2010 npr quourum sensing and bacterial biofilms.pdf

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Quorum sensing and bacterial biofilms

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 biofilms including the chemistry of their

constituents and signalling compounds that mediate or inhibit the formation of biofilms. Systems are

described with special emphasis, in which quorum sensing molecules (autoinducers) trigger the

formation of biofilms. In the first 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 first discovery of an AHL

autoinducer fromVibrio fischeria large and further increasing number of different AHL structures

from Gram-negative bacteria have been identified. This review gives a summary of all known AHL

autoinducers and producing bacterial species. A few systems are discussed, where biofilm formation is

suppressed by enzymatic degradation of AHL molecules or interference of secondary metabolites fromother 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 biofilm formation are discussed for the Vibriogenus that has

been extensively investigated.

1 Introduction

2 Chemistry of biofilms

3 Biosynthesis ofN-acyl-L-homoserine lactones

4 Detection and identification of N-acyl-L-homoserine

lactones

5 The role of N-acyl-L-homoserine lactones in biofilm

formation6 Antagonists ofN-acyl-L-homoserine lactones in nature

6.1 Enzymatic degradation of AHLs

6.2 Secondary metabolites

6.3 Biofilm inhibition by azithromycin

7 A novel genus-specific 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 flasks in laboratories around the

world, where bacteria are grown in liquid media. Antonie van

Leeuwenhoek, who first recognised the presence of animal-

cules in rain water and saliva, also encountered planktonic

bacteria. He was again the first to find a sessile form of bacteria in

dental plaque, where the microorganisms live in a biofilm, but

van Leeuwenhoek didnt realise the difference between these two

forms of bacterial life with his simple microscope. Today it is

known that bacterial biofilms are widespread in nature and are

formed by nearly all bacterial species. Biofilms enable bacteria to

grow on surfaces in a self-produced polymeric matrix. These

biofilms 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 biofilm 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 biofilms 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-specific as the

nature of itsN-acyl chain varies from species to species. The firstsignal molecule of this type was identified fromVibrio fischerias

N-(30-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-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 significant 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: [email protected]

This journal is The Royal Society of Chemistry 2010 Nat. Prod. Rep., 2010, 27, 343369 | 343

REVIEW www.rsc.org/npr | Natural Product Reports


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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-

specific 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 in3, a methylene as inN-butyryl-L-homoserine

lactone (C4-HSL, 1) from Pseudomonas aeruginosa,3 or

a 3-hydroxy group as inN-((30

R)-hydroxybutyryl)-L-homoserinelactone ((R)-3-OH-C4-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-C6-HSL system

in V. fischeri a second AHL autoinduction cascade synthesises

and detects N-octanoyl-L-homoserine lactone (C8-HSL, 1).5

Furthermore, N-(30-oxododecanoyl)-L-homoserine lactone

(3-oxo-C12-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 defined by a termi-

nology as used for fatty acids. Generally, Cn-HSL indicates

a saturatedN-alkanoyl group and iso-Cnis used as an identifier

for aniso-branched,i.e.u-1 methyl branched N-alkanoyl group

with n carbons. Oxygen functionalities in the 3-position of the

attachedN-alkanoyls are mentioned using the 3-oxo and 3-OH

prefixes for N-3-oxoalkanoyl and N-3-hydroxyalkanoyl deriva-

tives, respectively. The usage of Cn:m descriptors indicates the

presence of m double bonds in an N-alkanoyl chain with n

carbons, but saturated chains follow the Cn and not the Cn:0terminology unless the usage of Cn:0is required for unambiguity.

The positions and E or Z configurations as well as the

R stereochemistry of 3-OH functions (S stereochemistry hasnever been described so far) are denoted by the usual stereo-

chemical descriptors as prefixes. To give an example, the AHL

molecule from Rhizobium leguminosarum, N-((30R,70Z)-30-

hydroxytetradec-70-enoyl)-L-homoserine lactone (6), also known

as small bacteriocin, is briefly named (3R,7Z)-3-OH-C14:1-

HSL following this terminology.

Besides the mentioned luminescence in V. fischeri 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 conjugaltransfer 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 biofilm formation in

P. aeruginosa,1315 and many more are expressed by quorum

sensing control. The focus of this review will, in the first instance,

be on the mechanisms and chemical signals that are involved in

the formation of biofilms.

Knowledge about the mechanisms of bacterial biofilm

formation is particularly important, because biofilms constitute

many persistent and chronic infections with inherent resistance

to antibiotics.16 Biofilm infections are frequently seen onimplantations 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 fibrosis

patients and forms a biofilm in the sputum.13 These chronic

infections result in progressive lung damage and finally death by

respiratory failure for most cystic fibrosis patients, and due to the

biofilmarchitecture of the P. aeruginosa colonies in these patients

even long-term antibiotic therapy does not eradicate the infective

agent.18 Biofilm 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 ofProf. Dr Rolf Muller 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


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P. aeruginosa is operating through the two N-acylhomoserine

lactone autoinducers C4-HSL (1) and 3-oxo-C12-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-C12-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 rhlsystem, RhlI catalysesthe synthesis of the signalling molecule C4-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

TheluxI-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 fromV. fischeri, the las operon fromP. aeruginosa, orthetra operon inA. tumefaciens.2428

In this review a brief summary of the accumulated knowledge

about the chemistry of biofilms 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 configurations 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 identification of AHLs. At the end of thischapter 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 biofilms (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 biofilm 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 biofilms can be controlled.31 Specific aspects of the

biofilm formation 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

modified peptides as autoinduction signals that are involved in

the formation of biofilms. This type of autoinduction system will

not be discussed here, but has been described in previous

reviews.34,35

2 Chemistry of biofilms

Bacterial biofilms 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 ofStigmatella

aurantiaca and sporulation of Streptomyces coelicolor that are

controlled by stigmolone and the A-factor, respectively.3638

The biofilm matrix typically consists of extracellular poly-

saccharides (also termed exopolysaccharides, EPS), and can alsocomprise proteins and DNA, but the precise structure and

composition of biofilms strongly vary with the resident species

and environmental conditions. Before going into the details



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about the structure and mode of action of signalling molecules

that initiate biofilm formation, this chapter provides a brief

introduction to the chemistry of biofilms.

