Vipin Chandra Kalia Editor

Quorum Sensing vs Quorum Quenching: A Battle with No End in Sight

Vipin Chandra KaliaEditor

Quorum Sensing vsQuorum Quenching:A Battle with No Endin Sight

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EditorVipin Chandra KaliaMicrobial Biotechnology and GenomicsCSIR-Institute of Genomics and Integrative BiologyDelhi, India

ISBN 978-81-322-1981-1 ISBN 978-81-322-1982-8 (eBook)DOI 10.1007/978-81-322-1982-8Springer New Delhi Heidelberg New York Dordrecht London

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Contents

Microbes: The Most Friendly Beings? . . . . . . . . . . . . . . . . . . . . . . . . . . 1Vipin C. Kalia

Part I Quorum Sensing Mediated Processes

Evolution of MDRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Ashima Kushwaha Bhardwaj and Kittappa Vinothkumar

Biofilms: Maintenance, Development, and Disassemblyof Bacterial Communities Are Determined by QS Cascades . . . . . . 23Hadas Ganin, Eliane Hadas Yardeni, and Ilana Kolodkin-Gal

Quorum Sensing in Pathogenesis and Virulence . . . . . . . . . . . . . . . . . 39Pragasam Viswanathan, Suneeva S.C., and Prasanth Rathinam

Quorum Sensing in Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . 51Jie Gao, Anzhou Ma, Xuliang Zhuang, and Guoqiang Zhuang

Quorum Sensing in Competence and Sporulation . . . . . . . . . . . . . . . 61Navneet Rai, Rewa Rai, and K.V. Venkatesh

How Important Is the Absolute Configuration to Bacteria QuorumSensing and Quorum Quenching? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Francisca Diana da Silva Araújo, Armando Mateus Pomini,and Anita Jocelyne Marsaioli

Part II Quorum Sensing Systems in Microbes

Quorum-Sensing Systems in Pseudomonas . . . . . . . . . . . . . . . . . . . . . . 73Jamuna Bai Aswathanarayan and V. Ravishankar Rai

Quorum Sensing in Escherichia coli: Interkingdom, Inter-and Intraspecies Dialogues, and a Suicide-Inducing Peptide . . . . . . 85Bloom-Ackermann Zohar and Ilana Kolodkin-Gal

Quorum Sensing in Acinetobacter baumannii . . . . . . . . . . . . . . . . . . . 101Nidhi Bhargava, Prince Sharma, and Neena Capalash

Quorum Sensing Systems in Aeromonas spp. . . . . . . . . . . . . . . . . . . . . 115Weihua Chu, Wei Zhu, and Xiyi Zhuang

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viii Contents

Rhizobial Extracellular Signaling Molecules and Their Functionsin Symbiotic Interactions with Legumes . . . . . . . . . . . . . . . . . . . . . . . . 123Walter Giordano

Quorum Sensing Systems in Clostridia . . . . . . . . . . . . . . . . . . . . . . . . . 133Charles Darkoh and Godfred Ameyaw Asiedu

Quorum-Sensing Systems in Enterococci . . . . . . . . . . . . . . . . . . . . . . . 155Ravindra Pal Singh and Jiro Nakayama

Quorum-Sensing Systems in Bacillus . . . . . . . . . . . . . . . . . . . . . . . . . . 165Lalit K. Singh, Neha Dhasmana, and Yogendra Singh

Part III Detectors for Quorum Sensing Signals

Quorum Sensing Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173Navneet Rai, Rewa Rai, and K.V. Venkatesh

Caenorhabditis elegans as an In Vivo Non-Mammalian ModelSystem to Study Quorum Sensing in Pathogens . . . . . . . . . . . . . . . . . 185Sajal Sarabhai, Neena Capalash, and Prince Sharma

Strategies for Silencing Bacterial Communication . . . . . . . . . . . . . . . 197Kristina Ivanova, Margarida M. Fernandes, and Tzanko Tzanov

Part IV Natural Quorum Sensing Inhibitors

Silencing Bacterial Communication Through EnzymaticQuorum-Sensing Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219Manuel Romero, Celia Mayer, Andrea Muras, and Ana Otero

Fungal Quorum Sensing Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237Rohit Sharma and Kamlesh Jangid

Marine Organisms as Source of Quorum Sensing Inhibitors . . . . . . 259Fohad Mabood Husain and Iqbal Ahmad

Plant Quorum Sensing Inhibitors: Food, Medicinal Plants,and Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269Pragasam Viswanathan, Prasanth Rathinam, and Suneeva S.C.

Part V Synthetic Quorum Sensing Inhibitors

Synthetic Quorum Sensing Inhibitors: Signal Analogues . . . . . . . . . 285Dimpy Kalia

Synthetic Quorum Sensing Inhibitors (QSIs) Blocking ReceptorSignaling or Signal Molecule Biosynthesis in Pseudomonasaeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303Christine K. Maurer, Cenbin Lu, Martin Empting,and Rolf W. Hartmann

Contents ix

Development of Quorum-Sensing Inhibitors Targeting the fsrSystem of Enterococcus faecalis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319Ravindra Pal Singh and Jiro Nakayama

Part VI Alternative Strategies as Quorum Sensing Inhibitors

An Alternative Strategy as Quorum-Sensing Inhibitor:Pheromone-Guided Antimicrobial Peptides . . . . . . . . . . . . . . . . . . . . . 327Yung-Hua Li and Xiao-Lin Tian

Alternative Strategies to Target Quorum Sensing(QS): Combination of QS Inhibitors with Antibioticsand Nanotechnological Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335Divakara S.S.M. Uppu, Chandradhish Ghosh, and Jayanta Haldar

Heterologous Expression of Quorum Sensing Inhibitory Genesin Diverse Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343Prasun Kumar, Shikha Koul, Sanjay K.S. Patel, Jung-Kul Lee,and Vipin C. Kalia

Part VII Biotechnological Applications of QuorumSensing Inhibitors

Potential Applications of Quorum Sensing Inhibitors in DiverseFields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359Vipin C. Kalia and Prasun Kumar

Biotechnological Applications of Quorum-Sensing Inhibitorsin Aquacultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371Faseela Hamza, Ameeta Ravi Kumar, and Smita Zinjarde

The Battle: Quorum-Sensing Inhibitors Versus Evolutionof Bacterial Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385Vipin C. Kalia and Prasun Kumar

About the Editor

Dr. Vipin Chandra Kalia ispresently working as ChiefScientist, Microbial Biotechnologyand Genomics, CSIR-Institute ofGenomics and Integrative Biology,Delhi. He is a Professor, AcSIR whoobtained his M.Sc. and Ph.D. inGenetics, from Indian AgriculturalResearch Institute, New Delhi. Hehas been elected as: (1) Fellow ofthe Association of Microbiologistsof India (FAMI), and (2) Fellow

of the National Academy of Sciences (FNASc). His main areas ofresearch are microbial biodiversity, bioenergy, biopolymers, genomics,microbial evolution, quorum sensing, quorum quenching, drug discoveryand antimicrobials. He has published 65 papers in scientific journals suchas (1) Nature Biotechnology, (2) Biotechnology Advances, (3) Trendsin Biotechnology, (4) Critical Reviews in Microbiology, (5) BioresourceTechnology, (6) PLoS ONE, and (vii) BMC Genomics. His works have beencited 1750 times with an h index of 23 and an i10 index of 36. He is presentlythe editor in chief of the Indian Journal of Microbiology and editor of (1)Journal of Microbiology & Biotechnology (Korea), (2). Appl. Biochem. &Biotechnology (USA), (3) International Scholarly Res. Network RenewableEnergy, (4) Dataset Papers in Microbiology, and (5) PLoS ONE. He is alife member of the following scientific societies: (1) Society of BiologicalChemists of India (2) Society for Plant Biochemistry and Biotechnology,India; (3) Association of Microbiologists of India; (4) Indian ScienceCongress Association; (5) BioEnergy Society of India, and (6) the BiotechResearch Society of India (BRSI). He is also a member of the AmericanSociety for Microbiology. He can be contacted at: vckalia@igib.res.in;vc_kalia@yahoo.co.in

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Biofilms: Maintenance, Development,and Disassembly of BacterialCommunities Are Determined by QSCascades

Hadas Ganin, Eliane Hadas Yardeni, and Ilana Kolodkin-Gal

Introduction

Unicellular organisms use a variety ofmechanisms to coordinate activity withincommunities, called biofilms, and across speciesto accomplish complex multicellular processes(Aguilar et al. 2007; Kolter and Greenberg2006; Miller and Bassler 2001; Stoodley et al.2002). Informed by chemical communication,motile cells of the myxobacteria and filamentouscells of the streptomycetes organize themselvesinto conspicuous multicellular structures thatcarry out specialized tasks in spore formationand dispersal. Furthermore, most bacteria haveevolved elaborate mechanisms for adhering tosolid surfaces and thereby establishing complexcommunities referred to as biofilms. Biofilmscan be viewed as differentiated communitiesin which an extracellular matrix holds thecells together in the multicellular community.Bacterial biofilms are of a high significance inagricultural (Chen et al. 2013), environmental(Cha et al. 2012; Sanchez 2011), and clinical(Bryers 2008; Costerton et al. 1999) settings. Inmany instances they provide beneficial effects toother organisms. Such is the case for biofilmsof Bacillus subtilis that form on the surfaceof plant roots, thereby preventing the growth

H. Ganin • E.H. Yardeni • I. Kolodkin-Gal (�)Department of Molecular Genetics, Weizmann Instituteof Science, Rehovot, Israele-mail: Ilana.Kolodkin-Gal@weizmann.ac.il

of fungal pathogens (Nagorska et al. 2007).However, in other situations, bacterial biofilmscan have deadly effects; in a clinical context,biofilms in human hosts are inherently resistantto antimicrobial agents (Costerton et al. 1999)and are thus the cause of many persistent andchronic bacterial infections.

For decades, it has been mysterious how bac-teria in these biofilm communities communicatewith each other to coordinate their behavior. Thischapter sheds new light on cell-to-cell signalingduring the development of a bacterial biofilms inthe most prominent models of Gram-negative andGram-positive bacteria.

Quorum sensing is an efficient type ofcell-to-cell communication between bacteria.This process is concentration dependent andregulated by small chemical signals produced bybacteria. These small molecules are termed auto-inducers, and when bacterial concentration ishigh enough, their concentration raises up to athreshold concentration in which different genesare being transcribes and expressed by the groupof bacteria (Miller and Bassler 2001).

Below we are going to discuss QS cascadesdetermining the fates of bacterial biofilmsbelonging to five fascinating examples. Threebelong to the family of Gram-negative bacteria,generally communicating via small diffusibleorganic molecules such as homoserine lactones:(1) the deadly opportunistic Gram-negativepathogen, Pseudomonas aeruginosa; (2a)the Gram-negative pathogen Vibrio cholerae,and his immediate classic QS model, (2b)

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the Gram-negative Vibrio fischeri. Two areGram-positives, generally communicating viapeptide autoinducers: the agriculturally relevantbacterium (3) Bacillus subtilis and the Gram-positive pathogen (4) Staphylococcus aureus.

