Mutation design and strain background influence the phenotype of Escherichia coli luxS mutants

Mutation design and strain background influence thephenotype of Escherichia coli luxS mutants

Richard Haigh,1 Brijesh Kumar,2 Sara Sandrini2 andPrimrose Freestone2*Departments of 1Genetics and 2Infection, Immunity andInflammation, University of Leicester, Leicester, UK.

Summary

Previous analyses of luxS in Escherichia coli haveused different strain backgrounds and design formatsto produce the luxS mutation, resulting in luxSmutants with confusingly dissimilar phenotypes. Thisstudy therefore investigates the roles that strain back-ground and mutational design strategy have upon thephenotype of the pathogenic E. coli luxS mutant. Weinactivated luxS in three E. coli backgrounds: enter-opathogenic E. coli E2348-69, and enterohaemor-rhagic strains Sakai and NCTC12900. To investigatethe influence of mutational design strategy, four muta-tion formats were used: antibiotic resistance insertionmethodologies as previously employed, using tetracy-cline and chloramphenicol resistance cassettes, andnon-polar strategies creating deletion and prematuretermination mutations. Our study showed that theE. coli luxS phenotype was markedly dependent onstrain background: in some strains disruption of luxScaused significant metabolic stress or no stress at all.How the luxS mutation was constructed also shapedits phenotype: non-polar mutants were very similar towild type, while mutations made using the antibioticinsertion methodologies produced phenotypes defec-tive in growth and virulence. Proteomic profiling of ourluxS mutants showed only a few proteins were differ-entially expressed and those that were altered sug-gested a metabolic rather than communication role forthe E. coli luxS gene product.

Introduction

Work over the past two decades has established that manybacteria use sophisticated communication systems toco-ordinate multiple biological activities, and among thesesystems those involving quorum-sensing have been

shown to be key virulence regulators in both Gram-negative and Gram-positive pathogens (Ng and Bassler,2009; Antunes et al., 2010). The LuxS protein was initiallycharacterized in the fish pathogen Vibrio harveyi where it isinvolved in a quorum sensing response system whichregulates production of luminescence (Nealson et al.,1970; Miller and Bassler, 2001). luxS also has generalmicrobiological interest because of its role in the activatedmethyl cycle where it recycles the toxic intermediateS-adenosylhomocysteine (SAH) (Winzer et al., 2002a;Vendeville et al., 2005). LuxS’s metabolic activity catalys-ing the breakdown of S-ribosylhomocysteine to homo-cysteine also produces 4,5-dihydroxy-2,3-pentanedione(DPD); this molecule then spontaneously rearrangesleading to the production of the autoinducer 2 molecule(AI-2), which has been proposed to be a general inter-species quorum sensing signal (Miller and Bassler, 2001;Ng and Bassler, 2009; Pereira et al., 2013). Ascertainingthe role of luxS in relation to the virulence of bacterialpathogens has been a particular focus of interest for manyresearch groups for many years (Winzer et al., 2002a;2003; Sun et al., 2004; Vendeville et al., 2005; Antuneset al., 2010). Studies of the effects of disruption of luxS onpathogenicity have involved diverse bacterial species suchas Erwinia carotovora (Coulthurst et al., 2006), Lactobacil-lus rhamnosus (Lebeer et al., 2007), Porphyromonasgingivalis (Burgess et al., 2002), Neisseria meningitidis(Winzer et al., 2002b; Schauder et al., 2005; Heurlier et al.,2009), Staphylococcus aureus (Doherty et al., 2006) andStreptococcus mutans (Burne and Wen, 2004). The major-ity of investigations have however concentrated on the roleof luxS in the infectivity of human enteric pathogens. Theseinclude Campylobacter jejuni (He et al., 2008), Escherichiacoli (Sperandio et al., 1999; 2001; 2003; Surette andBassler, 1998; Surette et al., 1999; DeLisa et al., 2001;Sircili et al., 2004; Wang et al., 2005a; 2005b; Walterset al., 2006; Soni et al., 2007; Kim et al., 2010) Helicobac-ter pylori (Doherty et al., 2010), Salmonella enterica(Surette and Bassler, 1998; Surette et al., 1999; Soni et al.,2008; Perrett et al., 2009; Jesudhasan et al., 2010; Yohanand John, 2010) and Vibrio cholerae (Miller and Bassler,2001). The experimental work in these species has led toAI-2 being proposed as a universal signal for both inter-species communication and virulence regulation (Watersand Bassler, 2005; Bassler and Losick, 2006; Ng and

Accepted 12 April, 2013. *For correspondence. E-mail [email protected]; Tel. (+44) (0)116 2525656; Fax (+44) (0)116 2525030.

Molecular Microbiology (2013) 88(5), 951–969 ■ doi:10.1111/mmi.12237First published online 7 May 2013

© 2013 John Wiley & Sons Ltd

mailto:[email protected]

mailto:[email protected]

Bassler, 2009; Antunes et al., 2010). However, becausedisruption of the activated methyl cycle will also affectgeneral fitness, which may then in turn modulate thevirulence of bacteria (for instance see Lebeer et al., 2007),it becomes unclear as to what is cause and what is effect.As such there has evolved over the last decade (as moreand more analyses of luxS disruption in bacteria are pub-lished) a debate as to the exact role of LuxS and AI-2 inintercellular communication and metabolism (Winzer et al.,2003; Sun et al., 2004; Vendeville et al., 2005; Rezzonicoand Duffy, 2008; Pereira et al., 2013).

A wider regulatory role for luxS in pathogenic E. coli wasfirst proposed by Sperandio et al. (2001) who used tran-scriptional profiling to show that mutation of luxS in anE. coli O157:H7 strain affected expression of around 400genes (nearly 10% of the genome); these included manyvirulence genes, such as those involved in motility, locus ofenterocyte effacement (LEE), fimbriae and chemotaxis. Afurther study by DeLisa et al. (2001) examining the effect ofquorum sensing in an E. coli W3310 luxS mutant found that242 genes (5.6% of the genome) were differentially tran-scribed after the addition of conditioned medium contain-ing AI-2. These effects upon gene expression led to thesuggestion that luxS and AI-2 had a global regulatory rolein E. coli O157:H7 (Sperandio et al., 2001). But, in markedcontrast to these previous studies, when Wang et al.(2005a) used microarrays to examine the effects of muta-tion of the luxS gene in E. coli K12, they found that very fewgenes (around 40) were differentially regulated by luxS.Importantly, most of the affected genes were involved inbasic metabolism. Although these new data seemed tocast doubt on luxS as a global regulator it was suggestedthat the new strain background used, E. coli K12, might bea likely source of the differences between these studies(Sperandio et al., 1999; 2001; 2003; DeLisa et al., 2001;Wang et al., 2005a). It must be noted that as well asdifferences in the genetic backgrounds of the E. coli strainsemployed, these profiling investigations also used differentmutant creation strategies: inactivation by a tetracyclineresistance insertion for the Sperandio et al. (2001) andDeLisa et al. (2001) studies, and luxS gene deletion andreplacement by a kanamycin resistance gene for the Wanget al. investigation (Wang et al., 2005a). To complicatethings further a later proteomic investigation by Soni et al.(2007), utilizing exactly the same O157:H7 luxS tetracy-cline resistance insertion mutant strain (VS94) used in theSperandio et al. (2001) microarray study, has providedmore conflicting evidence in that only a few of proteins,rather than hundreds, were seen to be differentiallyexpressed in this mutant.