As outlined above, EPSs usually belong to the major compo-

nents of bacterial biofilms. These EPSs can be composed of only

one type of monosaccharide or can be formed from various

monosaccharides that are usuallyO- or N-acylated, respectively.

A widespread EPS is poly-b-1,6-N-acetylglucosamine (7) that is

excreted by Actinobacillus pleuropneumoniae,39 Staphylococcusaureus,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 is8 fromErwinia 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 1H and 13C NMR

spectroscopy.42 Cellulose (9) is the most abundant organic

molecule in nature, and is also an important component of the

biofilm matrix of bacteria such as SalmonellaandE. coli.43,44 The

mucoid phenotype ofP. aeruginosa that causes chronic respira-tory infections in cystic fibrosis patients is characterised by an

overproduction of alginate (10),45,46 a polysaccharide that is

composed ofa-L-guluronic anda-L-mannuronic acid. For many

years 10 was generally believed to be a matrix component of

P. aeruginosa biofilms, but recent investigations revealed that

10 is indeed not a significant component of non-mucoid strains

such asP. aeruginosa PAO1 and PA14.47

Extracellular DNA is required for the formation of biofims in

P. aeruginosa PAO1, and young biofilms can be dissolved by

DNAse treatment, whereas mature biofilms are not affected by

DNAse, suggesting that the stability of the matrix in young

biofilms highly depends on extracellular DNA, whereas other

components are holding the cells together in aged biofilms.48 Incontrast, mature biofilms 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 ofP. aeruginosa PAO1 biofilms, gene inac-

tivation experiments in a putative EPS biosynthetic gene cluster

(psl, polysaccharide synthesis locus) revealed the critical role of

an unidentified EPS for the biofilm matrix.50,51 Similar investi-

gations identified two loci in P. aeruginosa PA14 and ZK2870,psl

and pelthat are involved in the production of a mannose-rich

and a glucose-rich matrix material, respectively. Maturation of

P. aeruginosa biofilms requires at least one of these EPSs, and

either of the two matrix components is sufficient for the forma-tion of a mature biofilm.52,53 Recent investigations revealed that

extracellular DNA in P. aeruginosa PAO1 biofilms 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 ofN-acyl-L-homoserine lactones

Early feeding experiments with 15N-labelled methionine sug-gestedS-adenosyl-L-methionine (SAM) as a precursor for AHL

biosynthesis.55 Winans and coworkers showed by an incubation

experiment with the purified recombinant AHL synthase TraI

fromA. tumefaciensthat bacteria synthesise the AHL molecules

from acyl carrier protein (ACP) bound fatty acyl derivatives, but

not fatty acyl-CoA derivatives, and SAM (Scheme 1).56 The same

observations have been made independently and in the same year

by Greenberg and coworkers on the purified LuxI protein from

V. fischeri.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)configured, although the absolute configuration has only been

experimentally established in a few cases: by circular dichroism

for (S)-3-oxo-C6-HSL ((S)-3) from Pectobacterium car-

otovorum,7 by chiral GC for (S)-C6-HSL from Erwinia psidiias

well as (S)-C6-HSL and (S)-C7-HSL fromPantoea ananatis,5961

and, using both methods, to elucidate the Sconfiguration of C4-

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 theamino group of SAM can be of varying chain length, saturated

or unsaturated in different positions with Eor Z double bond

geometry, oxidised in the 3-position (3-OH or 3-oxo), and methyl

branched, depending on the substrate specificity of the LuxI-type

protein. In summary, the variety of the N-acyl chains represents

a broad range of modifications that are generated in fatty acid

biosynthesis. The LuxI homologue often shows a preference for

one specific 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 pathogenYersinia pestisproduces mainly

3-oxo-C8-HSL in combination with the two carbonsshorter 3-oxo-C6-HSL (3) and the two carbons longer 3-oxo-

C10-HSL. In addition, C8-HSL (2) and traces of C6-HSL with

Scheme 1 LuxI-catalysed synthesis ofN-acyl-L-homoserine lactones from fatty acyl-ACP derivatives and S-adenosyl-L-methionine (SAM).



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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 reflects its preferred

substrate, but especially in the case of AHL synthases with

a broad substrate specificity, the relative abundance of the

different AHL products may also feature the available fatty

acyl-ACP pool.

Random mutagenesis of the AHL synthases LuxI fromV. fischeriand RhlI fromP. aeruginosaidentified 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-specific 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 identified by

substrate modelling into a hydrophobic cavity spanned by

several conserved residues. Site-directed mutagenesis of an activesite tyrosine residue to alanine (T140A) altered the enzymes

substrate specificity from 3-oxo-C6-HSL (3) to C6-HSL.

According to the model the OH-function of the tyrosine residue

is crucial for the selection of the 3-oxo-C6-ACP by the formation

of a hydrogen bond to the 3-oxo group of this substrate. The

crystal structure of LasI fromP. aeruginosarevealed an enlarged

hydrophobic binding pocket for the uptake of the 3-oxo-C12-

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. carotovorumchanged the

substrate specificity from a 3-oxo-C6-HSL (3) toa 3-oxo-C8-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 IIfatty acid synthase system.68 The pathway starts from acetyl-

CoA that is selected and transferred to the ACP by the (malonyl-)-

acyltransferase (MAT, Scheme 2). Chain elongation proceeds

viathe 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 thistwo-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

Cn-HSL, 3-oxo-Cn-HSL, (R)-3-OH-Cn-HSL, and (2E)-Cn:1-

HSL types. AHL molecules of the (2E)-Cn:1-HSL type

are relatively rare, possibly because the reactivity of the

Scheme 2 Fatty acid biosynthesis and biosynthesis ofN-acyl-L-homoserine lactones.