Chemical Communication CascadesRegulate Biofilm Developmentin Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative,opportunistic human pathogen and is one of themost common bacteria found in nosocomial andlife-threatening infections of immunocompro-mised people (Hentzer et al. 2003b). Patients withcystic fibrosis (CF), burn victims, and patientswith implanted medical devices (Sadikot et al.2005) are especially sensitive to get infected bythis bacterium. The threat of P. aeruginosa reliesin its ability to produce diverse virulence factorssuch as elastase, alkaline protease, exotoxin A,rhamnolipids, pyocyanin, and biofilm formationwhich will be further discussed here in thischapter. P. aeruginosa uses two main QS systemsto control its pathogenicity: the Las and Rhlsystems in addition to other regulators which willbe discussed later (Pesci et al. 1997) (Schusteret al. 2003a).

The P. aeruginosaLasI–LasR/RhlI–RhlR Systems

The lasR–lasI system consists of the lasI genewhich produces the signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) that is detected by a transcriptionalregulatory protein LasR, and the rhlR–rhlIsystem (also called vsmR–vsmI) in a similar wayproduces and recognizes the signaling moleculeN-butyryl-L-homoserine lactone (C4-HSL) thatis detected by the transcriptional regulator RhlR(Latifi et al. 1996; Pesci et al. 1997). The lasand rhl systems are organized in hierarchy.The LasI–LasR system controls the expression

of lasI for autoregulation and also activates theRhlI–RhlR system by activating the productionof RhlR (Koch et al. 2005; Ochsner et al. 1994).The role of these quorum-sensing systems inP. aeruginosa was described, for the first time,by Davies and colleagues in 1998 (Davies et al.1998). Biofilm formation of wild-type (WT)P. aeruginosa PAO1 and a lasI–rhlI doublemutant that makes neither of the quorum-sensing signals was analyzed. Though both themutant and the wild type adhered to the surfaceand made a biofilm, the mutant biofilm wasthinner and about 20 % of the WT thickness.The WT formed characteristic microcoloniescomposed of groups of cells separated bywater channels, whereas the mutant appearedto grow rather as continuous sheets on theglass surface. Thus, although lasI–rhlI were notinvolved in the initial attachment and growthstages of biofilm formation, the P. aeruginosaquorum-sensing systems jointly participated inthe subsequent biofilm differentiation process.The flat biofilms of the quorum-sensing mutantwere susceptible to treatment with the surfactantsodium dodecyl sulfate, while the structuredbiofilms were resistant. The authors concludedthat las-regulated functions were required forbiofilm formation.

Subsequent research indicated that quorumsensing’s role in P. aeruginosa biofilm formationwas not always as dramatic (Kirisits andParsek 2006). Yet, Purevdorj and colleaguesshowed that although under high flow conditions,both wild-type and quorum-sensing mutantstrains formed structured biofilms, the biofilmsdiffered in their microscopic appearance(Purevdorj et al. 2002). Independently, it wasdemonstrated that AHL signal analogues calledfuranones, known to inhibit P. aeruginosa quo-rum sensing, impaired biofilm development whenadded to the growth medium (Hentzer et al. 2002;Hentzer et al. 2003a). In addition, it was shownthat a lasR–rhlR double mutant strain producedbiofilms which are more susceptible to theclinically relevant antibiotic tobramycin than theisogenic wild-type strain (Bjarnsholt et al. 2005).

Biofilms: Maintenance, Development, and Disassembly of Bacterial Communities Are. . . 25

Notably, the las- and rhl-based quorum-sensing systems regulate many differentfunctions, and their control of these functions canchange depending on environmental conditions.Supporting this notion, several different quorum-sensing-regulated functions have been shownto impact biofilm formation at different stages;the quorum-sensing-regulated surfactant rhamno-lipid is necessary for maintaining the open spacesbetween cell aggregates in structured biofilms(Davey et al. 2003). In addition, rhamnolipidproduction may aid in the formation of maturemushroom structures (Lequette and Greenberg2005). Another quorum-sensing-regulated factorshown to contribute to biofilm formation isthe siderophore pyoverdine. Siderophores aresmall, high-affinity iron chelators secreted bybacteria. In Pseudomonas pyoverdine is criticalfor acquiring iron, and mutants unable tomake pyoverdine formed flat biofilms, whilean isogenic wild-type strain formed structuredbiofilms (Banin et al. 2005). P. aeruginosa alsouses quorum sensing to regulate the productionof two sugar-binding lectins, LecA and LecB,which are secreted from the cell. These lectinsare expressed in biofilms and both lecA and lecBmutant strains formed aberrant biofilms (Diggleet al. 2006; Tielker et al. 2005).

Since so many different quorum-sensing-regulated functions affect biofilm development,it is especially clear that in Pseudomonas bothcritical behaviors are intervened.

Several open questions remain to be answeredto explain the discrepancies between differentstudies. One is whether quorum-sensing responsemay not be induced or active in biofilms grownunder these conditions. It was shown by DeKievitand and colleagues that the expression oflasI and rhlI is highest near the attachment sur-face and decreases toward the periphery of thebiofilm (De Kievit et al. 2001). This spatiotem-poral distribution of quorum-sensing autoinduc-ers producing community members suggests thatthere may be conditions, such as high liquid flow,where signal concentrations may not reach aninducing level in the biofilm.

The PQS System

The PQS system which is comprises from thePseudomonas quinolone signal (PQS); 2-heptyl-3-hydroxy-4-quinolone, the synthase PqsH andthe response regulator PqsR (also called MvfR).The PQS structure is very similar to the Pyocompounds, which had been identified as antibi-otics in 1945 (Dietrich et al. 2006; Hays et al.1945) and shown to belong to the family of4-quinolones in 1952 (Wells 1952; Wells et al.1952).

The three QS systems in P. aeruginosa arearranged in a temporal manner, with AHLs andPQS being released in the early and late expo-nential phase, respectively (Lepine et al. 2003).The expression of the PQS requires LasR, andthe PQS in turn induces transcription of rhlI.These data indicate that the PQS is an additionallink between the Las and Rhl circuits (Miller andBassler 2001). Thus, the PQS initiates the Rhlcascade by allowing the production of the RhlI-derived autoinducer only after the establishmentof the LasI–LasR signaling cascade (Pesci et al.1999a).

The Las, Rhl, and Pqs systems mutuallyregulate the production of virulence factorssuch as elastase, alkaline protease, exotoxin A,rhamnolipids, pyocyanin biofilm formation, andothers (Smith and Iglewski 2003). Specifically,PqsR/MvfR positively regulates the productionof a number of virulence factors as wellas the expression of PA2274, a putativemonooxygenase, and the mexGHI–opmD operonthat encodes proton-driven efflux pumps ofthe resistance-nodulation-cell division (RND)transporter super family (Deziel et al. 2005;Dietrich et al. 2006). It is thought that thisresponse is mediated through PqsE and the PQS(Deziel et al. 2005). P. aeruginosa releases a 4-quinolone signal molecule into the extracellularmilieu, as culture supernatants were found tocontain approximately 6 �M (Pesci et al. 1999b).

Importantly, the PQS has recently beenisolated from the lungs of CF patients infectedwith P. aeruginosa (Collier et al. 2002;

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Guina et al. 2003), and the presence of themolecule in vivo may be a factor in allowingP. aeruginosa to develop or maintain a chronicstate (Collier et al. 2002), involving biofilmformation. In support of this hypothesis, it wasshown that PQS concentrations of 60 �M andabove significantly enhanced surface coverageand biofilm formation of PAO1 on stainless steelcoupons. The PQS expression was shown tobe involved in DNA release in P. aeruginosabiofilms, and the expression of the pqsA reporteroccurred specifically in the microcolonies in theearly phase of biofilm formation (Allesen-Holmet al. 2006; Yang et al. 2007). A later studyhas suggested that the pqsA gene is expressedspecifically in the stalk-forming subpopulation,suggesting strongly that a subpopulation ofquorum-sensing producers dramatically affectsthe development of the biofilm structure as awhole (Yang et al. 2009).

Phenazines

P. aeruginosa releases phenazines which are agroup of small heterocyclic, redox-active com-pounds that are toxic to both prokaryotes andeukaryotes (Mavrodi et al. 2006; Mazzola et al.1992; Price-Whelan et al. 2006).

Phenazines are important virulence factors(Lau et al. 2004) that serve as antibiotics towardmicrobial competitors (Baron and Rowe 1981)and damage mammalian cells (Britigan et al.1992). Phenazines can benefit P. aeruginosa byserving as signaling molecules (Dietrich et al.2006), regulating persister cell formation (Mokeret al.), influencing colony morphology (Dietrichet al. 2008; Kempes et al. 2014), and promotingiron acquisition and biofilm development(Glasser et al. 2014; Wang et al. 2011). Likequinolones, phenazines are excreted from cellsat specific points following exponential growth(Diggle et al. 2003). The phenazine pyocyanin(PYO) is a terminal signaling molecule inthe P. aeruginosa QS network (Dietrich et al.2006). D. K. Newman and coworkers reportedthat micromolar concentrations of phenazinescan support anaerobic survival by transferring

electrons to an extracellular oxidant (Wang et al.2010). This is a critical observation for devel-oping biofilms. Indeed a phenazine-null mutantmakes an especially rugose morphology. Using avariety of approaches, it was demonstrated thatthe rugose morphotype increases colony surfacearea and access to oxygen for resident bacteriawhen phenazines and other electron acceptorsare absent (Dietrich et al. 2008). Consistentwith this, the production of phenazines ormedium amendment with the alternate electronacceptor nitrate promotes colony smoothness.Furthermore, in the phenazine-null mutant, anincrease in the cellular NADH/NADC coincideswith the onset of wrinkling and a decreaseoccurs as wrinkles develop (Dietrich et al. 2013).This pioneering work was followed by severalindependent works that suggested that indeedcolony wrinkling is an adaptation that supportsredox balancing in response to electron acceptorlimitation (Kolodkin-Gal et al. 2013; Okegbeet al. 2014). Furthermore, it provided a novelmissing link between QS and redox balancing.

QscR

QscR is an orphan LuxR homolog that does nothave a partner LuxI homolog, although QscRcan bind the AI 3-oxo-C12-HSL (Lequette et al.2006; Lintz et al. 2011; Oinuma and Greenberg2011).