Although luxS mutants have been used in multiple inves-tigations of E. coli virulence (Sperandio et al., 1999; 2001;2003; Surette and Bassler, 1998; Surette et al., 1999;DeLisa et al., 2001; Sircili et al., 2004; Wang et al.,

2005a,b; Clarke et al., 2006; Walters et al., 2006; Soniet al., 2007; Kim et al., 2010) there has still been nocomprehensive investigation into why the original Speran-dio and Wang reports produced such conflicting results.The current study attempts to resolve this confusion byinvestigating the importance of the mechanics used tocreate a luxS mutation. We examined the physiologicaleffects of mutation of luxS in three well-established patho-genic E. coli backgrounds: enteropathogenic E. coli(EPEC) strain E2348-69, and enterohaemorrhagic E. coli(EHEC) strains Sakai and NCTC12900. To resolve theissue of the influence of mutational design on the pheno-type of the E. coli luxS mutants, four mutation formats wereused to create the luxS mutations. These included two ofthe previously used insertion methodologies [using eithertetracycline (Sperandio et al., 2001) or chloramphenicol(Sircili et al., 2004) antibiotic resistance cassettes], andtwo novel non-polar mutations which created eitherframeshift or deletion mutants. Our results showed that thedesign strategy used to make the mutant and the strainbackground that hosts it both significantly influence theresulting luxS mutant phenotype. This has major implica-tions for more than 10 years of LuxS research, as itsuggests that the early LuxS studies were conducted usingmutants with atypical or even artefactual phenotypes.

Results

Design and construction of the E. coli luxS mutants

Our interest was initially drawn to LuxS due to reports ofthe effects of a luxS mutation upon both EHEC and EPECvirulence mechanisms (Sperandio et al., 1999; 2001), andalso because of the proposed links of luxS to catecho-lamine signalling (Sperandio et al., 2003). In a preliminaryinvestigation we constructed a complete in-frame deletionmutant of luxS in an O157:H7 strain (Sakai) (Fig. 1Ashows the genetic lineage of our luxS strains and theprimer sites used to create the mutations, and Fig. 1Bshows the deletion formats). However, upon testing thedeletion mutant failed to demonstrate many of thedecreased virulence phenotypes previously reported forE. coli luxS (these investigations are detailed later in thisreport). In order to investigate this anomaly further weconstructed a second mutation in which the luxS gene isintact but where a premature stop codon (TAA) has beenintroduced at codon 4 (Fig. 1B). Both of these mutationswere introduced into enteropathogenic E. coli strainE2348-69, and the toxin minus E. coli O157:H7 strainsSakai and NCTC12900; these strains have been desig-nated Deletion (Del) and Termination (TAA) respectively.For comparison the plasmids pVS72 and pVS98, whichwere used to construct the original O157:H7 luxS mutantsfrom which the global role of luxS originated (Sperandio

952 R. Haigh, B. Kumar, S. Sandrini and P. Freestone ■

© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 88, 951–969

et al., 1999; 2001; 2003; Sircili et al., 2004) were obtainedand these were also used to create luxS mutants in thethree E. coli strain backgrounds. Plasmid pVS72, whichwas used to create strain VS94 (a luxS mutant of strain86-24 (Sperandio et al., 1999; 2001; 2003) is an insertionmutant (at luxS codon 78) containing the tetracyclineresistance gene (in the current study this mutation formathas been designated Tet). The second luxS mutant con-struct (pVS98) is an insertion mutation at the same pointin the luxS gene but containing the chloramphenicolresistance gene (Sircili et al., 2004) (which we have heretermed Cm). All four of the mutant luxS genes were trans-ferred into their respective host strains using the sacB-dependent positive-selection system.

Different E. coli strains with different luxS mutationsshow different growth phenotypes

Typical growth response data obtained for the three setsof E. coli luxS mutants in both rich and minimal media areshown in Fig. 2. Most of the luxS mutants grew similarly towild type in rich media (Luria broth), although in both theE2348-69 and Sakai backgrounds the Tet mutant consist-ently showed a slightly longer lag phase (Fig. 2A, panelA). In DMEM, a medium often used for bacterial virulencestudies, (Fig. 2A, panel B) the E2348-69 Tet luxS mutant

displayed a marked growth defect which was not demon-strated by the corresponding Tet mutants in the Sakai orNCTC12900 backgrounds; however, all of the other luxSmutants in each of three strains showed essentially wild-type growth profiles in DMEM. In the more metabolicallychallenging glucose-supplemented M9 minimal medium(Fig. 2A, panel C), differences in growth profiles for boththe Tet and Cm luxS mutants were observed and theinfluences of strain background also became apparent. Inthe Sakai strain, the Tet and Cm mutants showed markedgrowth defects which were not exhibited by either theframeshift or termination mutants; whereas in strainE2348-69 only the Tet mutant grew poorly. Even moreatypical growth was observed in the Tet and Cm mutantswhen the strains were grown in M9 supplemented withpoorer carbon sources such as succinate (data notshown). In contrast to the E2348-69 and Sakai strainbackgrounds, all of the NCTC12900 luxS mutants grewsimilarly to their wild-type parent regardless of the culturemedia used.

Since our luxS mutants were deficient in the productionof AI-2 we investigated whether the aberrant growth of theTet and Cm mutants could be corrected by the addition ofexogenous AI-2. Figure 2B shows that the M9 mediumgrowth profiles of the Tet and Cm mutants in eitherE2348-69 or Sakai were not improved by the addition of

Fig. 1. The E. coli luxS locus and mutationdesign strategies. Panel (A) shows the geneorganization within the luxS regions in theE. coli E2348-69 and the O157:H7 lineagebackgrounds and the location of the primersused for the construction of the luxSmutations. Schematic outlines of thestrategies used for the four mutant types areshown in (B).

Escherichia coli luxS mutants 953


conditioned media from an AI-2 producing strain. Collec-tively, the results in Fig. 2A and B demonstrate that bothstrain background and mutation design can significantlyinfluence the growth characteristics of E. coli luxSmutants, and that lack of AI-2 is not the obvious underly-ing reason for the phenotypes observed.