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a,b-unsaturated enoyl-ACP substrates is different from the

reactivity of the saturated acyl-ACP substrates. Therefore, the

generation of (2E)-Cn:1-HSLs from 3-OH-Cn-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 aerobicbiosynthesis 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)-C10:1-ACP, (5Z)-C12:1-ACP, (7Z)-C14:1-ACP, (9Z)-C16:1-

ACP, and (11Z)-C18:1-ACP. The aerobic pathway proceeds first

via generation of the saturated carbon chain and subsequent

dehydration usually by a D9-desaturase yielding (9Z)-Cn: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 proceedsvia

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 configuration of the 3-hydroxyacyl-CoA inter-

mediate that isSconfigured in the catabolic pathway, in contrast

to (R)-3-hydroxyacyl-ACP intermediates in fatty acid biosyn-

thesis.68,70 Therefore, the absolute configurations of 3-OH-Cn-HSLs point to their origin from fatty acid biosynthesis or

b-oxidation, respectively. The stereochemistry of the autoinducer

from V. harveyiwas determined by a chiral NMR shift reagent as

(R)-3-OH-C4-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 configuration of small bacteriocin from R. legumi-

nosarum was elucidated using a chiral NMR shift reagent as

(3R,7Z)-3-OH-C14:1-HSL (6).72 Recently, the autoinducers

3-OH-C8-HSL (11) from Aeromonas culicicola and 3-OH-C10-

HSL (12) from Phaeobacter gallaeciensis have been established to

be enantiomerically pure and R configured by methanolysis andseparation 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-C8-HSL, whereas specific

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 arecomparably 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 foranteiso-odd AHL derivatives. The putative occur-

rence ofanteiso-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 knownAHL 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 Cn-

HSLs identified so far are indeed methyl branched, especially if

compound identification was only based on low resolution

techniques such as thin layer chromatography (TLC).

4 Detection and identification ofN-acyl-L-

homoserine lactones

Several different bioassays, spectroscopic, and chromatographic

methods have been used for the detection and identification ofAHLs in culture extracts from bacteria. In Tables 24 an effort

was made to summarise all AHLs that have been identified until

today, including information about the producing species, the

main component in complex AHL mixtures, and the methods

that have been applied for compound identification. Curiously,

there is one report about an AHL from a cyanobacterium in the

literature, whereas all other AHLs identified 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 biofilms in the lungs of

cystic fibrosis (CF) patients.13,76 The assay is based on the uptake

of radiolabelled [1-14C]-L-methionine that is incorporated viaSAM 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 quantification 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 specific 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



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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 promoteror 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. ThetraG::lacZfusion reporter inA. tumefaciensresponds

to Cn-HSLs with a chain length fromn 612 and to 3-oxo-Cn-

HSLs (n 412). A similarEnsifer melilotiRm41 reporter strain

complements this pattern and is capable of specifically detecting

long chain AHLs (Cn-HSL:n 1216, 3-oxo-Cn-HSL:n 14).82

This reporter system is particularly interesting, because it

contains asinI::lacZfusion derived from the sinIgene including

thesinIpromoter region that encodes the AHL synthase for long

chain AHLs. As a side effect of this transcriptional fusion the

production ofsinI-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. Inconsequence, the sinI::lacZfusion suppresses the natural AHL

backgroud inE. melilotiand results inb-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-C6-HSL (3) in

V. fischeri, rhlR: RhlR is the detector protein for C4-HSL (1)

inP. aeruginosa, andlasR: LasR responds to 3-oxo-C12-HSL (4)

inP. aeruginosa) and the promoter regions of the respective luxI

homologues (PluxI, PrhlI, and PlasI) have been fused to the lux-

CDABEoperon fromPhotobacterium luminescensthat responds

with bioluminescence upon transcriptional activation.83 In theseconstructs 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. coliJM109 that does not produce any

AHL signals. The reporter strains respond to AHLs with

different chain lengths and oxidation states at C-3 of theN-acyl

chain (Cn-HSL: n 412, 3-oxo-Cn-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 fluorescent protein (GFP). Plasmid pJBA132 contains

aluxR-PluxI::gfp(ASV) fusion construct that was introduced into

E. coliMT102.84 Although the range of AHL molecules detect-able by the resulting reporter strain has not been systematically

investigated, several AHLs including C6-HSL, C8-HSL (2),

3-oxo-C6-HSL (3), and 3-oxo-C12-HSL (4) can be detected with

high sensitivity. The related plasmids pKR-C12 and pAS-

C8 contain Plac::lasR and PlasB::gfp(ASV) or Plac::cepR and

PcepI::gfp(ASV) fusions, respectively, using genes from the

Fig. 1 TheA. tumefaciensNT1 (pDCI41E33)traG::lacZfusion reporter

strain.

Table 1 AHL reporter strains

Reporter strain Genotype Phenotype Cn-HSL 3-oxo-Cn-HSL Ref.

A. tumefaciensNT1 (pDCI41E33) traG::lacZ b-galactosidase activity n 612 n 412 80,81

E. melilotiRm41 (pVIKSinIsub) sinI::lacZ b-galactosidase activity n 1216 n 14 82E. coliJM109 (pSB401) luxR-PluxI::luxCDABE bioluminescence n 412 n 414 83E. coliJM109 (pSB406) rhlR-PrhlI::luxCDABE bioluminescence n 412 n 414 83E. coliJM109 (pSB1075) lasR-PlasI::luxCDABE bioluminescence n 412 n 414 83E. coli MT102 (pJBA132) luxR-PluxI::gfp(ASV) green fluorescence n 68 n 612 84P. aeruginosaPAO-JP2 (pKR-C12) Plac::lasR green fluorescence not sensitive n 1012 85,86

PlasB::gfp(ASV)B. cepacia H111-I (pKR-C12) Plac::lasR green fluorescence n 612 n 612 85,86

PlasB::gfp(ASV)P. aeruginosa PAO-JP2 (pAS-C8) Plac::cepR green fluorescence n 612 n 1012 85,86

PcepI::gfp(ASV)B. cepacia H111-I (pAS-C8) Plac::cepR green fluorescence n 612 n 612 85,86

PcepI::gfp(ASV)C. violaceumCV026 cviI::mini-Tn5 AHL production impaired n 48 (n 1014) n 48 (n 1014) 87



8/27

P. aeruginosa lassystem and theluxRandluxIhomologuescepR

and cepIfrom Burkholderia cepacia (the major product of CepI is

C8-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 AHLmolecules occur, if one plasmid is transformed into different

reporter strains, e. g. theP. aeruginosa PAO1-JP2 reporter con-

taining plasmid pKR-C12 does not detect any Cn-HSLs, whereas

the same plasmid in the B. cepacia H111-I reporter enables

sensitivity against Cn-HSLs (n 612).