QscR forms mixed dimers with LasR andRhlR and inactives them (Ledgham et al. 2003).QscR has broader signal specificity than LasRand can respond to some non-self-signals.Synthetic 3OHC10 activated QscR much morestrongly than 3OC12 did (Ha et al. 2012).The acyl side chain of C2 is ten-carbon aswith that of C10-HSL, although it is a non-self-produced signal. The bacterial speciesP. fluorescens, Burkholderia vietnamiensis,and Roseobacter gallaeciensis are capableof producing signals such as C10, C12, and3OHC10, which can preferentially activateQscR to LasR (Wagner-Dobler et al. 2005). Thedifference between QscR and LasR is the broadersignal specificity, suggesting that QscR might

Biofilms: Maintenance, Development, and Disassembly of Bacterial Communities Are. . . 27

respond to extrinsic signals by autoactivating itsown expression. In this situation, P. aeruginosacan preferentially activate QscR. The QscRregulon may be turned on independently of theLasR system in the presence of its preferredsignal. The earlier and stronger activation ofQscR may antagonize the conventional QSsignaling pathway led by LasR and RhlR. Themechanism of C2 inhibition of PAO1 biofilmformation is through repression of the Las andRhl systems by QscR (Weng et al. 2014). QscRactivation was also able to block the antibiotictolerance of biofilms and, when combined withantibiotics, abolish biofilm formation completely.This indicates a possible QSCr-based treatmentstrategy for P. aeruginosa biofilms.

Parallel QS Cascades InitiateDispersal in Vibrio Biofilms

Vibrio species are natural inhabitants of aquaticenvironments and form symbiotic or pathogenicrelationships with eukaryotic hosts. Recentstudies reveal that the ability of Vibrio toform biofilms depends on specific structuralgenes (flagella, pili, and exopolysaccharidebiosynthesis) and regulatory processes (two-component regulators, quorum sensing, and c-di-GMP signaling (Ng and Bassler 2009b; Tischlerand Camilli 2004)). While many Vibrio speciesare free living, a small group can form pathogenicor symbiotic interactions with eukaryotic hosts.These Vibrios change modes between growthwithin their hosts and prolonged survival inaquatic habitats (Yildiz and Visick 2009).Adaptation of Vibrio species to changes in theaquatic ecosystem and changes of their hosts iscritical to their survival and colonization success.One key factor for environmental survival is theability to form biofilms.

Vibrio cholerae

V. cholerae is a Gram-negative bacterium, whichusually inhabits natural aquatic environmentsand is best known as the causative agent of the

human disease cholera, and its QS mechanismshave been investigated extensively in recentyears. This pathogen triggers the disease cholerain humans, which is characterized by acuteenteric infection and severe diarrhea (Yildiz andVisick 2009), and it is a major cause of death indeveloping countries. Factors that are importantfor V. cholerae virulence are the choleraenterotoxin (CT) (enterotoxin is a protein toxinreleased by a microorganism in the intestine),the intestinal colonization factor known as thetoxin coregulated pilus (TCP) and the regulatoryprotein ToxR, which regulates their expression.The expression of CT and TCP in vivo is affectedby environmental signals such as optimumtemperature, sunlight, and osmotic conditions(Faruque et al. 1998; Lee et al. 1999). V. cholerauses cell-to-cell communication to controlpathogenicity and biofilm formation (Milleret al. 2002) (Zhu et al. 2002). Several structuralcomponents play a cardinal role in pathogenicityand in biofilm formation. For example, thetype IV pilus, MSHA, which is responsiblefor mannose-sensitive hemagglutination by V.cholerae El Tor, has been implicated in biofilmformation on nonnutritive, abiotic surfaces(Watnick et al. 1999), as well as in hostcolonization. Also, the colony morphology ofthe wrinkled form of V. cholerae El Tor, whichforms thicker biofilms than non-wrinkled ElTor, results from an exopolysaccharide (EPS)encoded by the vps locus (Yildiz and Schoolnik1999). Importantly, mutants lacking vps clustersexhibited a defect in intestinal colonization. Thespecific quorum-sensing systems most critical forbiofilm regulations are AI-2 and CAI-1.

Bassler and coworkers elucidated thestructure of V. cholerae’s autoinducer, (S)-3-hydroxytridecan-4-one (CAI-1), and demon-strated its control of virulence factor production(Higgins et al. 2007); another important study,by Kelly et al. showed that (S)-3-aminotridecan-4-one (amino-CAI-1) is the precursor of CAI-1produced by the synthase CqsA (Kelly et al.2009). Recently, Wei et al. proposed that3-aminotridec-2-en-4-one (Ea-CAI-1) is theprecursor of CAI-1 (Perez et al. 2012; Wei et al.2011).

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CAI-1 is produced by the enzyme CqsA andsensed by the receptor CqsS, and AI-2 is synthe-sized by the enzyme LuxS and its receptor is theLuxPQ complex (Higgins et al. 2007). BesidesCAI-1, V. cholerae also uses the autoinducer AI-2to control virulence and biofilm formation. TheCqsA/CqsS system is found in several Vibriospecies (Henke and Bassler 2004; Miller et al.2002), which suggests that it functions as anintragenus signal and used for communicationbetween Vibrios (Ng et al. 2011).

CAI-1 and AI-2 operate synergistically tocontrol gene regulation in V. cholerae, althoughCAI-1 was shown to be the dominant signal byHiggins et al. (Higgins et al. 2007).

At low cell density, when AI concentrationsare below the detection limit, CqsS acts as akinase and phosphorylates the response regulatorLuxO. As a result V. cholerae expresses virulencefactors and forms biofilms (Hammer and Bassler2003; Miller et al. 2002; Zhu and Mekalanos2003).

This pattern of gene expression enables hostcolonization and contributes to persistence in theenvironment. At high cell density, when AI con-centration is sufficient, CAI-1 binds CqsS, whichconverts from kinase to phosphatase, leading todephosphorylation and inactivation of LuxO andthus suppression of both the expression of vir-ulence factors and the formation of biofilms,through activation of the negative regulator HapRand repression of the positive regulator VpsT.Bassler and coworkers suggest that these eventsallow V. cholerae to exit from the host, reenter theenvironment in large numbers, and initiate a newcycle of infection (Higgins et al. 2007). We notethat in Gram-negative bacteria, CAI-1 is proba-bly the strongest known trigger of dispersal, thelast stage in a biofilm life cycle (Oppenheimer-Shaanan et al. 2013), allowing planktonic cellsto leave behind the differentiated colony andcolonize a new environment.

Vibrio fischeri

The best described QS system is the Luxsystem of Vibrio fischeri, a bioluminescent

Gram-negative bacterium. The V. fischeri QSmechanism consists of a synthase (LuxI) thatproduces the autoinducer signal, an acyl-homoserine lactone (AHL), 3-oxo-hexanoyl-homoserine lactone (3-oxo-C6-HSL) (Eberhardet al. 1981), and a transcriptional activator(LuxR)that recognizes the signal, leading to activationof genes in the lux operon (Engebrecht et al.1983). Sensor kinases LuxQ (in association withthe periplasmic protein LuxP), LuxN, and CqsS(not depicted), the histidine phosphotransferaseLuxU and the response regulator LuxO (Ngand Bassler 2009a) are involved in transmittingthe signal. Under low cell densities (low AIconcentrations), the Sensor Kinases exhibit netkinase activity and serve as phosphoryl donorsto LuxU, which serves as a phosphoryl donor tothe response regulator LuxO. Biofilm formationplays a key role in host colonization by V. fischeri(Nyholm et al. 2000; Yip et al. 2006). Vibriofischeri is known to promote biofilm formationthrough the symbiotic polysaccharide (syp) locus.The syp locus is a set of 18 genes thought tobe involved in the regulation, production, andtransport of a polysaccharide involved in biofilmformation (Shibata et al. 2012). It was recentlydemonstrated that syp is regulated by QS pathwayand more specifically by LuxU. The loss ofLuxQ and LuxU resulted in a delay in wrinkledcolony formation. In the aforementioned study,LuxU played a more critical role than LuxOin controlling biofilm formation suggesting thatLuxU may function independently of LuxO (Rayand Visick 2012).

Bacillus subtilis, Biofilmas aMulticellular Organism:Differentiation and ParacrineSignaling Orchestrated by QSCascades

Bacterial communities and bacterial biofilms, ahigher order community, thrive in their naturalhabitats as a result of their ability to respondaccordingly to environmental changes. Somemicroorganisms are capable of differentiatinginto subpopulations of phenotypically distinct but

Biofilms: Maintenance, Development, and Disassembly of Bacterial Communities Are. . . 29

genetically identical cells (Aguilar et al. 2007).These subpopulations of cells produce or respondto different signals and serve distinct functionswithin the community. A classic example is thesoil bacterium Bacillus subtilis that responds todifferent environmental cues by differentiatinginto subpopulations of specialized cell types,which coexist within a biofilm (Lopez et al.2009b). Each subpopulation must have the abilityto sense one particular extracellular signal andintegrate it with the rest, discarding irrelevantsignals. For this purpose, B. subtilis possessesat least three different master regulators, Spo0A,DegU, and ComA, that coordinate the activationand regulation of the developmental programsthat result in distinct cell fates within the biofilm(Vlamakis et al. 2013). The initiation of theproduction of the extracellular matrix, which isessential for biofilm formation, is carried out bya subpopulation of specialized cells in B. subtilis,activating a protein named SinI, derepressingSinR, the master regulator of the matrix genes(Chai et al. 2008; Kearns et al. 2005). All ofthe cells are encased within the matrix in amature biofilm. Thus, the matrix serves as a“public good,” benefiting the community as awhole (Vlamakis et al. 2008). Matrix-producingcells specialize to secrete the main componentsof the matrix through dedicated machinery:the extracellular polysaccharide (EPS) (Brandaet al. 2004) and the structural matrix-associatedproteins TasA (Branda et al. 2006), formingamyloid fibers (Romero et al. 2010), and BslA,forming a hydrophobic layer. The expression ofboth EPS and TasA is simultaneously triggeredupon induction of sinI. sinI induction commencesat low levels of Spo0A � P (Branda et al. 2001;Chai et al. 2008; Fujita et al. 2005). Low levelsof spo0A � P in the cell are reached by theaction of four membrane-bound and cytoplasmicsensor histidine kinases: KinA, KinB, KinC,and KinD (McLoon et al. 2011; Vlamakis et al.2013). KinA and KinB synergistically sense theredox state of the biofilm cells. KinB is activatedvia a redox switch involving interaction of itssecond transmembrane segment with one ormore cytochromes under conditions of reducedelectron transport. In parallel KinA is activated

by a decrease in the nicotinamide adeninedinucleotide (NADC)/NADH ratio via bindingof NAD(C) to the kinase in a PAS domain A-dependent manner (Kolodkin-Gal et al. 2013).KinD is a canonical membrane kinase with twotransmembrane segments connected by a 211-amino acid extracellular sensing domain thatis presumably involved in signal recognitionand binding to a specific extracellular signal.KinD was suggested to specifically respond tosmall secreted molecules produced by plants(Beauregard et al. 2013), as well as nonspecificsignals, such as osmotic pressure (Rubinsteinet al. 2012). The membrane kinase KinCharbors two transmembrane segments with noextracellular sensor domain. Instead, KinC hasa PAS–PAC sensor domain in the cytoplasmicregion of the kinase. PAS–PAC sensor domain ofKinC somehow senses the leakage of cytoplasmicpotassium cations (Lopez et al. 2009a). Diversesmall molecules that are able to form pores inthe membrane of the bacterium can induce thispotassium leakage. This triggers the phospho-rylation of Spo0A � P, which leads to matrixproduction. Because of the nature of the stimulus,the various small molecules identified that inducematrix production via KinC differ vastly intheir molecular structure that share the abilityto cause potassium leakage by making pores inthe membrane of B. subtilis (Lopez et al. 2009a).The most important small molecule describedto trigger matrix production via KinC is theself-generated lipopeptide, surfactin (Arima et al.1968; Lopez et al. 2009b). Surfactin production isvia a nonribosomal peptide synthetase machinerytermed SrfAA–AB–AC–AD (henceforth, Srf). Inthis process, multidomain enzymes coordinatelycatalyze several of the reactions needed tosynthesize surfactin (Kluge et al. 1988).