Inter-kingdom signal (catecholamine) responsiveness isnot altered by luxS mutation

Sperandio et al. (2003) reported that mutation of luxSreduces the motility of E. coli, and that this can be restoredby addition of the circulatory catecholamine stresshormone adrenaline. We therefore investigated the cat-echolamine signalling responsiveness of our three sets ofluxS mutants and their parent strains to adrenaline, aswell as the catecholamines noradrenaline and dopamine(which in contrast to adrenaline, have been shown to be

normally present within the enteric nervous system).We carried out a series of catecholamine growth assaysusing an established serum-containing minimal medium(Freestone et al., 1999; 2008) (Fig. 3). Although theNCTC12900 Tet and Cm luxS mutants typically grew lesswell in serum-based medium, neither they nor any of theother 10 luxS mutant strains showed any significant differ-ences from their wild-type parents in their ability to respondto each of the three catecholamines. It is noteworthy that interms of the growth stimulation context, the apparent pref-erence of all of the wild-type E. coli and luxS mutant strainswas not for adrenaline, but instead noradrenaline anddopamine, the catecholamines that are found within thegut (Fig. 3) (Freestone et al., 2007). Figure 3 also clearlydemonstrates that LuxS is not required for catecholamineresponsiveness, and suggests that, at least in the contextof growth, there is no cross-over between intra-kingdomand inter-kingdom signalling pathways.

Fig. 2. Strain background and mutation format but not AI-2 influence the E. coli luxS mutant growth phenotype.A. Overnight cultures of the four luxS mutants of E. coli E2348-69, Sakai and NCTC12900 plus their wild-type parents prepared as describedin Experimental procedures were diluted 1:1000 into triplicate aliquots of fresh Luria broth (panel A), DMEM (panel B) or M9 minimal mediumsupplemented with 0.4% glucose (panel C). The time-courses shown are the means of triplicate growth profiles and are typical data from threeseparate sets of experiments. Key: blue, wild type; luxS mutants: black, TAA; purple, Del; red, Tet; green, Cm.B. The growth profiles of the E. coli E2348-69, Sakai and NCTC12900 wild type, Tet and Cm luxS mutants in M9 minimal medium in theabsence (solid line) or presence of 5 units ml−1 AI-2 (prepared from the wild-type parent of each strain). The TAA and Del mutant growthprofiles were unchanged in the presence of AI-2 and so are not shown. Key: blue, wild type; red, Tet luxS mutant; green, Cm luxS mutant.



Mutation of luxS does not affect surface attachmentbut may (in the case of some mutant formats)reduce motility

Inactivation of luxS has previously been reported to affectbiofilm formation in a variety of bacterial species(reviewed in Hardie and Heurlier, 2008). We thereforeexamined the first stage of biofilm formation, surfaceattachment, for our 12 luxS mutants following growth ineither DMEM or LB culture media (Fig. 4). It can be seenthat, for both of the media tested, there were no majordifferences in surface attachment between any of the luxSmutants and their parents thus suggesting that luxS is notrequired for the initial stages of biofilm formation in theseE. coli strains.

It has been reported that mutation of luxS affects themotility of E. coli, and that this can, to a degree, berestored by addition of an adrenergic catecholamine(Sperandio et al., 2003). We therefore investigated themotility of our strains in LB or DMEM motility media, eitherwith no supplementations or with physiological (5 μM) orhigher (50 μM) additions of noradrenaline (Fig. 5). For allthree strains neither the TAA nor Del luxS mutantsshowed any obvious defect in their motility in either of themedia tested. In the case of the Sakai Tet and Cm

mutants, motility in DMEM was moderately reduced(P > 0.001), but it was not affected in the richer LB. Forthe E2348-69 luxS Tet mutant, motility was significantlyreduced in both DMEM and LB, a finding in agreementwith the work of Sperandio et al. (2003). In the case ofNCTC12900, the Tet and Cm mutants both showed wild-type levels of motility. In marked contrast to previouslypublished results (Sperandio et al., 2003), addition of thecatecholamine did not restore the motility defect observedin the E2348-69 Tet mutant, nor did it significantlyenhance motility of any of the wild-type strains or otherluxS mutants.

Proteomic analysis demonstrates that the E2348-69 andSakai luxS mutants are metabolically stressed

Analyses of the effect of luxS mutation on the cytoplasmicand membrane proteomes for the three E. coli strainbackgrounds were conducted. No obvious differenceswere seen in either proteome fraction for any of theNCTC12900 luxS mutants when compared with wild type(data not shown). However, it was noticed in the mem-brane protein profiles for strain Sakai (Fig. 6A) andE2348-69 (Fig. 6B) that the stringent starvation protein A

Fig. 2. cont.



Fig. 3. Catecholamine responsiveness of E. coli luxS mutants. Overnight cultures of the luxS mutants of E. coli E2348-69, Sakai andNCTC12900 plus their wild-type parents were inoculated at approximately 102 cfu per ml into duplicate 1 ml aliquots of serum-SAPI containingadditions of the catecholamines and ferric nitrate as shown. Cultures were incubated for 18 h and enumerated for growth as described inExperimental procedures. The results shown are representative data from three separate experiments; individual data points showed variationof no more than 7%; n = 3. Key: hatch bar, no additions (control); white bar, 50 μM noradrenaline; horizontal stripe bar, 100 μM adrenaline;black bar, 50 μM dopamine; vertical hatch, 100 μM ferric nitrate (included as a positive control, and to determine if the luxS mutants wereserum sensitive).



(SspA) was more highly expressed in all of the luxSstrains when compared with their wild-type parents. In thecase of E2348-69 levels of the SspA protein were highestin the Tet mutant. In E. coli induction of the stringentstarvation protein is often associated with metabolicstress, such as that experienced during stationary phase,or following perturbation of amino acid or carbohydratemetabolism (Hansen et al., 2005). Interestingly theexpression of glutamate decarboxylase (Gad), an amino

acid metabolism enzyme, was significantly reduced in thecytoplasmic proteomes of the Sakai Tet and Cm luxSmutants (Fig. 6A), but was unaffected in the TAA and Delmutant backgrounds. In the case of E2348-69 (Fig. 6B),the Tet mutant but not the Cm, TAA or Del mutants dis-played reduced expression of Gad.

E. coli glutamate decarboxylase mutants are typicallysensitive to low-pH environments (Blankenthorn et al.,1999; Foster, 2001). Since our data (Fig. 6) showed that

Fig. 4. Influence of luxS mutation on E. coli biofilm formation. The histograms show attachment ability of the four luxS mutants of E. coliE2348-69, Sakai and NCTC12900 and their wild-type parents after culture in Luria broth (LB) or DMEM. The results shown are means oftriplicate analyses, and are representative data from at least three separate sets of experiments.