Another simple, but very efficient and extensively used

reporter strain is Chromobacterium violaceum CV026.87 The

wildtype ofC. violaceum ATCC 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 (Cn-HSL: n 48, 3-oxo-Cn-HSL: n 48) bya restored pigment production.C. violaceumCV026 can also be

used for the detection of long chain AHLs (Cn-HSL: n 1014,

3-oxo-Cn-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 identifica-

tion. Nevertheless, TLC is extensively used, since today several

Cn-HSLs (n 4, 6, 7, 8, 10, 12, 14) and 3-OH-Cn-HSLs (n 6, 8,

10, 12, 14) are commercially available. For the detection of

AHLs after the chromatographic procedure the TLC plates areoverlaid 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 identifications.

Gas chromatography in combination with mass spectrometry

(GC-MS) has been applied for the identification of several AHLs

from a large number of alphaproteobacteria.73,88 Characteristicfragment ions can be used to trace back the different classes of

AHLs in culture extracts. The mass spectra of 3-OH-Cn-HSLs

such as 11 are dominated by fragment ions at m/z 102 and m/z

172 representing the ion C4H8NO2+ originating from the

homoserine lactone moiety and the ion C7H10NO4+ formed by

a-cleavage, respectively, whereas the fragment ions at m/z

102 and m/z 143 (C6H9NO3+, McLafferty rearrangement) are

typical for Cn-HSLs such as C8-HSL (2). For the quantification

of 3-oxo-Cn-HSLs in complex culture extracts a useful derivati-

sation method has been developed.89 The 3-oxo-Cn-HSLs are

transformed into their pentafluorobenzyloxime (PFBO)

derivatives that exist asEand Zdiastereomers, 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 (C7H2F5) representing the

pentafluorobenzyl group and atm/zM-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 config-

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 disulfide (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 Cn: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 configuration(s) is possible by an

E/Z-selective synthesis. Using this approach the structures of

(7Z)-C14:1-HSL (15) and (2E,9Z)-C16:2-HSL (16) have been fully

established.73,88

The mass spectra of methyl-branched AHLs are very similar tothe mass spectra of their unbranched counterparts, but methyl

branched AHLs elute with significantly shorter retention times

from the GC column compared to unbranched ones making

GC-MS analysis a useful tool for the identification 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 first methyl branched AHLs

iso-C9-HSL (17) and 3-OH-iso-C9-HSL (18) fromA. culicicola.73



9/27

Another extensively used chromatographic method for the

identification 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 themost powerful technique and has been used for the structure

elucidation of several AHL molecules. The first structurally

characterised AHL autoinducers have been identified as 3-oxo-

C6-HSL (3) fromV. fischeri,1 followed by the planar structure of

(R)-3-OH-C4-HSL (5) from the related bacterium V. harveyiand

C8-HSL (2) as the second messenger ofV. fischeri.4,5 After these

initial isolations and characterisations of AHLs from the genus

Vibrio, several other AHL derivatives have been identified by

purification and NMR spectroscopy from a broad range of

Gram-negative bacteria including C4-HSL (1) from P. aerugi-

nosa, S. liquefaciens, and Pantoea sp.,3,10,62,94 3-oxo-C6-HSL (3)

fromP. carotovorum,7 3-oxo-C8-HSL (19) fromA. tumefaciens,8

and 3-oxo-C12-HSL (4) fromP. aeruginosa.6

NMR spectroscopy

is also a very powerful method for the localisation and deter-

mination of the configuration of C]C double bonds in the

N-acyl chain, highlighted by the structure elucidations of

(5Z)-C12:1-HSL (20) and (5Z)-3-oxo-C12:1-HSL (21) fromMeso-

rhizobium sp.,95 (7Z)-C14:1-HSL (15) from Rhodobacter sphaer-

oides,96 and of (3R,7Z)-3-OH-C14:1-HSL (6) from R.

leguminosarum,11,72 that is also termed smallbacteriocin. The

structures of two unsaturated AHLs from Methylobacterium

extorquenshave only been solved in part as (Z)-C14:1-HSL (22)and (2E,Z)-C14:2-HSL (23) with unidentified localisation of the Z

double bonds.97 In a few cases the NMR-spectroscopic structure

elucidation of 3-oxo-Cn-HSLs has been assisted by the applica-

tion of IR spectroscopy.1,7,8

5 The role ofN-acyl-L-homoserine lactones in

biofilm formation

First hints for the involvement of AHLs in the formation of

biofilms have been obtained by their detection in aquatic

biofilms.187 As outlined in chapter 2, biofilms are aggregates ofmicroorganisms that adhere to a solid surface in a matrix

composed of extracellular biopolymers. Biofilms 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-C6-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 identified that is functionally involved in

the virulence by several mechanisms.190 The EPS capsules,

efficiently 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 freeflow 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 biofilms. These pathogens are particularly

problematic among cystic fibrosis (CF) patients. CF is caused

by a genetic defect195 that affects a chloride ion channel regu-

lating the transport of chloride ions and, as a consequence, of

fluids 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 isP. aeruginosa that forms a biofilm in the lungs ofCF patients in an AHL-regulated process.14,15 Another impor-

tant opportunistic pathogen in CF lung infections is Bur-

kholderia cepacia that forms biofilms 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 biofilm-