Once produced, surfactin causes the leakageof potassium with the formation of pores in themembrane (Sheppard et al. 1991), and that issensed as a trigger to the subpopulation of matrixproducers to differentiate. Surfactin thus servesas an autoinducer signal. Surfactin is recognizedby its specific mode of action, generating poresin the membrane, promoting membrane leak-age rather than by its chemistry, and offering

30 H. Ganin et al.

Fig. 1 QS signals and biofilm formation by Pseudomonas aeruginosa

an efficient strategy to allow sensing of quitea large repertoire of signals. This mechanismallows B. subtilis to respond not only to self-produced molecules but also to natural productssecreted by other soil-dwelling organisms. Thismechanism may also suggest that ECM secretionmay have developed primarily as a defense strat-egy versus neighboring enemies.

The production of the quorum-sensingmolecule surfactin is tightly regulated by anotherquorum-sensing pathway, mediated by thebacterial pheromone ComX, and the subsequentphosphorylation of the response regulatorComA (Roggiani and Dubnau 1993). ComA � Pactivates the expression of the operon responsiblefor surfactin production (Magnuson et al. 1994).Only after ComX is sensed and surfactin isproduced can surfactin go on to trigger matrixproduction via activation of KinC, suggesting theneed for several sequential cascades for properbiofilm development (Lopez et al. 2009d).

The activation of ComA is at some extentcontrolled by bimodal regulation because onlya subpopulation of cells senses ComX and be-comes surfactin producers (Lopez et al. 2009d).The subpopulation of surfactin producers is dif-ferent from the matrix producers, responding tosurfactin. Therefore, surfactin acts as a unidirec-tional signal in which one population producesthe molecule and another population respondsto it by producing an extracellular matrix. Thismechanism adds sophistication to the concept of“quorum sensing,” where all cells are physiolog-ically similar and thus able to produce the signaland response (Bassler and Losick 2006; Millerand Bassler 2001). In the case of surfactin, thesignaling can be referred to as paracrine signaling

because there is a producing cell that is distinctfrom the nearby cell that can sense the signal.

The “paracrine signaling” system of B. subtiliscan be compared with other autocrine quorum-sensing signaling systems described previouslyin bacteria. For example, as we discussed, Pseu-domonas aeruginosa possesses two interrelatedacyl-homoserine lactone quorum-sensing signal-ing systems. These systems, the LasR–LasI sys-tem and the RhlR–RhlI system, are global regula-tors of the expression of a large number of genesinvolved in diverse developmental processes. Theanalysis of quorum-induced genes suggests thatthe gene expression is sequential and time depen-dent (certain genes are activated early in growth,most genes are activated during the transition,and some genes are activated at the stationaryphase) (Schuster et al. 2003b) (Fig. 1).

QS Signaling Promotes Cannibalism,Tightly Linked with BiofilmFormation

Bacillus subtilis responds to nutrient depletionby sporulating, a developmental process that re-sults in the formation of two distinct cell types(McKenney et al. 2013). However, sporulation isa time- and energy-consuming process. B. subtilisdelays the commitment to initiate sporulationunder nutrient-limited conditions by forming asubpopulation of cells termed cannibals, obtain-ing nutrients by lysing their surrounding sensitivecells. Cannibal cells secrete two peptide toxins,Skf and Sdp, while at the same time express-ing the immunity machinery to resist the actionof these toxins. The toxins kill their sensitive

Biofilms: Maintenance, Development, and Disassembly of Bacterial Communities Are. . . 31

siblings in a process termed cannibalism becausethe dead cells can be used as food to temporar-ily overcome the nutritional limitation and de-lay the onset of sporulation (Ellermeier et al.2006; Engelberg-Kulka et al. 2006; Gonzalez-Pastor et al. 2003). The expression of the Skfand Sdp toxins is positively regulated by thetranscriptional regulator Spo0A, when the cellhas low levels of Spo0A � P (Fujita et al. 2005).Spo0A � P directly induces the expression of theoperon responsible for Skf production (skfA–H)and indirectly, by repressing the repressor AbrB,induces the expression of the operon responsi-ble for Sdp production (sdpABC). Because bothcannibalism and matrix production are triggeredby low levels of Spo0A � P, the expression ofcannibalism and matrix production have beenreported to occur in the same subpopulation ofcells. This subpopulation specializes to producethe extracellular matrix required for biofilm for-mation at the same time that the cannibalismtoxins and the immunity to the action of the tox-ins are expressed. Additionally, as described formatrix production, the differentiation of cannibalcells is also triggered by the quorum-sensingsignal surfactin (Lopez et al. 2009c). Becauseonly cells that have achieved high-enough levelsof Spo0A � P express the cannibalism genes, anycells that do not have Spo0A � P will be sensitiveto the toxins and lyse. The nutrients released bythese lysed cells are used to promote the growthof the matrix producers/cannibals, because thoseare the only cells immune to the action of thecannibalism toxins. In this way, the representa-tion of matrix producers within the communityincreases, allowing them to thrive at the expenseof the rest of the cell types (sporulation is delayedand the other cell types are killed). Cannibalismin B. subtilis may also play a role in regulatingthe differentiation of matrix producers/cannibals.The subpopulation of cells that produce surfactin(the molecule responsible for the differentiationof matrix producers/cannibals) will also benefitfrom cannibalism. Because a fraction of surfactinproducers will ultimately differentiate into com-petent cells, they might take up the DNA releasedwhen cells are killed by the cannibalism toxins(Lopez et al. 2009c, d).

QS Signaling Through AI-2 ControlsBiofilm Formation

As mentioned previously, LuxS-/AI-2-dependentQSS has been proposed to act as a universal lexi-con that mediates intra- and interspecific bacterialbehavior. B. subtilis luxS produces active AI-2able to mediate the interspecific activation of lightproduction in Vibrio harveyi. It was demonstratedthat in B. subtilis, luxS expression was negativelyregulated by the master regulatory proteins ofbiofilm development, SinR and Spo0A. B. sub-tilis cells, from the undomesticated natto strain,required the LuxS-dependent QSS to form robustand differentiated biofilms and also to swarm onsolid surfaces. Furthermore, LuxS activity wasrequired for the formation of complex colonies.AI-2 production and spore morphogenesis werespatially regulated at different sites of the de-veloping architectonically complex colony (Lom-bardia et al. 2006). Though the research of thisQS cascade in Bacillus is still scarce, it is highlyfeasible that AI-2 behaves as a morphogen thatcoordinates the social behavior and biofilm de-velopment of B. subtilis.

Bacillus subtilis Biofilms as aModelfor Multicellularity Regulated by QSCascades

Differentiation of distinct cells types in B. sub-tilis is necessary for the proper development ofthe bacterial community. This differentiation isregulated, at least partially, by sensing severalextracellular signals. Most of these signals areproduced by B. subtilis itself. Secretion and sens-ing of these extracellular signals might regulatethe timing of development in concordance withthe surroundings.

Staphylococcus aureus: A Cross TalkBetween the AIP Pheromoneand the Agr Regulon

Biofilm formation plays a critical role in manydevice-related infections, infective endocarditis,

32 H. Ganin et al.

urinary tract infections, and acute septic arthritisthrough pathogens such as Staphylococcusaureus. S. aureus forms complex and highlyheterogeneous communities in the presence ofglucose to enhance as a result of acidificationof the media caused by increased excretion ofmetabolites. Other supplements such as serumand high salt were also reported to induce biofilmformation. Once a biofilm forms, strikingly,over 60 % of the total cells become phenotypicvariants, making heterogeneity at the molecularlevel in the staphylococci, perhaps the highest ofall Gram-positive bacteria.

Biofilms are known to be heterogeneous struc-tures, with channels running throughout that fa-cilitate the transport of nutrients and water. Thecells are held together by extracellular matrix thatincludes the following:(a) polysaccharides similar or identical to

staphylococcal polysaccharide intercellularadhesion polysaccharides (PIA) (Mack et al.1992). PIA-related polymers are producedby Staphylococcus epidermidis and S. aureus(PNAG) (Foreman et al. 2013);

(b) proteins, mostly surface adhesins, such asBap and SasG (Roche et al. 2003); and

(c) extracellular DNA (eDNA) are the primarymatrix components.

The reason for these assignments is straight-forward; enzymes that degrade each of these ma-terials, such as polysaccharide hydrolases, pro-teases, and DNases, can disassemble staphylo-coccal biofilms.

Staphylococci regulate biofilm formation anddispersal using the agr quorum-sensing system.The agr system responds to the extracellularconcentration of an autoinducing peptide (AIP)signal (Boles and Horswill 2008), which is acyclic thiolactone-containing peptide of varyingamino acid composition depending on the strain(McDowell et al. 2001). Once the local AIPconcentration reaches a critical level, usuallyin the low nanomolar range, AIP binds tothe membrane-bound receptor domain of theAgrC histidine kinase, activating the AgrCAtwo-component system. This activation alters

global gene expression and leads to increaseddetachment cells from a mature biofilm andreturns them to a planktonic state, completingthe biofilm life cycle (Boles and Horswill 2008,2011). Agr activation can result in increasedlevels of staphylococcal proteases that cut cellsurface proteins and disrupt cell–cell interactionswithin the biofilm, and proteases can also beadded exogenously to cause biofilm dispersal.Matrix-degrading materials, such as dispersin B,can lead to biofilm disassembly by weakeningthe structural integrity of the biofilm matrix(Boles and Horswill 2011; Tegmark et al. 1998;Tsompanidou et al. 2011).

The agr system controls staphylococcalbiofilm formation in several scenarios. S. aureusbiofilm formation in some in vivo modelswas pronounced in agr mutants accumulatedsuggesting that the quorum-sensing mechanismwas inhibitory toward biofilm formation.Importantly, pockets of agr-activated S. aureuscells within an established biofilm were observedto detach under in vitro flow conditions, whilethe agr inactive cells remained in the biofilm(Dai et al. 2012). Exogenous AIP addition wasfound to activate the agr system throughout amature biofilm, leading to complete disassemblyand conversion of biofilm-associated cells backto a planktonic phenotype. Thus, QS via theAIP system triggers a biofilm disassemblymechanism (Boles and Horswill 2008). Notably,across the staphylococci, activation of theagr system by QS is known to induce theexpression of phenol-soluble modulins (PSMs).PSMs are surfactant-like molecules and have animportant role in the structuring of staphylococcibiofilms, a property achieved by their sharedphysicochemical properties. PSM expressioncan also result in biofilm dispersal (i.e., thedetachment of cells or cellular clusters frombiofilms), which is a key mechanism leadingto the systemic dissemination of infectionsinvolving biofilms (Periasamy et al. 2012). Thus,the AIP-mediated QS system may have a dualrole in the structuring of biofilms, as well in theirdispersal.