Fig. 5. Influence of luxS mutation and catecholamines on E. coli motility. Motility assays in Luria broth agar (LB) or DMEM agar wereperformed as described in Experimental procedures. Panel (A) shows the motility of the luxS mutants of E. coli E2348-69, Sakai andNCTC12900 in LB and (B), motility in DMEM. The catecholamine noradrenaline was included at 5 μM and 50 μM to investigate if it affectedmotility of the luxS mutants. The data shown are means of triplicate analyses; n = 3.



expression of the Gad protein was affected in some of theTet and Cm luxS strains we investigated the acid sensi-tivity of all of our mutants by analysis of their growth in LBwhich had been adjusted to pH 5 or pH 4 (Fig. 7). TheE2348-69 Tet mutant demonstrated increased acid sensi-tivity, in the form of reduced growth at pH 5, and showedeven more inhibition at pH 4. The E2348-69 Cm mutantgrew as wild type at pH 5, but showed slightly increasedacid sensitivity at pH 4. In contrast the E2348-69 non-polar TAA and Del mutants both showed acid stressresponses similar to wild type. All of the Sakai strainswere innately more acid resistant than E2348-69 (Fig. 7)

and the growth profiles observed at both pH values weresimilar for wild type and all luxS mutants despite thereduced expression of glutamate decarboxylase in the Tetand Cm mutants. Although NCTC12900 had wild-typelevels of Gad in both the parent and luxS mutants (datanot shown) they all showed greater acid sensitivity thanE2348-69, and similar growth profiles were obtained atboth pH 5 and pH 4 for both parental and luxS strains.Collectively, this indicates that there could be a non-gad-related acid sensitivity deficiency specific to NCTC12900.Overall the data also suggests that the acid sensitivityseen in the E2348-69 Tet and Cm mutants must be

Fig. 6. Proteomic analysis of E. coli luxS mutants. The 12% SDS-PAGE gels show the membrane and cytoplasmic protein profiles (left andright hand gels respectively) of strain Sakai (A) and E2348-69 (B). The arrows indicate the position of glutamate decarboxylase (Gad) and thestringent starvation protein A (SspA).



caused by physiological defects other than those causedby lowered expression of the Gad protein.

MicA and its possible role in the luxS phenotype

It has recently been shown in E. coli that a trans-actingsmall RNA, MicA (Coornaert et al., 2010), is located in theintergenic region between the luxS and gshA genes. Fur-thermore MicA is located entirely within, and transcribedin the opposite direction to, the luxS transcript (Coornaertet al., 2010; Udekwu, 2010) (illustrated in Fig. 1).Recently, Udekwu (2010) has shown that the luxS mRNAexists as three transcripts of different sizes, and that MicAaffects the abundance of each of these transcripts. Giventhe position of micA with respect to luxS we considered

whether disregulation of micA might be involved in theluxS mutation phenotype. The Tet antibiotic cassetteinsertion mutant used in this study was created using thepVS72 plasmid (Sperandio et al., 1999; 2001; 2003;Walters et al., 2006; Soni et al., 2007) and our DNAsequencing of pVS72 has shown that the tetracyclineresistance gene is inserted in the reverse orientation withrespect to the luxS gene (Fig. 1). This arrangementstrongly suggested that any mutant made using pVS72could be polar with respect to genes both upstream aswell as downstream of luxS; therefore the luxS::Tet muta-tion would be predicted to affect MicA expression. SinceMicA has been shown to regulate a number of genes inE. coli and other microbial species (Coornaert et al.,2010; Kint et al., 2010) we investigated whether MicA

Fig. 7. Acid resistance of E. coli luxS mutants. The E2348-69, Sakai and NCTC12900 wild-type and luxS mutant strains were grown in LBbuffered to pH 5 or pH 4 as described in Experimental procedures. The time-courses shown are the means of triplicate growth profiles and aretypical data from three separate sets of experiments; n = 3. Key: blue, wild type; luxS mutants: black, TAA; purple, Del; red, Tet; green, Cm.



levels were altered in our luxS mutants. Northern blotanalysis of micA sRNA taken from exponential-phase cul-tures showed that MicA levels were apparently unaffectedfor each of the luxS mutants in the Sakai and NCTC12900backgrounds (data not shown). In contrast, in E2348-69,while the TAA, Del and Cm luxS mutants showed essen-tially wild-type MicA levels, the expression of the sRNAwas significantly reduced in the Tet mutant (P < 0.05)(Fig. 8A).

Given that MicA has been shown to downregulate theglobal two-component regulator phoPQ (Coornaert et al.,2010) we investigated if the various defects in growth andother phenotypes that we had observed in the Sakai andE2348-69 Tet luxS mutant backgrounds could be repli-cated by perturbation of MicA expression levels. Func-tional MicA levels were increased in wild-type E2348-69,using the pmicA plasmid which overexpresses MicA(Coornaert et al., 2010). Since MicA regulates OmpAexpression (Coornaert et al., 2010), we saw not unexpect-edly a clear reduction in OmpA levels in the MicA over

producing mutant membrane proteome (Fig. 8B). TheMicA overexpressing E2348-69 strain also produced agrowth defect in DMEM cultures (Fig. 8C) similar to thatseen with the Tet luxS mutant suggesting that some of thephysiological defects previously observed in these strainsmay have been related to an increase in MicA levels;similar effects were seen in the Sakai and NCTC12900strains carrying the plasmid (data not shown). However,the introduction of plasmid pmicAmut, which contains amutant MicA that is unable to repress phoPQ, also pro-duced growth defects when the MicAmut was overex-pressed in E2348-69 (Fig. 8D); this indicates that theseMicA-dependent effects are probably not caused directlythrough regulation of the PhoPQ system. Recently Kintet al. (2010) found that the reduced biofilm formation phe-notype associated with a S. enterica serovar TyphimuriumluxS deletion mutant was actually caused by perturbationof MicA expression, rather than by the loss of the luxStranscript itself. However, our analyses of bacterial attach-ment in E. coli strains overexpressing either MicA or

Fig. 8. micA and its role in the luxS phenotype. Panel (A) shows a Northern blot of the expression levels of MicA in the E2348-69 wild typeand TAA, Del, Tet and Cm luxS mutants. The membrane protein profiles of wild-type E2348-69 and MicA and mutantMicA overexpressingstrains are shown in (B). Panels (C) and (D) are growth profiles in LB of E2348-69 overexpressing a functional MicA (C, +pmicA) oroverexpressing a mutant MicA (D, +pmutmicA); the time-courses shown are the means of triplicate data sets (n = 3). Key: OmpA (outermembrane protein A), SspA (stringent starvation protein A). Note that the highly expressed protein running above the 19 kDa marker in the gelin (D) is the E2348-69 MicAmut strain is the OmpX protein.



MicAmut did not find any reduction in biofilm formationwhen compared with wild type (data not shown).