producer.198 The formation of biofilms has also been shown to be

AHL dependent in the fish pathogen Aeromonas hydrophila,199

the soil bacterium Ensifer meliloti,200 and the plant associate

Methylbacterium extorquens.201



10/27

Table2

Cn:m-HSL

Species

Taxona

4:0

5:0

6:0

7:0

iso-7:0iso-7:18:0

9:0

iso-9:010:0

12:0

12:1

14:0

14:1

14:2

16:0

16:116:218:018:118:2

AcidithiobacillusferrooxidansATCC19

85998

g

+c,f

+c,f


27099

g

+c,f

+c,f

AcidithiobacillusferrooxidansDSMZ58

398

g

+c,f

+c,f

AeromonasculicicolaMTCC324973

g

+d

,f

+d

,f

+d

,f

+d

,f

+d

,f

Aeromonashydrophila96-3-35100

g

+b

,c,f

+b

AeromonashydrophilaA1101

g

+b

,c,f

+b

,c,f

AeromonashydrophilaATCC7966100

g

+c,f

Aeromonassalmonicida02-9-1

100

g

+b

,c,f

+b

,c,f

+c,f

AeromonassalmonicidaNCIMB1102101

g

+b

,c,f

+b

,c,f

AeromonassalmonicidaNCIMB1110100

g

+b

,c,f

+b

AgrobacteriumtumefaciensK7948

a

+c,f

AgrobacteriumvitisF2/5102

a

+b

,d,f

+b

,d,f

+d

,f

+d

,f

AzospirillumlipoferumB518103

a

+b

,c,f

+b

,c,f

AzospirillumlipoferumTVV3103

a

+b

,c,f

+b

,c,f

Brucellamelitensis16M104

a

+c,f

Burkholderiasp.CBMB40105

b

+b

+b

+b

+b

BurkholderiacenocepaciaK56-2106,107

b

+b

+b

,c,d,f

BurkholderiacepaciaATCC1085678

b

+c

BurkholderiacepaciaATCC1775978

b

+c

BurkholderiacepaciaATCC2541678,84,1

08

b

+b

+b

,c

BurkholderiacepaciaBC778

b

+c

BurkholderiacepaciaC125778

b

+c


b

+c


b

+c


b

+c

BurkholderiacepaciaCEP50978

b

+c

BurkholderiacepaciaDM5018084

b

+b

+b

BurkholderiacepaciaH111(R-6282)109

b

+b

+b

BurkholderiacepaciaJ41578

b

+c

BurkholderiacepaciaJA-7

110

b

+d

,f

+d

,f

+d

,f

+d

,f

BurkholderiacepaciaK56-278,86

b

+c,d,f

BurkholderiacepaciaLA-3

111

b

+e,f

+e,f

BurkholderiacepaciaLA-10110

b

+d

,f

+d

,f

+d

,f

+d

,f

BurkholderiacepaciaLMG1222109

b

+b

+b

BurkholderiacepaciaLMG18943109

b

+b

+b

BurkholderiacepaciaPC18478

b

+c

BurkholderiaglumaeBGR1112,113

b

+b

,f

+b

,f

BurkholderiamalleiATCC23344114116

b

+c

+c,f

+c,f

BurkholderiamultivoransATCC176167

8

b

+c

BurkholderiamultivoransC157678

b

+c

BurkholderiamultivoransC196278

b

+c

BurkholderiaplantariiATCC43733117

b

+b

+b

Burkholderiapseudomallei008118

b

+c

BurkholderiapseudomalleiDD503119

b

+c,f

+c,f

BurkholderiapseudomalleiKHW120

b

+c

BurkholderiapseudomalleiPP844121

b

+c,f

+c,f

BurkholderiastabilisATCC3525478

b

+c

BurkholderiastabilisLMG0700078

b

+c


b

+c


b

+b

+b


b

+c

BurkholderiathailandensisDW503122

b

+b

,c,f

+b

,c,f

+b

,c,f



11/27

Table2

(Contd.)