Biofilms: Maintenance, Development, and Disassembly of Bacterial Communities Are. . . 33

Conclusion

The plasticity of transiting between unicellularand multicellular lifestyle renders bacterial cellssimilar to many other types of living cells, whichare capable of unicellular existence, yet gen-erally reside within multicellular communities.Biofilms offer their member cells several ben-efits: they protect their residents from environ-mental insults and assaults, improve their attach-ment to many different hosts, and allow effi-cient access to oxygen and nutrients (Chen et al.2012; Costerton et al. 1987; Dietrich et al. 2013;Kolodkin-Gal et al. 2013). Importantly, the for-mation, maintenance, and disassembly of struc-tured multicellular communities critically dependupon the chemical communication between cells.Those chemical autoinducers are various: Theycan be HSLs determining the maturation of Pseu-domonas biofilms, or CAI-1 signal triggeringVibrio to disperse, ComX pheromone initiatingcomplex development of Bacillus biofilms, orthe AI-P autoinducers controlling the stabilityof staphylococcal communities. But, whateverthe signal is, it seems that bacterial multicellu-lar communities critically depend on a chemicalcross talk between resident bacteria.

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Quorum Sensing in Escherichia coli:Interkingdom, Inter- and IntraspeciesDialogues, and a Suicide-InducingPeptide

Bloom-Ackermann Zohar and Ilana Kolodkin-Gal

Introduction

An emerging theme in microbiology is the abilityof bacteria to communicate with one another viaquorum-sensing signal molecules (Bassler andLosick 2006; Camilli and Bassler 2006; Fuquaet al. 1996; Waters and Bassler 2005). Quorumsensing provides a mechanism for bacteria tomonitor one another’s presence and to modu-late gene expression in response to populationdensity. In the simplest scenario, accumulationof a threshold autoinducer concentration, whichis correlated with increasing population density,initiates a signal transduction cascade that cul-minates in a population-wide alteration in geneexpression. Our text brought here is highlightingthe recent development in the study of quorum-sensing behaviors in E. coli. E. coli is a rod-shaped bacterium from the family Enterobac-teriaceae. It is able to grow both aerobicallyand anaerobically, preferably at 37 ıC, and caneither be nonmotile or motile. Besides beingprominent and a fascinating model organism,Escherichia coli can be an innocuous resident ofthe gastrointestinal tract or cause significant diar-rheal and extraintestinal diseases (Croxen et al.2013). Genome sizes of E. coli can differ bya million base pairs between commensals and

B.-A. Zohar • I. Kolodkin-Gal (�)Department of Molecular Genetics, Weizmann Instituteof Science, Rehovot, Israele-mail: Ilana.Kolodkin-Gal@weizmann.ac.il

pathogenic variants, and this extra-genetic con-tent can contain virulence and fitness genes. Thepathogenic ability of E. coli is therefore largelyafforded by the flexible gene pool through thegain and loss of genetic material at a number ofhot spots throughout the genome (Touchon et al.2009).

Quite surprisingly, this Gram-negative bac-terium, which has been intensively investigatedfor over 60 years and is the most widely stud-ied prokaryotic model system, is poorly under-stood and investigated for its social behaviorsand more particularly for quorum-sensing path-ways. The relative meagerness of data regardingthe quorum-sensing pathways participating in theregulation of group behaviors in E. coli mayalso relate to some critical riddles regarding theexact mechanism of pathogenicity, for example,the regulation of attaching and effacing lesionsand acid resistance during the persistent cattleinfection by enterohemorrhagic E. coli (EHEC)that causes severe foodborne disease (Kanamaruet al. 2000).

This review focuses on the major quorum-sensing systems comprehensively studied in E.coli. We chose to divide them into the followingfive categories:(I) SdiA-mediated signaling [SdiA is a LuxR

homolog, a receptor for homoserinelactones]

(II) Indole signaling, mediated by the self-produced effector indole

(III) AI-2 signaling, mediated by an autoinducerproduced by the enzyme LuxS

V.C. Kalia (ed.), Quorum Sensing vs Quorum Quenching: A Battle with No End in Sight,DOI 10.1007/978-81-322-1982-8__9, © Springer India 2015

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Fig. 1 Self-produced QS signals as well as signals pro-duced by epithelial cells and neighbor gut bacteria are in-tegrated by Escherichia coli. The QscEF two-componentsystem integrates self-produced autoinducer names AI-3, and the hormones produced by the mammalian host(epinephrine and norepinephrine) represented as hexagons(Fig. 1). Self and neighbor bacteria-produced AI-2 repre-

sented as diamond is sensed by LasR. Self-produced EDFrepresented by a star is sensed by the toxin MazF, andfinally, SdiA senses HSLs produced by neighbor bacteriarepresented by triangles. The overall gene expression andbehavior of E. coli, residing in the gut, represents anoutcome of integration of all these signals

(IV) EDF signaling conveyed by a self-producedpeptide that triggers the activation of toxin–antitoxin systems

(V) AI-3/epinephrine/norepinephrine signalingpathway, involved in host–bacteria commu-nication

The majority of these signaling systems areinvolved in interspecies communication, andthe AI-3/epinephrine/norepinephrine signalingsystem is also involved in interkingdomcommunication (Fig. 1).

SdiA Quorum-Sensing System:Sensing Bacterial Neighbors in theGastrointestinal Track

In Gram-negative bacteria, the most studied typeof quorum-sensing (QS) systems are LuxI/LuxRhomologs. The LuxI homolog synthesizes a QSsignal molecule, and the LuxR homolog, thesignal receptor, binds the signal and responds

by regulating gene transcription (Engebrechtand Silverman 1984; Fuqua et al. 1996).LuxI homologs produce a spectrum of relatedN-acyl-homoserine lactone (acyl-HSL) signalmolecules. The acyl-HSLs have a conservedhomoserine lactone ring connected by an amidelinkage to a variably structured acyl side chain.The acyl side chain can vary in length, rangingfrom 4 to 18 carbons, can be substituted witha carbonyl or hydroxyl group on the thirdcarbon, and may or may not be saturated. Eachindividual LuxI produces a type of acyl-HSLspecific for detection by its cognate LuxR.These autoinducers are synthesized in thecytoplasm during exponential growth and candiffuse passively through the bacterial membrane,accumulating both inside and outside of the cell.When the concentration of AHLs reaches thestimulatory level, the AHLs are bound by LuxR-type protein molecules (Hanzelka and Greenberg1995). The LuxR–AHL complexes activate thetranscription of quorum-sensing-regulated target

Quorum Sensing in Escherichia coli: Interkingdom, Inter- and Intraspecies Dialogues. . . 87

genes by binding to the appropriate promoters(Hanzelka and Greenberg 1995). Most LuxR-type proteins require binding of AHL as a foldingswitch that stabilizes them; as in the absence ofthe signal, they are targeted to degradation (Zhuand Winans 2001).

In a singular exception, one branch of theproteobacteria phylum, within the Gammapro-teobacteria class, encodes an orphan LuxRhomolog named SdiA (Ahmer 2004). Thisbranch includes the medically relevant genusof Escherichia, Salmonella, Klebsiella, Shigella(Smith and Ahmer 2003), and Enterobacter(Swearingen et al. 2013). In these genomes, thereare no acyl-HSL synthase genes, reminiscent toLuxI. Furthermore, it has been experimentallyverified that Escherichia coli and Salmonella donot synthesize acyl-HSLs. While E. coli andSalmonella do not produce acyl-HSLs, theycan sense and respond to a variety of acyl-HSLs produced by other QS bacterial species,a phenomenon described as eavesdropping (Leeet al. 2009).

The sdiA (suppressor of cell division inhibitor)gene was first isolated as a positive regulator ofthe cell division genes ftsQAZ (Wang et al. 1991).When expressed from a multi-copy plasmid,SdiA suppresses the expression of a number ofchromosomally encoded cell division inhibitorsleading to an increase in cell division (Wanget al. 1991). SdiA acts as a positive transcriptionregulator by controlling the P2 promoter of theftsQAZ operon. The overexpression of the ftsQAZgene cluster increases bacterial cell division andblocks the action of endogenous cell divisioninhibitors (Wang et al. 1991). The effect ofchromosomal sdiA on chromosomal ftsQAZexpression was not examined, but sdiA mutantdoes not have any apparent cell division defects(Wang et al. 1991). Sitnikov and colleagues ex-plored the hypothesis that SdiA regulates ftsQAZin response to AHL, examining the activationof a plasmid-encoded ftsQ–lacZY fusion bya plasmid-encoded sdiA using synthetic AHL(3-oxo-C6-HSL, 3-OH-C4-HSL, or C12-HSL)(Sitnikov et al. 1996). A fourfold activation ofthe system was observed without AHL, yet uponaddition of AHL, there was a further increase in

the activation up to 7.5-fold. Experiments usingchromosomal sdiA or chromosomal fusions werenot reported (Sitnikov et al. 1996).

Later, an intriguing finding by Kanamaru andcolleagues (2000) linked sdiA-mediated signalingto pathogenicity in enterohemorrhagic E. coli(EHEC) O157:H7. Overexpression of sdiA (us-ing an overexpression vector) caused abnormalcell division and reduced adherence to culturedepithelial cells (Kanamaru et al. 2000). In addi-tion, SdiA reduced expression of virulence fac-tors, encoded on the enterocyte effacement (LEE)pathogenicity island (Lee et al. 2009). The signalsactivating sdiA remained unknown.

Additional link between SdiA-mediated sig-naling and intestinal colonization of calves byE. coli O157:H7 was provided by comparingwild-type and sdiA mutant strains. Transcrip-tome studies of SdiA–AHL signaling in EHECrevealed that SdiA–AHL signaling altered theexpression of 49 genes, including the LEE (lo-cus of enterocyte effacement) locus and GAD(glutamic acid decarboxylase) system. LEE is apathogenicity island, encoding for type III secre-tion system, and the associated chaperons andeffectors required attaching and effacing (AE)lesions in the large intestine. The GAD sys-tem is the most efficient acid resistance (AR)mechanism in E. coli (Castanie-Cornet et al.1999). Both are essential for EHEC colonizationof cattle and pathogenicity (Dziva et al. 2004;Sheng et al. 2006). Importantly, when oxo-C6-homoserine lactone was added, transcription ofthe LEE genes was decreased in the WT strainbut not in an sdiA mutant. These results suggestedthat AHLs repress transcription of the LEE genes,and, critically, this repression is mediated throughSdiA. In addition, Hughes and colleagues demon-strated, by electrophoretic mobility shift assays,that SdiA binds to the promoter of ler (LEE-encoded regulator). Ler (encoded by ler) is themaster regulator of the LEE locus. Thus, SdiA inthe presence of AHL appears to repress the LEEvirulence locus in E. coli O157:H7 by regulatingthe transcription of ler (Hughes et al. 2010).

