the overall expression of
GGDEF/EAL domain enzymes followed by a decreased c-di-
GMP concentration. The biolm transcriptional activator vpsT
is not induced, but rather blocked by HapR. Virulence and
biolm formation in V. cholerae are not only controlled by
autoinduction, but also by environmental signals such as
temperature and pH. The environmental signals modulate the
intracellular level of c-di-GMP via the incompletely characterised
VieSAB signaling system to co-regulate virulence and biolm
formation in V. cholerae.
The regulation of biolm formation and expression of viru-
lence factors is in sharp contrast to the quorumsensing control of
these traits in several other (pathogenic) bacteria. Usually, bio-
lm formation and virulence are favoured in dense populations,
whereas in V. cholerae both characteristics are specic to the low
cell density state and downregulated in the highly populous
phase, providing a perfect adaptation to the requirements of its
life cycle. During the low density aquatic stage V. cholerae forms
biolms on the surfaces of the fauna and ora that protect the
bacteria against several environmental stresses. These biolms
also protect V. cholerae in acidic environments and therefore aid
in the passage through the gastric barrier of the stomach, while
the quorum-sensing controlled detachment from biolms is
advantageous for the subsequent colonisation of the intestinal
epithelium.
310
The repressed virulence and biolm formation at
high cell densities enable V. cholerae to re-enter the environment
in large numbers to start a new infectious life cycle.
8 Conclusions
Bacterial biolms are remarkable structures in which unicellular
microorganims organise a multicellular way of life. The biolm
architecture is mainly built up from extracellular polysaccharides
providing the structural basis for improved nutrient supply and
protection against environmental stresses such as antibiotics. In
many bacteria the biosynthesis of these extracellular poly-
saccharides and thus biolm formation is a quorum sensing-
dependent process involving autoinducer molecules, but as in the
Vibrio genus autoinducers can also suppress biolm formation.
Indeed, autoinduction is not only responsible for biolm
formation, but also for many other processes such as expression
of virulence factors and the biosynthesis of antibiotics. In Gram-
negative bacteria AHLs are an important and well-investigated
class of species-specic autoinducers, whereas the furanosyl
borate diester is an inter-species signal that can be detected by
many diverse bacteria. Nature has developed different mecha-
nisms to interrupt bacterial communication, obviously to
specically disturb these AHL-dependent processes. The AHL
molecules can be enzymatically degraded, or the communicatory
channel can be blocked by autoinducer antagonists. Among the
most intriguing antagonistic secondary metabolites are bromi-
nated furanones from the macroalga Delisea pulchra that inter-
rupt cell-cell communication both via AHLs and the furanosyl
borate diester.
Downstream intracellular processing of the extracellular
bacterial quorum sensing signals can involve highly complex
regulatory cascades as demonstrated exemplarily for the Vibrio
genus, for which a profound knowledge has accumulated, giving
a clear picture of how the expression of virulence factors and the
formation of biolms is regulated by autoinducers within this
genus.
A clear and detailed picture about the mechanisms of biolm
formation is highly important to combat bacterial biolms,
where they are unwanted, e. g. for the development of antibiotics
with a new mode of action against important human pathogens
such as P. aeruginosa. This is of utmost importance, since the
formation of biolms by pathogenic bacteria signicantly
increases their tolerance against known antibiotics, and also
because the available antibiotics run the risk of becoming more
and more useless due to the emergence of multi-resistant strains.
The interruption of quorum sensing signalling by blockage of
autoinducer synthases or their cognate receptors may provide
a new and useful strategy to encounter bacterial biolm infec-
tions.
9 Acknowledgements
Funding by the Deutsche Forschungsgemeinschaft DFG as an
Emmy Noether Fellow and within the Transregional Collabo-
rative Research Centre Ecology, Physiology and Molecular
Biology of the Roseobacter clade is gratefully acknowledged. I
thank Prof. Dr Stefan Schulz for excellent support.
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Quorum sensing and bacterial biolms

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