Species

Taxona

4:0

5:0

6:0

7:0

iso-7:0iso-7:18:0

9:0

iso-9:010:0

12:0

12:1

14:0

14:1

14:2

16:0

16:116:218:018:118:2

BurkholderiaubonensisAB030584123

b

+c,f

BurkholderiavietnamensisC282278

b

+c

BurkholderiavietnamensisC917878

b

+c

BurkholderiavietnamensisFC369124

b

+b

+b

+b

BurkholderiavietnamensisFC44178

b

+c

BurkholderiavietnamensisG478,124,125,126

b

+b

,c,f

+b

,c,d,f

+b

,c,d,f

+b

,c,d,f

BurkholderiavietnamensisLMG109297

8,109

b

+b

+b

+b

,c

BurkholderiavietnamensisLMG162327

8

b

+c

BurkholderiavietnamensisPC25978

b

+c

BurkholderiavietnamensisR-921109

b

+c,f

+c,f

+c,f

+c,f

+c,f

BurkholderiavietnamensisTVV75127

b

+b

+b

ChromobacteriumviolaceumATCC3153287

b

+c,f

DinoroseobactershibaeDFL2788

a

+d

,f

+d

,f

EdwardsiellatardaNUF251128

g

+b

+b

EnsifermelilotiRm41(AK631)129,130

a

+c,d,f

+b

,f

+f

+f

EnsifermelilotiRm1021129,131

a

+b

+b

,c,f

+c,f

+c,d,f

+f

+f

+c,f

+c,f

EnsifermelilotiRm8530132

a

+d

,f

+d

,f

Erwiniachrysanthemi3937133

g

+f

+f

ErwiniapsidiiATCC4940659

g

+d

,f

+d,f

Gloeothecesp.PCC690975

cyano

+f

GluconacetobacterintermediusNCI1051134

a

+c,f

+c,f

+c,f

HalomonasanticariensisFP35127

a

+b

,d,f

+b

,d,f

+b

,d,f

+d

,f

HerbaspirillumfrisingenseDSM131301

35

b

+f

+f

JannaschiahelgolandensisHEL1073,88

a

+d

,f

+d

,f

+d

,f

ListonellaanguillarumNB10136

g

+b

,c,f

Mesorhizobiumsp.R8-Ret-T53-13d95

a

+c,f,g

Mesorhizobiumhuakuii93137

a

+b

MethylobacteriumextorquensAM197

a

+b

,c,f

+b

,c,f

+b

,c,f,g

+b

,c,f,g

NitrosomonaseuropaeaATCC19718138

b

+b

,d,f

+b

,d,f

+b

,d,f

OceanibulbusindolifexHEL7688

a

+d

,f

Pantoeasp.CBMAI73262

g

+d

,f,g

+d

,f

Pantoeaagglomeranspv.gypsophilae139

g

+f

+f

PantoeaananatisCCT648159

g

+d

,f

+d,f

+d

,f

ParacoccusdenitrificansATCC1774179

a

+c,f

PectobacteriumatrosepticumCFBP627

6140

g

+c,f

PectobacteriumcarotovorumSCC167

g

+c,f

+c,f

+c,f


7

g

+c,f

+c,f

+c,f

+c,f

+c,f

PhaeobactergallaeciensisT588

a

+d

,f

Pseudoalteromonassp.520P1141

g

+b

,c,f

PseudomonasaeruginosaPAO13,94

g

+c,d,f,g

+c,f

Pseudomonaschlororaphis3084142,143

g

+b

,c,f

+b

,c,f

+b

,c,f

PseudomonaschlororaphisGP72144

g

+b

+b

PseudomonaschlororaphisPCL1391142,

145

g

+b

+b

,c,f

+b

PseudomonascorrugataNCPPB2445146

g

+b

+b

Pseudomonasfluorescens27980

g

+b

PseudomonasfluorescensF113147

g

+c,f

+c,f

RalstoniasolanacearumAW1130

b

+b

RalstoniasolanacearumK6080,130

b

+b

Rhizobiumleguminosarumbv.phaseoli8401148150

a

+b

,c,f

+b,c

,f

+b

,c,f

Rhizobiumleguminosarumbv.phaseoli14482130

a

+b

Rhizobiumleguminosarumbv.

trifolii162E8130

a

+b

+b

Rhizobiumleguminosarumbv.viciae24

8148

a

+b

+b

+b



12/27

Table2

(Contd.)

Species

Taxona

4:0

5:0

6:0

7:0

iso-7:0iso-7:18:0

9:0

iso-9:010:0

12:0

12:1

14:0

14:1

14:2

16:0

16:116:218:018:118:2

Rhizobiumleguminosarumbv.viciae30

0148

a

+b

+b

+b

Rhizobiumleguminosarumbv.viciaeTO

M148

a

+b

+b

+b

RhodobactercapsulatusB1079

a

+c

+c

Rhodobactersphaeroides2.4.1

80,96

a

+b

+c,f,g

RoseobacterlitoralisDSM700188

a

+d

,f

RoseovariusmucosusDFL2488

a

+d

,f

+d

,f

RoseovariustoleransEL17288

a

+d

,f

+d

,f

Serratiasp.ATCC39006151

g

+b

+b

SerratialiquefaciensATCC27592152

g

+c,f

+c,f

+c,f

+c,f

+c,f

+c,f

SerratialiquefaciensMG110

g

+c,f,g

+c,f,g

SerratiamarescensSS-1

153

g

+b

,c,f

+b,c

,f

+b

,c,f

SerratiaplymuthicaHRO-C48154

g

+b

+b

StaleyaguttiformisLM0988

a

+d

,f

+d

,f

+d

,f

Vibriocampbellii155

g

+b

,d,f

VibriofisheriMJ-15

g

+c

+c,f,g

Vibriosalmonicida289100

g

+b

,c,f

VibriosalmonicidaNCIMB2262100

g

+b

,c,f

Vibriovulnificus156

g

+c,f

+c,f

Yersiniaenterocolitica90/54157

g

+b

YersiniaenterocoliticaNCTC10460158

g

+c,f

YersiniapestisKIM6+63

g

+c,f

+c,f

YersiniapseudotuberculosisYpIII159,160

g

+b

,c,f

+c,f

+b

,c,f

Yersiniaruckeri88-6-44100

g

+b

,c,f

YersiniaruckeriNCIMB1316100,161

g

+c,f

+b

,c,f

a

Taxon:a

alphaproteobacteria,

b

betaproteobacteria,g

gammaproteobacteria,cyano

cyanobacteria.

b

Identification

byTLC/biosensorassay.

cIdentificationbyHPLC.

d

Identificationby

GC.

eIdentificationbyCE.

fIdentifica

tionbyMS.

g

IdentificationbyNMRspectroscopy.

h

IRspectroscopy.