SdiA has an additional role in controllingbiofilm formation. Biofilms are bacterialmulticellular communities in close association

88 B.-A. Zohar and I. Kolodkin-Gal

with surfaces and interfaces, who acquirephenotypic resistance versus environmentalinsults and antimicrobials (Costerton et al. 1999).Overexpression of sdiA in E. coli at 37 ıC led toan increase in biofilm formation, and an isogenicsdiA null mutant showed a threefold decrease inbiofilm formation as compared to the wild type(Suzuki et al. 2002). Lee and colleagues foundthat an sdiA mutant of E. coli demonstrated a 51-fold increase in biofilm formation as comparedto the wild type at moderate temperatures. Thepresence of SdiA is required to reduce E. colibiofilm formation in the presence of AHLs aswell as in the presence of indole, which willbe discussed later (Lee et al. 2007). It was alsodemonstrated that SdiA plays a role in decreasingthe formation of extracellular matrix components,such as the curli fibers anchoring cell to cellwithin the biofilm, and regulates motility (Leeet al. 2009).

SdiA was also demonstrated to play a rolein antibiotic resistance. Overexpression of sdiAconfirmed resistance to mitomycin C (MMC)(Wei et al. 2001). The proposed mechanism forthis resistance was the activation of efflux pumpsby SdiA. It was also noted that, although overex-pression of sdiA resulted in increased resistanceto MMC, an sdiA mutation in the chromosomedid not increase sensitivity to MMC (Wei et al.2001).

In addition to mitomycin C, ectopic expressionof sdiA can confer resistance to quinolones andchloramphenicol, as well as a less pronouncedincrease in resistance to kanamycin and tetracy-cline (Rahmati et al. 2002). This sdiA-dependentresistance was found to depend on the drug effluxgenes acrAB. An sdiA mutant was reported tobe two- to threefold more sensitive to fluoro-quinolones than wild type but was not more sensi-tive to chloramphenicol, tetracycline, or nalidixicacid (Rahmati et al. 2002).

In E. coli K-12 (with chromosomal sdiA), thegadA gene was upregulated when C6-HSL wasadded; upregulation was noted at 30 ıC but notat 37 ıC (Van Houdt et al. 2006). gadA encodesglutamate decarboxylase A, involved in acid re-sistance (i.e., acid resistance system 2 [AR2]),which allows survival of the bacteria at extreme

pH values. The addition of C6-HSL increased thetolerance of the wild-type E. coli strain to pH 4,whereas the addition of AHL to an isogenic strainwith a deleted sdiA did not lead to acid toleranceand resulted in cell death (Van Houdt et al. 2006).Thus, SdiA is involved in acid resistance. Theauthors did not complement the mutant E. colistrain with an active sdiA gene followed by addi-tion of C6-HSL. Lee and colleagues showed thatoverexpression of SdiA downregulated the genesassociated with acid resistance, including gadA(Lee et al. 2007).

In summary, SdiA-dependent signaling playsvarious important roles in regulating socialand communal behaviors in pathogenic andnonpathogenic strains of E. coli. It is highlyunlikely that it is not dedicated to sense a signalwith physiological relevance. It was suggestedin several studies to sense a stationary-phase-related secondary metabolite, which remainsto be elucidated. However, both E. coli andSalmonella carry functional SdiA, but lack theLuxI homologs, and both inhabit the intestinalenvironment of humans and many other animals.We suggest that the simplest hypothesis is thatE. coli and Salmonella use SdiA to detectacyl-HSL production of the normal intestinalmicrobiota.

Indole Signaling

Indole production by E. coli during a stationarygrowth phase was reported in 1897 (Smith1897). Indole is an aromatic heterocyclicorganic compound, consisting of a six-memberedbenzene ring fused to a five-membered nitrogen-containing pyrrole ring. In bacteria, indole isproduced by the tnaA gene product, codingfor the enzyme tryptophanase, whose majorenzymatic reaction is the breakdown oftryptophan into ammonia, pyruvate, and indole.E. coli produces very little indole duringexponential growth. However, the expressionof tryptophanase is strongly upregulated by thestationary-phase sigma factor RpoS, so indoleproduction rises as cells approach the stationaryphase (Gaimster et al. 2014).

Quorum Sensing in Escherichia coli: Interkingdom, Inter- and Intraspecies Dialogues. . . 89

Indole is highly produced by E. coli, and atest for its presence has been regularly used as adiagnostic marker for the identification of E. coli(Wang et al. 2001). The identity of indole asa possible extracellular signaling molecule wasconfirmed by Wang and colleagues (2001) whodemonstrated that indole can act as a signalingmolecule activating the transcription of gabT,astD, and tnaAB genes (Wang et al. 2001). Acti-vation of the tnaAB operon is predicted to inducemore indole production potentially creating apositive feedback loop. The other two targets ofindole-mediated signaling, astD and gabT, areinvolved in pathways that degrade amino acids topyruvate or succinate providing energy to starv-ing cells. These results led to a speculation thatsignaling by indole may have a role in adaptationof bacterial cells to a nutrient-poor environmentwhere amino acid catabolism is an importantenergy source (Wang et al. 2001). Currently,multiple roles have and are been assigned forindole signaling in various aspects of bacterialphysiology. The biological function of indole hasbeen extensively studied in the past decade, anddiverse functions were reported for the molecule.

Multiple studies have shown that indole in-creases drug resistance by inducing the intrinsicxenobiotic exporter genes (mdtEF and acrD),acting via a two-component system (BaeSR andCpxAR) (Hirakawa et al. 2005). Hirakawa andcolleagues (2005) proposed a model suggestingthat indole first acts on the sensor kinases BaeSand CpxA; the signals are then transmitted tothe cognate response regulators which directlybind to different sites in the promoter regions ofthe exporter genes upregulating their expression.Further evidence for indole-induced drug resis-tance was introduced when the development ofantibiotic-resistant strains was studied. Lee andcolleagues found that a few highly resistant mu-tants rose in the population upon increasing levelsof antibiotic treatment. These mutants improvedthe survival of the population’s less-resistant con-stituents, in part by producing indole (Lee et al.2010). Following this research, Vega and col-leagues examined the hypothesis that indole sig-naling may trigger the formation of bacterialpersisters, a phenomenon in which a subset of an

isogenic bacterial population tolerates antibiotictreatment. Incubating E. coli cultures with indoleprior to treatment with high concentrations ofbactericidal antibiotics led to different degreesof persistence to different antibiotics (Vega et al.2012). Furthermore, the incubation with indoleincreased persistence to each of the tested antibi-otics by at least an order of magnitude indicat-ing that the protective effects of indole are notspecific to a single antibiotic and suggesting thatindole induces the transition to a persistent state(Vega et al. 2012).

Indole also controls group behavior such asbiofilm formation, i.e., a process whereby mi-croorganisms irreversibly attach to and grow ona surface and produce extracellular polymers thatfacilitate attachment. Using DNA microarrays,Ren and colleagues discovered that genes forthe synthesis of indole (tnaAL) were inducedby a stationary-phase signal (Ren et al. 2004b).Following this work, they revealed that the geneencoding tryptophanase, tnaA, was significantlyrepressed in 6-day-old E. coli biofilms in com-plex medium (Ren et al. 2004a). The differentialgene expression of two E. coli mutants, yliHand yceP, both exhibiting increased biofilm for-mation, demonstrated that indole probably in-hibits biofilm formation (Domka et al. 2006).The deletion of each gene leads to biofilms withlower intracellular indole concentrations, result-ing in a dramatic increase in biofilm formation.At the same time, the addition of extracellular in-dole reduced biofilm formation for these mutants(Domka et al. 2006).

In contrast, Di Martino and colleagues re-ported that indole induces biofilm formation inE. coli as tnaA deletion resulted in reduction inbiofilm formation which was restored by the ad-dition of indole (Martino et al. 2003). To explorethis contradiction, Lee and colleagues examinedthe role of indole in biofilms by performing DNAmicroarrays on mutants of genes that controlindole synthesis in E. coli (Lee et al. 2007). Theyrevealed that the effect of exogenous indole ismore significant in the presence of glucose, asglucose turns off endogenous indole productionresulting in a profound effect of the experimen-tal conditions on biofilm formation. In addition

90 B.-A. Zohar and I. Kolodkin-Gal

they discovered that indole negatively regulatesE. coli biofilm formation through SdiA by reduc-ing motility and by influencing acid resistance(Lee et al. 2007). An additional explanation forthe discrepancy observed in the effects of indoleon biofilm formation was the discovery that in-dole signaling occurs primarily at low tempera-tures (below 37 ıC) (Lee et al. 2008).

Indole has also been shown to influencebiofilm formation of enterohemorrhagic E. coliO157:H7 (EHEC) (Bansal et al. 2007) byinfluencing motility, acid resistance, chemotaxis(i.e., movement of an organism in response to achemical stimulus), and adherence to HeLa cells.The migration of EHEC to the epithelial cellsurface while primarily driven by epinephrineand norepinephrine occurs only in regions ofthe biofilm where indole concentrations arebelow the critical threshold. Accordingly, EHECcolonization occurs to a large extent in regions ofthe gastrointestinal tract that are not colonized bynonpathogenic E. coli (regions with low indolelevels). Moreover, since E. coli O157:H7 itselfsecretes indole, subsequent colonization willoccur only in places that are not already colonizedby the pathogen, thereby contributing to furthercolonization and the spread of infection (Bansalet al. 2007).

The relationship between indole and type IIIsecretion and the formation of A/E lesions inpathogenic Escherichia coli (EHEC) O157:H7was examined by Hirakawa and colleagues(2009). They revealed that indole increases theproduction and secretion of type III secretionsystem-mediated translocators, leading to anincrease in the formation of A/E lesions in HeLacells. Addition of indole restored and enhancedthe secretion of type III secretion system-mediated translocators as well as the formation ofA/E lesions by the tnaA deletion mutant EHEC.Taken together, the results reported by Bansaland colleagues and Hirakawa and colleaguesmay indicate that indole has dual roles in thevirulence of EHEC (Bansal et al. 2007; Hirakawaet al. 2009). These observations suggest thatthe virulence of EHEC is tightly regulated bythe concentration of the indole. While additionof 500–600 �M of indole decreased motility,

biofilm formation, and attachment to HeLa cells(Bansal et al. 2007), type III secretion-relatedprotein production and virulence phenotypes arestimulated by indole concentrations of 125 �M(Hirakawa et al. 2009). Indole concentrationin the enteric site may change dynamicallywith the amount of indole-producing entericbacteria the amount of commensal bacteria andthe environmental conditions leading to a tightcontrol of EHEC virulence.