Discussion

For two decades there has been extensive researchcarried out across many bacterial species investigatingthe function and role of the luxS gene; however, there stillremains an intensive debate as to whether its main role isin metabolism or the production of the AI-2 intercellularsignal. The presence of a cognate receptor for cell signal-ling products is generally accepted as indicative of a cel-lular communication process; however, only in Vibrio spp.has the classical LuxPQ receptor been clearly demon-strated (Winzer et al., 2003; Sun et al., 2004; Rezzonicoand Duffy, 2008; Rezzonico et al., 2012; Pereira et al.,2013). A second class of AI-2 receptor, the ABC trans-porter Lsr (LuxS-regulated) family, has been identified insome Enterobacteriacae and some Gram-positive bacte-ria (Rezzonico and Duffy, 2008; Rezzonico et al., 2012).However, while deletion of the lsrR or lsrK regulators inE. coli does affect some quorum sensing phenotypes,such as biofilm production, the pattern of altered generegulation seen in such mutants is quite dissimilar to theeffects of a luxS mutation (Li et al., 2007; Wood andBentley, 2007). With alternative AI-2 receptors still notidentified for many bacterial species (Rezzonico andDuffy, 2008; Rezzonico et al., 2012), it therefore remainsunclear whether the phenotypes associated with a luxSmutation in such bacteria are due to the lack of AI-2 (andtherefore impaired quorum sensing) or may result solelyfrom the metabolic imbalances caused by disruption of akey enzyme in the activated methyl cycle, or are perhapsa combination of both deficits.

In E. coli there has been extensive phenotypic analysisof luxS functionality; however, the interpretation of theseanalyses has been complicated by major differences instrain backgrounds and/or the methodology used tocreate the luxS mutants (Sperandio et al., 2001; Wanget al., 2005a). The current study was designed to deter-mine the importance that each of these parameters has indeveloping the E. coli luxS mutant phenotype. Our find-ings clearly indicate that both the design strategy used tocreate the mutants and the background of the strain usedto analyse the impact of the luxS mutation are majorinfluences on the resultant phenotype. We observed thatone luxS mutation, the Tet insertion, consistently pro-duced mutants whose phenotypes differed markedly fromthose possessed by other, potentially less polar, termina-tion and deletion mutants. This is significant, as our Tetmutants were made using the pVS72 plasmid, the con-struct originally used to create the strains analysed in themicroarray studies that led to the suggestion of a globalregulatory role for luxS in E. coli O157:H7 (Sperandio

et al., 2001). Our DNA sequencing of the luxS regions ofpVS72 revealed that during plasmid construction the tet-racycline resistance gene had inadvertently been insertedin the reverse orientation when compared with luxS(Fig. 1). This arrangement entails that any mutants con-structed with pVS72 would be much more likely to bepolar with respect to genes both upstream and down-stream of luxS, an observation that is consistent with theprevious inability to fully complement the VS94 Tet mutant(Sperandio et al., 2003). In contrast our DNA sequenceanalysis of the similar luxS::Cm mutant construct (pVS98)demonstrated that the antibiotic resistance gene here wasinserted in the forward orientation with respect to luxS andthus should be less likely to cause similar polar effects;however, our analyses of the Cm mutants revealed thatthey also showed phenotypes, quite similar to the Tetmutants, which were not observed in the deletion or ter-mination mutants. This implies that attempts to make luxSmutants using insertion methodologies may always resultin adverse polar effects.

It is clear from our proteomic data that mutation of luxS instrains Sakai and E2348-69 causes a significant metabolicstress; this is most evident in the upregulation ofmembrane-associated stringent starvation protein A thatoccurs in all four luxS mutants and in both strain back-grounds. The synthesis of glutamate decarboxylase wasalso observed to be reduced in the Sakai Tet and Cmmutants, and in the E2348-89 Tet mutant; this metabolicenzyme catalyses conversion of glutamate into γ-aminobutyric acid and CO2 and is important in bacterialacid resistance (Blankenthorn et al., 1999; Foster, 2001). Itis not immediately obvious whether the decrease in gluta-mate decarboxylase expression is the cause of theincrease in sensitivity to low pH seen in the E2348-69 Tetmutant as apparently the same lower protein levels did notaffect the acid resistance of the Tet and Cm mutants ofstrain Sakai. Additionally, while the NCTC12900 wild-typestrain was observed to be inherently more acid sensitivethan either of the other two E. coli strains, mutation of itsluxS did not seem to increase this sensitivity nor did it alterthe levels of glutamate carboxylase. These observationsdemonstrate that the inactivation of luxS can have a detri-mental metabolic effect in E. coli; however, they alsoemphasize that the genotypic background of the parentalstrain is a major influence on the phenotype of the resultantmutants. Furthermore, the proteomic data for our deletionand termination mutants show a striking similarity to thework of Soni et al. (2007) which pointed to only a smallnumber of protein changes in a luxS mutant that wereclearly linked to metabolism and appeared specifically tobe the result of disruption of the activated methyl cycle.

None of the E. coli luxS termination or deletion mutantsshowed any overt growth defects in any of the growthmedia tested, in contrast to a number of the Tet and Cm



mutants which grew poorly in minimal medium. The factthat addition of exogenous AI-2 did not restore wild-typelevels of growth to the Tet or Cm mutants suggests thatloss of autoinducer-2 signalling was not the sole reasonfor the phenotypes observed. Significantly we found thatneither the termination nor the deletion luxS mutants hadany of the motility or biofilm defects that were observed inthe previously described E. coli O157:H7 luxS Tet mutants(Sperandio et al., 2003). Previously Karavolos et al.(2008) found that deletion of the S. enterica serovar Typh-imurium luxS gene repositioned flagellar phase variation,and that this was not corrected by the addition of AI-2 butwas complemented by replacing the luxS gene in trans.This has now been explained by the discovery of thesRNA molecule MicA which is encoded in the oppositestrand to the luxS gene, and which when mutateddecreases aspects of Salmonella virulence such asbiofilm formation (Kint et al., 2010). In the case of E. coli,previous work with the AI-2 transport system (encoded bythe lsr operon) has indicated that quorum sensing isinvolved in the regulation of motility and biofilm production(Wood and Bentley, 2007); however, the results from ourassays with the ‘non-polar’ termination and deletionmutants, which retained full motility, clearly indicate thatluxS is not required/essential for motility regulation.Although we have as yet no mechanistic explanation forthe wild-type motility phenotype of our non-polar luxSmutations these results are consistent with the study ofWang et al. (2005a) which also saw no reduction in motil-ity or growth of an E. coli K12 luxS deletion mutation. Wedid however observe significant changes in growth char-acteristics and/or decreases in motility in some of theisogenic Tet and Cm luxS mutants; these differenceswould most logically be explained by these two mutationshaving polar effects where some of the phenotypes seenwere not directly due to the loss of LuxS activity. Wanget al. (2005a) postulated that the explanation for themarked differences in gene expression between their dataand those of the Sperandio et al. studies (Sperandioet al., 2001) might be due to the genetic differencesbetween the two E. coli strains used; however, it nowseems equally probable that the phenotypic differencesseen were due to the different mutation strategiesemployed in the two studies.