13/27

Table3

30-oxo-Cn:m-HSL

Species

Taxona

4:0

5:0

6

:0

7:0

8:0

9:0

10:0

10:1

11:2

12:0

12:1

13:2

14:0

16:0

16:1


27099

g

+c,f

+c,f

AcidithiobacillusthiooxidansDSMZ50498

g

+b


g

+b


g

+b

AgrobacteriumtumefaciensBo542130

a

+b

+b

AgrobacteriumtumefaciensC58130

a

+b

AgrobacteriumtumefaciensK7948

a

+c,f,g,h

AgrobacteriumtumefaciensNT1130

a

+b

AgrobacteriumvitisF2/5102

a

+d

,f

+d

,f

AzospirillumlipoferumB518103

a

+

b,c,f

+b

,c,f

AzospirillumlipoferumTVV3103

a

+b

,c,f

+b

,c,f

Burkholderiapseudomallei1026b119

b

+f

BurkholderiapseudomalleiPP844121

b

+c,f

BurkholderiavietnamensisG4125

b

+f

BurkholderiavietnamensisR-921109

b

+c,f

EnsifermelilotiRm41(AK631)129,130

a

+b

,f

+f

+f

+f


a

+c,f

+c,f


a

+d

,f

+d

,f

+d

,f

EnsifermelilotiYA280

a

+b

Erwiniachrysanthemi3937133

g

+

c,f

Erwiniachrysanthemipv.zeaeEC1162

g

+

b

Hafniaalvei163

g

+

b

Listonellaanguillarum90-11-287164

g

+b

,c,f

+c,f

ListonellaanguillarumNB10164,165

g

+b

,c,f

+c,f

Mesorhizobiumsp.R8-Ret-T53-13d95

a

+c,f

+c,f

+c,f,g

PantoeaagglomeransB6a163,166

g

+

b

+b

Pantoeastewartiissp.stewartiiDC283167

g

+

b

Pectobacteriumsp.A2JM168

g

+

b,c,f

PectobacteriumatrosepticumCFBP6276140

g

+

c,f

+c,f

+c,f

PectobacteriumatrosepticumSR8130

g

+

b

+b

PectobacteriumcarotovorumATCC390

487

g

+

f,g,h

PectobacteriumcarotovorumDM21051

30

g

+

b


g

+c,f

+

c,f

+c,f

+c,f


7

g

+

c,f

+c,f

+c,f

+c,f

+c,f

Pseudoalteromonassp.520P1141

g

+b

,c,f

Pseudomonasaeruginosa629489

g

+d

,f

+d

,f

+d

,f

+d

,f

PseudomonasaeruginosaPAO16,80

g

+

b

+b

+b

+b

,c,f,g

PseudomonascorrugataNCPPB2445146

g

+

b

PseudomonasputidaIsoF169,170

g

+

b

+b

+b

+b

PseudomonasputidaPCL1445171

g

+

b

+b

+b

+b

PseudomonasputidaWCS358172

g

+

b

+b

+b

+b

Pseudomonassyringaepv.coronofaciensPC27130

g

+

b

+b

Pseudomonassyringaepv.savastanoi1670130

g

+

b

+b

Pseudomonassyringaepv.syringaeB72

8a130

g

+

b

Pseudomonassyringaepv.

tabaci20248

0

g

+

b,f

+b

Rhizobiumsp.NGR234173

a

+b

RhizobiumetliCFN42174

a

+b


trifolii14480130

a

+b


trifolii162E8130

a

+b

SerratialiquefaciensATCC27592152

g

+

c,f

+c,f

+c,f

+c,f

+c,f

Serratialiquefaciens163

g

+

b

SerratiamarescensSS-1

153

g

+

b,c,f



14/27

6 Antagonists ofN-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 efficiently metabolised AHLs

were identified as Pseudomonas, Variovorax, Camomonas, and

Rhodococcus spp., respectively, and investigated in detail for

their substrate specificity 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 identified that catalyse the degradation of

quorum sensing messengers, termed quorum quenching 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 byPectobacterium 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-C6-HSL and 3-oxo-C8-HSL. ExpR1

is sensitive to 3-oxo-C8-HSL, whereas ExpR2 detects both 3-oxo-

C6-HSL and 3-oxo-C8-HSL.211 The AHL lactonase from Bacillus

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 ofN-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 from Bacillus 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. C4-HSL and 3-oxo-C12-HSL fromP. aeru-

ginosa.205 The substrate specificity of AHL lactonases was further

tested with the respective enzyme from Bacillus thuringiensis

against the two enantiomers of C6-HSL. This lactonase hydro-

lyses the lactone moiety of C6-L-HSL, whereas the enantiomeric

C6-D-HSL is not cleaved.206

Table3

(Contd.)

Species

Taxona

4:0

5:0

6:0

7:0

8:0

9:0

10:0

10:1

11:2

12:0

12:1

13:2

14:0

16:0

16:1

SerratiaplymuthicaHRO-C48154

g

+

b

SerratiaproteamaculansB5a166,175

g

+

b

Sinorhizobiumfredii191130

a

+b

Vibriocampbellii155

g

+

b,d,f

VibriofisheriMJ-11,80

g

+

b,f,g,h

+b

Vibriosalmonicida289100

g

+

b,c,f

VibriosalmonicidaNCIMB2262100

g

+

b,c,f

Vibriovulnificus156

g

+c,f

+c,f

+c,f

+c,f

Xanthomonascampestrispv.campestris

4546130

g

+b

Xanthomonascampestrispv.pelargoniX-5

130

g

+b

Xanthomonasoryzaepv.oryzicolaBLS

303130

g

+b

Yersiniaenterocolitica8081176

g

+

b

Yersiniaenterocolitica90/54157

g

+

b

+c,f

+c,f

+c,f

YersiniaenterocoliticaNCTC10460158

g

+

c,f

YersiniapestisKIM6+63

g

+

b,c,f

+b

,c,f

+c,f

YersiniapseudotuberculosisYpIII159,160

g

+c,f

+

b,c,f

+c,f

+c,f

+c,f

+c,f

Yersiniaruckeri88-6-44100

g

+b

,c,f

YersiniaruckeriNCIMB1316100,161

g

+

c,f

+c,f

+b

,c,f

+c,f

+c,f

+c,f

a

Taxon:a

alphaproteobacteria,

b

betaproteobacteria,g

gammaproteobact

eria.

b

IdentificationbyTLC/biosensorassa

y.cIdentificationbyHPLC.

d

Identification

byGC.

eIdentificationby

CE.

fIdentificationbyMS.

g

Identifica

tionbyNMRspectroscopy.

h

IRspectroscopy.



15/27


16/27

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 (Zn2+, Mn2+, Co2+, Cd2+) demon-

strated a direct involvement of the metal ion in the turnover ofAHL substrates by their binding.214 To date several AHL lac-

tonases have been identified fromArthrobactersp.,216 A. tumefa-

ciens,217,218andespeciallydiverseBacillusspp.206,207,210,212,215,219222

A. tumefaciens produces the autoinducer 3-oxo-C8-HSL to

control the conjugal transfer efficiency 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-C8-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 thegenome 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 rapidlyundergo 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 withPectobacteriumimmediately respond by increasing

the pH of the apoplastic fluid around the source of infection from

pH < 6.4 to pH > 8.2 that is accomplished by a rapid proton

influx into the cells.226

A second class of AHL-degrading enzymes was identified 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 thesole 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

identified from this species.208,227 However, such genes and

enzymes have subsequently been reported from other bacteria.