In summary, indole is one of most influen-tial cell-to-cell signaling systems that have beenidentified in E. coli. It may be metabolic innature or true “quorum-sensing” systems meantto coordinate the behavior of microbial popula-tions. It seems to play a cardinal role in regu-lating E. coli social behaviors, persistence, an-tibiotic resistance, and maybe most importantlyvirulence.

luxS/AI-2 System

AHLs represent the major class of known bacte-rial cell–cell signaling molecules. However, thebacterial repertoire of communication was sig-nificantly enhanced by the discovery of a familyof molecules generically termed autoinducer-2(AI-2). These families have been found to bewidespread in the bacterial world and to facilitateinterspecies communication. AI-2s are all derivedfrom a common precursor, 4,5-dihydroxy-2,3-pentanedione (DPD), the product of the LuxS en-zyme (Surette et al. 1999). DPD undergoes spon-taneous rearrangements to produce a collectionof interconverting molecules, some (and perhapsall) of which encode information (Xavier andBassler 2005a). Presumably, AI-2 interconver-sions allow bacteria to respond to endogenouslyproduced AI-2 and also to AI-2 produced by otherbacterial species in the vicinity. Thus, in contrastwith the specific dialogue-based communicationchannels provided by the HSLs, AI-2 representsa universal language. AI-2, often in conjunc-tion with an AHL or oligopeptide autoinducer,controls a variety of traits in different bacte-ria ranging from bioluminescence in V. harveyito growth in Bacillus anthracis to virulence in

Quorum Sensing in Escherichia coli: Interkingdom, Inter- and Intraspecies Dialogues. . . 91

Vibrio cholerae and many other clinically rele-vant pathogens. E. coli carries an active copy ofLuxS, the enzyme involved in the metabolismof S-adenosyl methionine (SAM); it convertsS-ribosyl homocysteine into homocysteine and4,5-dihydroxy-2,3-pentanedione (DPD).

In E. coli, expression of LuxS demonstrateda fascinating case of coupling the production ofquorum-sensing signals to the cell’s metabolicstate. As expected, the expression of luxS in-creases with bacterial growth (Li et al. 2006;Xavier and Bassler 2005b). However, luxS isalso induced at low pH, high osmolarity, and inthe presence of a preferred carbon source suchas glucose (Ahmer 2004). The synthesis anduptake of AI-2 are subject to catabolite repres-sion through a complex of cyclic AMP (cAMP)–CRP (catabolite regulation protein), which di-rectly stimulates transcription of the lsr (luxSregulated) operon and indirectly represses luxSexpression. The cAMP–CRP complex was shownto bind to a CRP binding site located in theupstream region of the lsr promoter and thatmutation in the CRP binding site abolishes thisstimulation (Wang et al. 2005a). The expressionof luxS and the production of AI-2 are regulatedat the posttranscriptional level by a small RNA(sRNA) cyaR, by direct binding with comple-mentary sequences in luxS mRNA, activatingits degradation (De Lay and Gottesman 2009).Given that cyaR is positively regulated by thecAMP–CRP complex, it is thus induced underconditions of low glucose, providing an expla-nation for the observed increase of LuxS in thepresence of glucose (De Lay and Gottesman2009). A second sRNA, micA, was found to affectthe length and transcript levels of luxS in anRNase III-dependent manner (Udekwu 2010), butwhether this regulation affects protein amountsand activity or explains the observed growth de-pendence of LuxS expression is not yet known(Udekwu 2010).

Alternative pathways for AI-2 formationwhich are LuxS independent were reportedby Li and colleagues, as AI-2 activity wasobserved from luxS-deficient extracts suppliedwith adenosine (Li et al. 2006). Similar resultswere observed by Tavender and colleagues

using an E. coli luxS mutant carrying additionalmutations that alter carbon fluxes, suggestingLuxS-independent formation of AI-2, viaspontaneous conversion of ribulose-5-phosphate(Tavender et al. 2008). The importance of thisroute for AI-2 production in E. coli may benegligible; it may, however, be responsible forthe AI-2-like signals reported for some higherorganisms or bacteria lacking luxS (Tavenderet al. 2008).

While the export mechanisms of AI-2 are notfully understood, the uptake of AI-2 by the Lsrtransport system has been extensively studied inmultiple organisms (Pereira et al. 2013; Wanget al. 2005a, b; Xavier and Bassler 2005b). TheAI-2 uptake system by the lsrACDBFG operonwas first elucidated by Taga and colleagues inSalmonella enterica serovar typhimurium (Tagaet al. 2001). The receptor for AI-2 is LsrB, ahigh-affinity substrate-binding periplasmic pro-tein which interacts with the membrane compo-nents of an ABC transport system. The trans-porter comprises a membrane channel formed bytwo transmembrane proteins, LsrC and LsrD, andan ATPase, LsrA, which provides the energy forthe transport of the AI-2 signal (Pereira et al.2013; Xavier et al. 2007). The transporter pro-teins are regulated by cyclic AMP/cyclic AMPreceptor protein and by the product of lsrK andlsrR, located immediately upstream of lsr and di-vergently transcribed in its own lsrRK operon (Liet al. 2007). Following uptake, AI-2 is phospho-rylated by the kinase LsrK to produce phospho-AI-2 (P-AI-2); the active molecule then binds andderepresses the lsr repressor LsrR (Wang et al.2005b). In the absence of P-AI-2, LsrR repressesthe transcription of both the lsr operon and lsrRKoperon, thus regulating its own expression andthat of LsrK (Pereira et al. 2013). This positivefeedback loop derives the uptake of AI-2, allow-ing a rapid uptake. The AI-2 is further processedby two additional genes within the lsr operon,LsrF and LsrG (Xavier et al. 2007). Recently,it was reported by Pereira and colleagues thatphosphoenolpyruvate phosphotransferase system(PTS) is required for Lsr activation and AI-2internalization (Pereira et al. 2012). The PTS cat-alyzes transport across the periplasmic membrane

92 B.-A. Zohar and I. Kolodkin-Gal

of a large range of compounds concomitant withtheir phosphorylation. The proposed mechanismsuggests that the initial uptake of AI-2 is PTSdependent, thereby allowing derepression of thelsr operon and the initiation of Lsr-dependentuptake of AI-2 (Pereira et al. 2012).

The uptake of AI-2 would lead to differentialgene expression regulating various socialbehaviors. Multiple studies have used DNAmicroarrays to show AI-2 controls 166 to 404genes, including those for chemotaxis, flagellarsynthesis, motility, and virulence factors inE. coli (DeLisa et al. 2001; Ren et al. 2004b;Sperandio et al. 2001). Gonzalez Barrios andcolleagues demonstrated that AI-2 stimulatesbiofilm formation and motility in five differentE. coli hosts (ATCC 25404, MG1655, BW25113,DH5’, and JM109) in two different media(Gonzalez Barrios et al. 2006). The induction wasdependent upon the presence of the LsrK enzyme,indicating that AI-2 signaling through the Lsrsystem was responsible for these phenotypes(Gonzalez Barrios et al. 2006). These resultsfurther explain previous reports by Sperandioand colleagues and Ren and colleagues indicatingthat AI-2 controls chemotaxis, flagellar synthesis,and motility in E. coli and that the quorum-sensing antagonist furanone was effective inpreventing the biofilms of E. coli by repressingthese same chemotaxis, flagellar synthesis, andmotility genes (Ren et al. 2004a, b; Sperandioet al. 2001). Further analysis of lsrR and lsrKmutant strains using microarrays increased theevidence for Lsr-dependent regulation of biofilmformation and motility (Li et al. 2007).

An additional role for AI-2 is as a chemoat-tractant (i.e., a chemical agent that induces move-ment of chemotactic cells) for E. coli K-12,a behavior dependent upon both the L-serinereceptor (Tsr) and LsrB (Englert et al. 2009;Hegde et al. 2011). It is hypothesized that LsrBbinding to AI-2 in the periplasm enables inter-action with the periplasmic domain of Tsr; theresulting signaling elicits downstream chemo-tactic responses, while the LsrC seems to bedispensable for this process (Hegde et al. 2011).Given that AI-2 is produced by many different

species of biofilm-forming bacteria, the proposedecological context in which AI-2 chemotaxis oc-curs may serve to recruit free-swimming, plank-tonic bacteria to biofilm (Hegde et al. 2011).Concentration-dependent chemoattraction of en-terohemorrhagic E. coli (EHEC) by AI-2 has alsobeen observed through the use of luxS mutantbacteria (Bansal et al. 2008). In this verotype, AI-2 also regulates pathogen motility and attachmentto HeLa cells (Bansal et al. 2008). Similarly,luxS mutant of enteropathogenic E. coli (EPEC)exhibits reduced motility compared to wild-typebacteria when in the presence of epithelial cells(Girón et al. 2002).

Overall, these studies show that in E. coli, luxSgene expression and AI-2 signaling are impor-tant for proper regulation of biofilm formation,motility, attachment to epithelial cells, and othercrucial virulence traits.

Peptide Signaling in E. coli, the EDF

In Gram-positive bacteria, autoinducers are short,usually modified peptides processed from pre-cursors. They are involved, for example, in thedevelopment of competence and sporulation in B.subtilis (Lazazzera 2001; Magnuson et al. 1994;Tortosa and Dubnau 1999) as well as in the vir-ulence response in S. aureus (Ji et al. 1997; Laz-dunski et al. 2004; Novick 2003) and in biofilmformation in Streptococcus mutans (Aspiras et al.2004). These oligopeptide autoinducers are ac-tively transported out of the cell, and they interactwith the external domains of membrane-boundsensor proteins. Signal transduction occurs bya phosphorylation cascade that activates a DNAbinding protein that controls transcription of tar-get genes. These autoinducers in Gram-positivebacteria are highly specific because each sen-sor oligopeptide selects for a given peptide sig-nal (Lyon and Novick 2004; Waters and Bassler2005). Unique case for peptide based signalinglinking Programmed Cell Death and QS existsin E. coli, several prokaryotic genetic moduleshave been described as systems that mediate pro-grammed cell death. Among these is the E. coli