The use of antibiotic resistances inserted into bacterialgenes to create mutants has always been a complexdecision because of the potential of the inserted resist-ance gene to adversely affect the expression of othergenes within the locality of the target gene (Link et al.,1997). With the advent of new mutation strategies, suchas those involving sacB (Donnenberg and Kaper, 1991) orλRed (Datsenko and Wanner, 2000), the need to useantibiotic resistance genes in E. coli has largely beeneliminated although it is still not entirely guaranteed that

all markerless mutations will be non-polar. In the case ofmutation of the luxS gene it has recently been demon-strated that there is a key regulatory sRNA, MicA, whichlies within the E. coli luxS promoter (Coornaert et al.,2010; Kint et al., 2010) and which would be an obvioustarget for polar effects of a luxS mutation. Examination ofRNA from exponentially growing bacteria found that allof our luxS mutant strains in Sakai and NCTC12900backgrounds had wild-type levels of MicA, and that inE2348-69 it was only the Tet mutant which showed sig-nificantly reduced MicA expression. As the reduction wasseen only in this one strain it could not be ruled out thatthe decrease in MicA expression in E2348-69 luxS::Tetwas actually as an indirect result of the observed cellulardisregulation rather than it being the origin of the polareffects. Further investigations using a plasmid system tooverexpress MicA in the wild-type E. coli strains demon-strated that alteration of MicA levels could indeed causegrowth defects similar to those observed with theE2348-69 and Sakai Tet mutants. Exactly how an excessof MicA is causing these changes in phenotype remainsunclear because the use of an alternative plasmidexpressing a variant sRNA, MicAmut, which does notrepress the MicA target phoPQ (Coornaert et al., 2010),also caused similar growth defects in the wild-type strains.The similarity in growth phenotypes between theE2348-69 and Sakai luxS::Tet strains and those strainsoverexpressing MicA strongly implicates MicA beinginvolved in the polar effects; however, it is difficult toreconcile this conclusion with the contrasting lack ofchanges in MicA levels observed in the Sakai Tet and Cmstrains. We do know however from Udekwu’s work(Udekwu, 2010) that MicA binds to multiple luxS mRNAtranscripts, and thus it is possible to hypothesize that thepolar effects seen are due to alterations in the levels of theluxS mRNAs which in turn could cause changes in therelative levels of free and luxS-target-bound MicA; unfor-tunately such an explanation is impossible to confirmusing the standard Northern blotting strategy currentlyemployed to measure total MicA levels.

This investigation has clearly demonstrated that thegenetic background of the host strain is a major factor indetermining the final phenotype of an E. coli luxS mutant.It has also become apparent that some of the insertionalmutation strategies used by previous studies to createluxS mutants can produce additional artefactual pheno-types in our strain backgrounds; this is clearly evidencedby the fact that not all of these ‘luxS’ phenotypes are seenwith our new ‘non-polar’ deletion and termination luxSmutations. Because of the impracticalities of working withShiga-toxin containing strains, such as 86-24, we did notreconstruct the original luxS::Tet strain VS94 (Sperandioet al., 1999; 2001) for this study. However, it seems rea-sonable to hypothesize, based upon the phenotypes of



our EPEC and toxin-minus EHEC luxS mutants, that asignificant proportion of the transcriptional and phenotypicchanges that Sperandio et al. and other groups(Sperandio et al., 1999; 2001; 2003; Sircili et al., 2004;Walters et al., 2006; Soni et al., 2007) have observed instrain VS94, and the other pVS72- and pVS98-derivativestrains, were actually the result of unforeseen polareffects, possibly involving the regulatory sRNA MicA. Webelieve that it is vital that quorum sensing researchersbecome aware of the problematic nature of using antibi-otic insertion mutants of luxS in order to avoid furtherconfusion over the exact role of AI-2 in E. coli virulencephenotypes. For example: in a very recent paper Wanget al. (2012) also attempted to address the question ofwhether phenotypes associated with an E. coli luxS muta-tion are quorum sensing based or are simply related todisruption of the activated methyl cycle. By using a recom-binant Pseudomonas aeruginosa SAH hydrolase, SahH,to independently complement the methylation disorderWang et al. were able to demonstrate that it was notdisruption of the activated methyl cycle which had causedthe decrease in expression of motility genes (fliA, fliC,motA, motB) observed in their luxS mutant. Then by over-expression of recombinant LuxS they were able to showrestoration of expression of the motility genes; from theseresults they naturally concluded that it was loss of quorumsensing via AI-2 that was the reason for the motility defectof the mutant. Unfortunately, the Wang et al. study usedthe E. coli K12 luxS mutant MDA12 (Tsao et al., 2011)which was constructed using the pVS72 Tet mutation.Therefore, based upon our work with non-polar luxSmutants, we would predict that that the motility defectobserved is the consequence of polar effects of theluxS::Tet mutation and is not due to the loss of AI-2 pro-duction by removal of LuxS activity. Wang et al.’s (2012)observation that overexpression of recombinant LuxS cancomplement the motility defect is still very interesting inthat it suggests that the polar effects of a luxS::Tet muta-tion can somehow be counteracted; however, for theirstudy to be truly informative it would need to be repeatedusing an E. coli K12 strain containing a non-polar luxSmutation.

In conclusion, we hope that this study may finally providea convincing explanation for the wide disparity in thephenotypes which have been reported for E. coli luxSmutants over the last 10 years. This study also provides anobject lesson in the problems that polar mutations cancause, especially when these mutations are constructed ingenes proposed to be involved in complex regulatorycircuits, such as quorum sensing, where simple comple-mentation might not always necessarily be expected to besuccessful/complete. We hope that the novel luxS muta-tions that we have created here will inform other researchgroups and allow them to go back and reassess previous

conclusions about the nature of LuxS’s involvement inquorum sensing in E. coli. Furthermore we would hope thatthe problems of mutation polarity and the difficulties inproperly complementing the phenotypes observed withsome E. coli luxS mutants will inform the work of research-ers making luxS mutations in other microorganisms.

Experimental procedures

Reagents

Serum (bovine), ferric nitrate, and the catecholaminesnoradrenaline, adrenaline and dopamine, and the antibioticsampicillin, tetracycline, streptomycin, kanamycin and chlo-ramphenicol were all purchased from Sigma Chemical(Poole, Dorset, UK). Bacterial culture media were obtainedfrom Oxoid. Taq DNA polymerases, restriction enzymes andall molecular biology reagents used were purchased fromNew England BioLabs or Promega, UK.

Strains and plasmids

All strains and plasmids used or created in this study areshown in Table 1. The plasmids pVS72 and pVS94 used toconstruct the Tet and Cm mutants were obtained as a kind giftfrom Dr Vanessa Sperandio (Sperandio et al., 2001; Sirciliet al., 2004) and the plasmids pBRlac, pmicA and pmicAmut(Coornaert et al., 2010) used to make the MicA over produc-ing and mutant MicA strains were a kind gift of Dr MaudeGuillier.