The gene aiiD was cloned from the biofilm isolate Ralstonia

eutropha XJ12A and XJ12B (betaproteobacteria) and

sequenced. The purified gene product AiiD catalyses the rapid

conversion of 3-oxo-C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-

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 otheracylases. Site-directed mutagenesis of Gly-232 and Ser-233

Table 5 Analysis of functional residues in AHL lactonases by site-specific mutagenesis

Enzyme Activityd References

WT 1.00H104S,a H104Ab 1.00, 0.01 207,210H106S,a H106Ab 0.61, 0.02 207,210D108E,a D108S,a D108Ab 0.91, 0.00, 0.02 207,210H109S,a H109Ab 0.00, 0.16 207,210

H169S,a

H169Ab

0.61, 0.01 207,210D191A,b D191Lb 0.02, 0.01 207Y194F,b Y194Ac 0.33, strong reduction 207,215H235Ab 0.15 207

a Experiment carried out with the AHL lactonase AiiA fromBacillussp.240B1. b AiiA fromBacillus thuringiensissubsp.kurstakiHD263. c AiiAfrom Bacillus cereus Y2. d Relative activity compared to the respectivewildtype enzyme.

Fig. 2 Catalytic mechanism for 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



17/27

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

derivativevia the acylase mechanism. In contrast to R. eutropha

the initial product L-homoserine lactone is hydrolysed toL-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 purified,

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-C12-HSL, but not of C4-HSL.203,229

Both L-homoserine lactone messengers are produced by the

wildtype, and therefore the expression of these AHL acylases inthe wildtype seems to be tightly regulated to allow the

production of 3-oxo-C12-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. tumefaciensby 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 specificity 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

Streptomycessp. M664 secretes the AHL acylase AhlM into the

culture medium that efficiently degrades long-chain AHLs,

whereas C6-HSL and 3-oxo-C6-HSL are only slowly metab-olised, and C4-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-Cn-HSLs into the respective 3-OH-Cn-

HSLs.209

A combined mechanism for the degradation ofN-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 toN-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,

purified, 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. fischeri by a blockage of the receptor site of the LuxRhomologue,239 and24 inhibits AHL-regulated luminescence and

toxin production in a virulent strain ofV. 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 luxIgene, it

possesses the LuxR homologue SdiA and responds to AHLs such

as C6-HSL and 3-oxo-C6-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 biofilm formation in E. coliand the expression of virulence inV. harveyiby interference with

the AI-2 system, making compound 24 a non-specific auto-

induction antagonist.244246 Indole (28) that is secreted into the

medium by E. coli in the stationary phase, decreases biofilm

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 biofilms in E. coli, whereas

isatin (31) induces biofilm formation.248 The two brominated

tryptophane-derived alkaloids32and33have 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



18/27

sativa produces L-canavanine (36) that interferes with the

quorum sensing system of its nitrogen-fixing symbiont Ensifer

melilotiand inhibits the expression of genes for the production of

exopolysaccharides in the bacterium.251

Three AHL inhibitors, floridoside (37), betonicine (38), and

isethionic acid (39), have been isolated from the red alga Ahn-

feltiopsis flabelliformis 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 biofilms inP. aeruginosa, whereas41 is inhibitory.

The sesquiterpene lactones 4247 from Acanthospermum hispi-

dum all inhibit the production of biofilms by P. aeruginosa.253

The diterpenoid salvipisone (48) isolated from roots of Salviasclarea inhibits the biofilm 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 of48 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 biofilm formation

of the plant pathogen P. aeruginosa.255

6.3 Biofilm inhibition by azithromycin

Antibiotic treatment of bacterial infections is complicated by the

formation of mature biofilms in terms of the resistance of

bacterial cells within the biofilm matrix. Therefore it is of high

importance to prevent the formation of biofilms during infec-

tions to enable an effective antibiotic therapy. A promising

possibility to treat biofilm infections is the suppression of biofilm

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

low risk. Several compounds presented in chapter 6.2 are efficient

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 forclinical use and marketed by Pfizer. 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 significant effect against Gram-

negative bacteria such as P. aeruginosa, e. g. the minimum

inhibitory concentration (MIC) of 50 is above 1 mg mL1.

However, at sub-inhibitory concentrations azithromycin has

a beneficial effect on cystic fibrosis patients.257,258 At these levels

the antibiotic has been shown to retard the production of bio-

films, to reduce virulence factor production, and to interrupt

quorum sensing.259262 In spite of this promising perspective



19/27


20/27


21/27


22/27

pathways via the second messenger c-di-GMP provides an

explanation for this finding, 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 proteinsare altered, resulting in a net increase of the intracellular

c-di-GMP pool. This finally causes the expression ofvps genes

and the formation of biofilms. 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 biofilm transcriptional activator vpsT

is not induced, but rather blocked by HapR. Virulence and

biofilm formation in V. cholerae are not only controlled by

autoinduction, but also by environmental signals such as

temperature and pH. The environmental signals modulate theintracellular level of c-di-GMP via the incompletely characterised

VieSAB signaling system to co-regulate virulence and biofilm

formation inV. cholerae.

The regulation of biofilm formation and expression of viru-

lence factors is in sharp contrast to the quorum sensing control of

these traits in several other (pathogenic) bacteria. Usually, bio-

film formation and virulence are favoured in dense populations,

whereas inV. choleraeboth characteristics are specific 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. choleraeforms

biofilms on the surfaces of the fauna and flora that protect the

bacteria against several environmental stresses. These biofilmsalso protectV. choleraein acidic environments and therefore aid

in the passage through the gastric barrier of the stomach, while

the quorum-sensing controlled detachment from biofilms is

advantageous for the subsequent colonisation of the intestinal

epithelium.310 The repressed virulence and biofilm formation at

high cell densities enable V. choleraeto re-enter the environment

in large numbers to start a new infectious life cycle.

8 Conclusions

Bacterial biofilms are remarkable structures in which unicellular

microorganims organise a multicellular way of life. The biofilm

architecture is mainly built up from extracellular polysaccharidesproviding the structural basis for improved nutrient supply and

protection against environmental stresses such as antibiotics. In

2010 npr quourum sensing and bacterial biofilms.pdf

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