Quorum Sensing in Escherichia coli: Interkingdom, Inter- and Intraspecies Dialogues. . . 93

toxin–antitoxin module mazEF, which is the firstbacterial programmed cell death system that wasdescribed. mazF encodes a stable toxin, MazF,and mazE encodes a labile antitoxin, MazE, thatprevents the lethal effect of MazF. Thus, anystressful condition that prevents the expressionof the chromosomally borne mazEF module willlead to the reduction of MazE in the cell, permit-ting toxin MazF to act freely (Engelberg-Kulkaet al. 2006). Such conditions include (1) short-term inhibition of transcription and/or translationby antibiotics such as rifampin, chlorampheni-col, and spectinomycin (Sat et al. 2001); (2) theoverproduction of ppGpp, which inhibits mazEFtranscription (Aizenman et al. 1996); and (3)DNA damage caused by thymine starvation (Satet al. 2003) as well as by DNA-damaging agents,such as mitomycin C or nalidixic acid (Hazanet al. 2004). These antibiotics and stressful con-ditions that are well known to cause bacterialcell death have been found to act through themazEF module. Clearly, a system that causesany given cell to die is not advantageous to thatparticular cell. On the other hand, the death of anindividual cell may be advantageous for the bac-terial population, enduring stressful conditionsas a whole. E. coli mazEF-mediated cell deathcould only be observed in cultures with highpopulation density. Therefore, it was assumedthat cell death probably requires a “quorum sens-ing,” a process in which a secreted autoinducerallows cells to “measure” cell density of themedium. This quorum-sensing process involves asecreted signaling molecule that was designatedthe extracellular death f actor (EDF). EDF wasdetermined to be an oligopeptide. Thus, it wasdifferent from other molecules so far describedto participate in quorum sensing in E. coli: AHL,AI-2, and indole. The characterization of thechemical nature of EDF revealed that EDF is alinear pentapeptide NNWNN. Each of the fiveamino acids in EDF is important for its mazEF-mediated killing activity, and the terminal as-paragines are the most crucial (Kolodkin-Galet al. 2007). The quorum-sensing process in-volved in mazEF-mediated cell death and thequorum-sensing peptide EDF are particularly in-

teresting not only because no other peptide hasapparently been reported to be involved in quo-rum sensing in E. coli but also because EDFappears to be a distinct type of molecule re-lated to the quorum-sensing peptides of gram-positive bacteria. An additional surprising find-ing was the source of the pentapeptide. Manyself-generated, secreted signaling molecules inbacteria are small molecules, including peptides.So the fact that the “extracellular death factor”is a peptide was no surprise. But the peptidesignals described in earlier studies are encodedin small genes that generate prepeptides, whichare processed to yield the signaling molecule. TheE. coli peptide is derived from the degradation ofglucose-6-phosphate dehydrogenase, a metabolicenzyme. Although the exact pathway leading tothe production of this pentapeptide remains to bedefined, it seems reasonable that it is made whenthe bacterium is under an apparent internal stress.Such stress can be due to the onset of starvationthat can generate this signal, priming the popu-lation such that some cells can be killed throughthe programmed cell death pathway when con-fronted with other stresses (Kolter 2007). Thezwf product, carrying the sequence NNWDN,may generate the full NNWNN sequence onlyby subsequent amidation step. Amidation mayoccur either before or after the cleavage of theprecursor by one of E. coli’s proteases. Theinvolvement of Asn synthetase A, an enzymethat can perform such a modification (Humbertand Simoni 1980), was implied; deleting thegene asnA prevented production of active EDF.In the mid-log phase only, the rate-limiting en-zyme in the pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), E. coli Zwf,is apparently cleaved between its catalytic andstructural domains to release an internal pen-tapeptide, NNWDN. Amidation of the third as-paragine residue is presumed to yield the killerpentapeptide, NNWNN; exposure to recombinantNNWNN, together with a transient stress (e.g.,10 min rifampicin), which activates MazEF, re-sults in significant, but not complete, loss ofviability. Other stresses that enforce MazF sta-bilization or activate RelA to synthesize ppGpp

94 B.-A. Zohar and I. Kolodkin-Gal

might also activate this pathway (Kolodkin-Galand Engelberg-Kulka 2008; Kolodkin-Gal et al.2007).

More recently, the mode of action for EDF wasrevealed (Belitsky et al. 2011). It was shown thatEDF specifically affects the endoribonucleolyticactivities of MazF, the mRNA interfering toxin,and of an homologous toxin ChpBK. In vitro,EDF significantly amplified the endoribonucle-olytic activities of both MazF and ChpBK. EDFalso overcame the inhibitory activities of theantitoxin MazE over the toxin MazF and of theantitoxin ChpBI over the toxin ChpBK. EDF andMazF were directly interacting, and peptide–protein modeling showed parallel contactsbetween EDF–MazF and MazE–MazF. Thus,the EDF system is a quorum-sensing system ofmany novel components, both at the level of thepeptide autoinducer, derived by proteolysis ofmetabolic protein, and at the mechanistic level,where once accumulated, the peptide carriescatalytic properties.

Very recently, Kumar and colleagues havefurther demonstrated that the E. coli mazEF-mediated cell death could be triggered by QSpeptides from the supernatants (SN) of theGram-positive bacterium Bacillus subtilis andthe Gram-negative bacterium Pseudomonasaeruginosa. A hexapeptide RGQQNE wasproduced by Bacillus, and three peptides,a nonapeptide INEQTVVTK and two hex-adecapeptides VEVSDDGSGGNTSLSQ andAPKLSDGAAAGYVTKA, were produced byPseudomonas aeruginosa. When added to dilutedE. coli cultures, each of these peptides acted as aninterspecies EDF that triggered mazEF-mediateddeath. Furthermore, though their sequences arevery different, each of these EDFs amplified theendoribonucleolytic activity of E. coli MazF,probably by interacting with different sites onE. coli MazF. Therefore, the group of QS peptideswas expended to several additional differentpeptides. This family provides the first exampleof quorum-sensing molecules participating ininterspecies bacterial cell death. Furthermore,each of these peptides provides the basis of a newclass of antibiotics triggering death by actingfrom outside the cell (Kumar et al. 2013).

AI-3/Epinephrine/NorepinephrineSystem

Prokaryotes and eukaryotes coexist in bothcommensal and pathogenic relationships formillions of years, consequently coevolving tosense and respond to each other’s signalingmolecules (Karavolos et al. 2013). Interkingdomchemical signaling plays an important role inthe relationships forged between bacteria andanimals. Such interkingdom signaling processesled to high-jacking by bacterial pathogens, suchas enterohemorrhagic Escherichia coli (EHEC),to activate virulence genes, colonize the host,and initiate the disease process (Clarke et al.2006; Walters and Sperandio 2006). EHECcolonizes the large bowel and causes a lesionon intestinal epithelial cells (AE lesions). Thegenes involved in the formation of the AElesion are encoded within the chromosomalLEE pathogenicity island, which is present inEHEC but absent in commensal and K-12 E. coli(Sperandio et al. 2003). The majority of theLEE genes are organized in five operons (LEE1–5), containing a transcriptional activator (Ler)essential for the expression of the LEE genesin addition to the type three secretion systemand virulence effectors (Sperandio et al. 2001;Sperandio et al. 2003). Upon reaching the humancolon, EHEC senses the autoinducer-3 (AI-3)produced by the microbial gastrointestinal floraand epinephrine and norepinephrine, produced bythe host, through histidine sensor kinases (HKs)in their membrane (Clarke et al. 2006). HKsconstitute the predominant family of signalingproteins in bacteria, usually acting in concert witha response regulator (RR) protein constituting atwo-component system (Hughes et al. 2009).Two HKs, QseC and QseE, both part of thetwo-component systems (QseCB and QseEF)characterized in E. coli have been reported tosense AI-3, epinephrine, and norepinephrine.Upon sensing AI-3, QseC initiates the signalingcascade that will activate the flagella regulon,leading to swimming motility, which may aidEHEC to reach the intestinal epithelial layer.QseC activates transcription of the master

Quorum Sensing in Escherichia coli: Interkingdom, Inter- and Intraspecies Dialogues. . . 95

regulators of the flagellar regulon, directlythrough QseB as well as activating AE lesionformation and Shiga toxin expression. While theactivation of the flagellar regulon is dependenton QseB’s phosphorylation state (Clarke andSperandio 2005), the expression of the LEE andShiga toxin genes is not regulated by QseB.As EHEC approaches the epithelium and startsforming AE lesions, it is exposed to epinephrineand/or norepinephrine; AE lesion formationand the commencement of bloody diarrheamay increase EHEC exposure to epinephrineand norepinephrine, further upregulatingexpression of virulence genes in EHEC (Hugheset al. 2009).

A second two-component system, the QseEFsystem (Reading et al. 2007), where QseE is theHK and QseF is the RR was shown to also reg-ulate virulence in EHEC. QseE can also respondto the host hormone epinephrine like QseC but,in contrast, does not sense the bacterial signalAI-3. The QseEF system is not involved in theregulation of flagella and motility but plays animportant role in activating genes necessary forAE lesion formation (Reading et al. 2007) andalso activates expression of Shiga toxin.

Norepinephrine has been reported to inducebacterial growth (Freestone et al. 2000), in addi-tion to a role in activates virulence, e.g., Shigatoxin expression in E. coli (Lyte et al. 1996) byan unknown mechanism of induction. Becauseepinephrine and norepinephrine exert a profoundeffect on the host physiology and immune sys-tem, the ability to sense these hormones by bac-teria may facilitate gauging the fitness of the host.

Following their earlier studies (Clarke et al.2006; Clarke and Sperandio 2005) (Hughes et al.2009; Walters et al. 2006), the Sperandio grouprecently discovered a novel two-componentsignal transduction system, named FusKR,where FusK is the histidine sensor kinaseand FusR the response regulator. FusK sensesfucose and controls expression of virulence andmetabolic genes. This fucose-sensing system isrequired for robust EHEC colonization of themammalian intestine (Pacheco et al. 2012). Theproposed model is that fucose freed from themucus layer by a member of the microbiota,

Bacteroides thetaiotaomicron, inhibits LEEexpression, relieving the pathogen from themetabolic burden of expressing the type IIIsecretion system and giving it a competitivegrowth advantage in the lumen of the gut(Pacheco et al. 2012). Once EHEC approachesthe mucosal surface, adrenergic metabolitesderepress the LEE, initiating its adherencemechanisms. B. thetaiotaomicron was able torepress ler expression when incubated withmucin, a derivative of mucus that contains boundfucose, suggesting that the fucosidases producedby B. thetaiotaomicron cleave fucose from mucinto directly repress virulence expression of EHEC(Keeney and Finlay 2013).

The existence of multiple evidences forinterkingdom communication by bacterialpathogens, relying on the host adrenergicnetwork, may hence reflect the culmination ofmany millions of years of receptor structuralevolution to accommodate these signals andorchestrate the best survival response. Theseinteractions are delicately balanced, and it isclear that in most circumstances, they can benefitthe pathogen.

Summary

The existence and importance of quorum-sensing signaling in E. coli have been establishedthroughout the last decades. Some scenarios inwhich this occurs in nature, however, are stilllargely unknown and many questions remain.First, it is clear that the gastrointestinal tractsof most animals, excluding the bovine rumen,do not contain AHL. It appears that EHECuses sdiA in the bovine rumen to repress theLEE pathogenicity island and to increase acidresistance. What organism(s) are producing theAHLs and what role do the AHLs play in therumen community? Is detection of bacteria inthe rumen the only scenario in which sdiAprovides a benefit to EHEC? Second, it hasbeen embroiled that LuxS synthesizes AI-2,as well as an autoinducer coined AI-2, whichremains structurally unsolved. Thus, LuxScan govern bacterial behaviors such as the

96 B.-A. Zohar and I. Kolodkin-Gal

secretion of virulence factors, biofilm formation,and swarming motility. Can novel approachesto interrupt LuxS sensing be recognized asnext-generation antimicrobials? How doesinterspecies communication from neighborgastrointestinal tract resident [both Gram-negative and Gram-positive bacteria] use thispathway to communicate with E. coli? Andlastly, how prominent is the role of interkingdomcommunication is in shaping the behaviors ofE. coli in vivo? Clearly, the study of QS and QSsensing by Escherichia coli has only just begun.

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