Construction of plasmids for luxS mutagenesis

For this study two new E. coli luxS mutations (a completein-frame deletion and a premature termination construct)were generated in a sacB-dependent positive-selectionsuicide vector, pRDH10, for the construction of non-polarluxS mutants. Initially, oligonucleotide primers (see Fig. 1 andTable 2) were designed to PCR-amplify the 350 bp regionupstream of, and including, the E. coli O157:H7 luxS startcodon (luxS1 and luxS2 in Fig. 1) and the 350 bp regiondownstream of, and including, the stop codon (luxS3 andluxS4). In EPEC strain E23496-9 the luxS downstream regionis variant due to a Tn200 insertion and thus an alternative350 bp downstream region was amplified using primersluxS3 and luxS4E. The upstream and downstream O157:H7luxS amplified fragments were then separately cloned intothe pUC18NotI vector to create plasmids pRDH112 andpRDH113 respectively. The luxS downstream fragment wasthen excised from pRDH113 using PstI and SphI andre-ligated into pRDH112 to create the O157:H7 ΔluxS mutantplasmid pRDH118. Next, the ΔluxS mutant fragment wasexcised from pRDH118 using BamHI and SphI and ligatedinto complementary restriction sites within the Tetr gene ofplasmid pRDH10 in order to create pRDH119. All pRDH10-derived plasmids (oriR6K) were propagated in the replicationpermissive strain SY327λpir. Subsequently the E2348-69ΔluxS mutant plasmid pRDH126 was constructed by excisionof the O157:H7 luxS downstream region (luxS3–luxS4) from



pRDH119 using PstI and SphI and replacement with theE2348–69 luxS downstream fragment (luxS3–luxS4E). Inboth constructs the result was a complete in-frame deletionwith the luxS gene reduced to encoding a four amino acidpolypeptide (MLQI). For construction of the premature termi-nation mutation the primers luxS3A and luxS4A were used toPCR-amplify a 350 bp internal luxS fragment which containsthe region just downstream of the start codon (Fig. 1). Primer

luxS3A contains a basepair mismatch such that it creates aTTA to TAA mutation (Table 2) at codon 4 of the luxS geneleaving the transcript intact, but causing premature termina-tion of translation and therefore a greatly shortened polypep-tide (MLQ). The luxS::TAA mutation construction plasmidpRDH127 was created by excision of the luxS downstreamregion (luxS3–luxS4) from pRDH119 using PstI and SphI andreplacement with the luxS::TAA fragment (luxS3A–luxS4A).

Table 1. Bacterial strains and plasmids.

Strain or plasmid Derivations, or relevant characteristic(s) Source or reference

E. coli strainsE2348-69 (WT) EPEC O127:H6, Strepr Haigh (1999)E2348-69 (Del) O127:H6, Strepr, ΔluxS 3-1: everything deleted except 4 codons and stop codon

of luxS using pRDH126This study

E2348-69 (TAA) O127:H6, Strepr, luxS::TAA 1-11: frameshift at codon 2 by introduction of TAAcodon within luxS using pRDH127

This study

E2348-69 (Tet ) O127:H6, Strepr, luxS::Tet 1-1 insertion of Tetr within luxS using pVS72 This studyE2348-69 (Cm) O127:H6, Strepr, insertion of Cmr within luxS using pVS98 This studySakai (WT) EHEC, O157:H7, tox− Kanr Sasakawa laboratorySakai (Del) ΔluxS 2-4 This studySakai (TAA) luxS::TAA 2-1 This studySakai (Tet) luxS::Tet 1-2 This studySakai (Cm) luxS::Cm 1-4 This studyNCTC12900 (WT) EHEC, O157:H7, tox−, Strepr NCTCNCTC12900 (Del) ΔluxS 1-5 This studyNCTC12900 (TAA) luxS::TAA 2-2 This studyNCTC12900 (Tet) luxS::Tet 1-3 This studyNCTC12900 (Cm) luxS::Cm 1-5 This studyDH5α F−, Δ80lacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK

− mK+), relA1,

Δ(lacIZYA-argF), U169Hanahan (1983)

SY327λpir F−, araD, Δ(lac, pro), argE, recA56, rifr, nalA Miller and Mekalanos (1988)SM10 λpir thi1, thr1, leuB6, supE44, tonA21, lacY1, recA::RP4-2-Tc::Mu Kmr, λpir Simon et al. (1983)

PlasmidspUC18NotI Ampr, ColE1, bla lacI, lacZ Miller and Mekalanos (1988)pRDH10 Cmr, Tetr, sacRB, mobRP4, ori R6K Haigh (1999)pRDH112 Ampr, pUC18NotI containing O157:H7 luxS 5' region (luxS1–luxS2; BamHI–PstI ) This studypRDH113 Ampr, pUC18NotI containing O157:H7 luxS 3' region (luxS3–luxS4; PstI–SphI) This studypRDH118 Ampr, pUC18NotI containing O157:H7 ΔluxS construct (BamHI–SphI) This studypRDH119 Cmr, pRDH10 Tet::ΔluxS (O157:H7) This studypRDH126 Cmr, pRDH10 Tet::ΔluxS (E2348-69) This studypRDH127 Cmr, pRDH10 Tet::luxS::TAA This studypVS72 Ampr, Tetr, luxS::Tet, mobRP4, ori R6K, sacB Sperandio et al. (1999)pVS98 Ampr, Cmr, luxS::Cm, mobRP4, ori R6K, sacB Sircili et al. (2004)pBRlac Ampr Guillier and Gottesman (2006)pmicA Ampr, pBRlac + mica Coornaert et al. (2010)pmicAmut Ampr, pBRlac + mutmicA Coornaert et al. (2010)

Table 2. Primers used.

Primers Sequence Source or citation

luxS1 TTGGCGGAtCcGGCAAAGCG This studyluxS2 CTAACtgCaGCATTTAGCCACCTC This studyluxS3 TTGCAGGAACTGCAgATTTAGTCAG This studyluxS4 GCAgCATGCGCAGTTGACTG This studyluxS4E ACAGCaTgCTCAAAATACAGCC This studyluxS3A ATGCtGcaGTaAGATAGCTTCACAGTC This studyluxS4A TCAGCAtGcCTTCCATTGCCGC This studyMicA Bio-CCAAAATTTCATCTCTGAATTCAGGGATGATGATAACAAATG Coornaert et al. (2010)SsrA Bio-CGCCACTAACAAACTAGCCTGATTAAGTTTTAACGCTTCA Coornaert et al. (2010)

Lower case: mismatches; underlined: introduced restriction sites; bold: introduced stop codon.



At all steps the mutation constructs were verified by DNAsequencing prior to further work.

luxS mutagenesis of E. coli strains

All mutations were introduced into our E. coli strains usingthe established sacB-dependent positive-selection system(Donnenberg and Kaper, 1991). Each of the plasmidspRDH118, pRDH119 (ΔluxS), pRDH127 (luxS::TAA), pVS72(luxS::Tet) and pVS94 (luxS::Cm) were transformed into thereplication-permissive, mobilization positive, strain SM10λpir.Plate conjugations were performed with donor strains andeach of the three recipient strains, and duplicate merodiploidclones were isolated for each using both strain and plasmidantibiotic resistances (see Table 1) for selection. Merodip-loids were then propagated overnight in LB without selectionbefore being plated onto sucrose selection plates (Luria agarwithout NaCl plus 6% sucrose) at 30°C to identify resolvedmerodiploids. Resolved merodiploids were confirmed by lossof the plasmid antibiotic resistance, and from these recom-binants the luxS mutants were identified either by gain ofantibiotic resistance (luxS::Tet, luxS::Cm) or by using PCRamplification and DNA sequencing (ΔluxS, luxS::TAA). Initialattempts to obtain merodiploids in any of the strains withpVS72 using a standard tetracycline concentration of 20 μgml−1 were unsuccessful; however, after titrating the antibioticdown to 4 μg ml−1 we were finally able to create luxS::Tetmutants. This unusually low level of tetracycline resistancewas subsequently explained when DNA sequencing of thepVS72 construct (Sperandio et al., 1999; 2001) revealed thatthe Tet gene is cloned in the reverse orientation with respectto the luxS promoter. Preparation of AI-2 for use in growthassays, and confirmation of the AI-2-negative status of theluxS mutants used in this study used the protocols describedby Sivakumar et al. (2011).

Growth and acid stress resistance assays

For the analysis of the growth of strains in different culturemedia, cultures of the E. coli wild type and luxS mutants werefirst grown overnight in Luria broth (LB) media, washed in thetest culture media, and then diluted (1:1000) into fresh testmedia (LB, DMEM or M9 minimal medium supplemented with0.4% glucose as carbon source). Growth assays took placeaerobically at 37°C in triplicate 200 μl volumes in 96-wellplates, and were monitored for growth increases using aVarioskan shaking spectrophotometer running at 180 r.p.m.(Transgalactic); measurements of optical density (600 nm)were taken every 15 min.

To examine the acid stress resistance of our strains, theovernight LB cultures of E. coli luxS mutants or their wild-typeparents were diluted to 1:1000 in LB, adjusted either to pH 4or to pH 5 using sodium citrate as buffer, and grown in 96-wellplates as described above. The plates were incubated aero-bically with shaking at 37°C for 24 h in a Varioskan Spectro-photometer (Transgalactic); measurements of optical density(600 nm) were recorded every 15 min.

For analysis of catecholamine responsiveness, bacteriawere inoculated at low cell density (50–100 cfu per ml)into serum-SAPI (a host-like serum-supplemented minimal

medium) in order to more closely approximate in vivo condi-tions and the numbers of bacteria present at the beginning ofan infection (Freestone et al., 1999; 2008). Bacterial inocu-lum sizes were determined by pour-plate analysis, and cul-tures incubated statically at 37°C in a humidified 5% CO2

incubator. After incubation bacterial numbers were enumer-ated using serial dilution and plating on Luria agar.

Attachment and motility assays

Quantification of bacterial initial attachment to polystyrenewas performed using the crystal violet staining method ofMerritt et al. (2005). Motility assays were performed in DMEMor LB media solidified with 0.3% agar. To determine if stresshormones could restore motility of the luxS mutants(Sperandio et al., 2003) exponentially growing cultures wereinoculated (5 μl of culture) onto DMEM agar and LB agarcontaining 5 μM or 50 μM noradrenaline (NE); controls con-sisted of an equivalent volume of distilled water. Inoculatedplates were incubated statically at 37°C for 18 h for DMEMagar and 8 h for LB agar. Motility halos were measured byexamining the turbidity of the growth zone extending from theinoculation point of the culture.

Proteome analysis

Bacterial cultures were harvested by centrifugation for 10 minat 4000 r.p.m., 4°C, washed twice in 100 mM Tris-HCl pH 7.5,and lysed by sonication. Cell debris was removed fromprotein lysates by centrifugation at 10 min at 10 000 g. Theresulting supernatant containing total cellular proteins wascentrifuged at 50 000 g for 10 min to separate membraneproteins from cytosolic proteins, using a TLA100 rotor in aBeckman TL-100 ultracentrifuge. The membrane proteinpellet was resuspended in 100 mM Tris pH 7.5 andre-pelleted by centrifugation at 50 000 g for 10 min. Proteinconcentrations of the cytosolic and membrane protein frac-tions were measured using a Nanodrop spectrophotometer(Nanodrop 1000, Thermo Scientific) set at 280 nm. Proteinswere separated by electrophoresis in 12% SDS-PAGE gels.Protein profiles were visualized using colloidal CoomassieBlue staining and recorded using a GS-710 CalibratedImaging Densitometer connected to Quantity One software(BIO-RAD, UK). Protein sequencing was performed by theUniversity of Leicester in house Protein Nucleic Acid Chem-istry Laboratory using in-gel tryptic digestion followed bymass spectroscopy.

Analysis of micA expression levels

RNA extraction was performed on E. coli strains grown toexponential phase in LB (Coornaert et al., 2010) using theTotal RNA Purification Kit (Novagen). RNA levels were esti-mated using a Nanodrop 1000 Spectrophotometer (ThermoScientific) set at 260 nm. RNA samples (3 μg per lane) wereseparated on 8% 7 × 8 cm polyacrylamide TBE-Urea gels(acrylamide/bis-acrylamide ratio 19:1) with no stacking gel.The gel was electrophoresed at a constant voltage of 70 V for2.5 h. RNA was then transferred onto a pre-wetted HybondN+ membrane (Amersham) using a Trans Blot SD, Semi Dry



Transfer Cell-SU273 (BIO-RAD) set at 200 mA for 2 h. Afterblotting, RNA was UV cross-linked to the membrane. Mem-branes were equilibrated in Ultrahyb hybridization solutionaccording to the manufacturer’s instructions (Ultrahyb,Ambion). Blots were probed with biotin-labelled oligonucleo-tides corresponding to MicA, and SsrA as a loading levelcontrol (Coornaert et al., 2010) (Table 2). Detection of probebinding to target RNA molecules on blots were achievedusing the Ambion Brightstar Detection kit’s procedures(Ambion) with extended washing times.

Statistics

Growth analyses were performed in triplicate and all experi-ments were performed on at least three separate occasions;unless stated otherwise, numerical data shown areexpressed as mean ± SD. Where appropriate, statisticalanalysis was first performed using one-way ANOVA, and ifsignificant, an unpaired t-test. Statistical significance wasindicated by a P-value of less than 0.05.

Acknowledgements

Richard Haigh and Brijesh Kumar contributed equally to thismanuscript. We would like to thank Dr Maude Guillier for hergenerous advice with setting up the MicA Northern blotting.

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Mutation design and strain background influence the phenotype of Escherichia coli luxS mutants