Hindawi Publishing Corporation e Scientific World Journal Volume 2014, Article ID 593546, 14 pages http://dx.doi.org/10.1155/2014/593546 Research Article Characterization of the algC Gene Expression Pattern in the Multidrug Resistant Acinetobacter baumannii AIIMS 7 and Correlation with Biofilm Development on Abiotic Surface PraveenK.Sahu,1,2PavithraS.Iyer,1SagarH.Barage,3 KailasD.Sonawane,4andBaluA.Chopade1,5 1InstituteofBioinformaticsandBiotechnology,UniversityofPune,Pune411007,India 2IspatGeneralHospital,SAIL,Rourkela769005,India 3DepartmentofBiotechnology,ShivajiUniversity,Kolhapur416004,India 4StructuralBioinformaticsUnit,DepartmentofBiochemistry,ShivajiUniversity,Kolhapur416004,India 5Dr.BabasahebAmbedkarMarathwadaUniversity,Aurangabad431001,India CorrespondenceshouldbeaddressedtoPraveenK.Sahu;[email protected] Received30July2014;Revised10November2014;Accepted10November2014;Published3December2014 AcademicEditor:JoseCorreaBasurto Copyright©2014PraveenK.Sahuetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense, whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. RelativequantificationofalgCgeneexpressionwasevaluatedinthemultidrugresistantstrainAcinetobacterbaumanniiAIIMS 7 biofilm (3 to 96h, on polystyrene surface) compared to the planktonic counterparts. Comparison revealed differential algC expressionpatternwithmaximum81.59-foldincreaseinbiofilmcellsversus3.24-foldinplanktoniccells(𝑃 < 0.05).Expression levelsstronglycorrelatedwithspecificbiofilmstages(scaleof3to96h),coincidingmaximumatinitialsurfaceattachmentstage(9h) andbiofilmmaturationstage(48h).Cloning,heterologousexpression,andbioinformaticsanalysesindicatedalgCgeneproductas thebifunctionalenzymephosphomannomutase/phosphoglucomutase(PMM/PGM)of∼53kDasize,whichaugmentedbiofilms significantlyinalgCclonescomparedtocontrols(lackingalgCgene),furtherlocalizedbyscanningelectronmicroscopy.Moreover, moleculardynamicsanalysisonthethree-dimensionalstructureofPMM/PGM(simulatedupto10ns)revealedenzymestructureas stableandsimilartothatinP.aeruginosa(synthesisofalginateandlipopolysaccharidecore)andinvolvedinconstitutionofbiofilm EPS(extracellular polymericsubstances).Our observationondifferential expressionpatternofalgC havingstrongcorrelation withimportantbiofilmstages,scanningelectron-microscopicevidenceofbiofilmaugmentationtakentogetherwithpredictive enzymefunctionsviamoleculardynamic(MD)simulation,proposesanewbasisofA.baumanniiAIIMS7biofilmdevelopment oninanimatesurfaces. 1.Introduction biofilmformation[7]oneitherabiotic[8,9]orbioticsurfaces [10,11].BiofilmformationisavirulencetraitinA.baumannii In recent years, Acinetobacter baumannii has been listed as whichisofmultifactorialnature[4,12].Theprocessofbiofilm oneofthemostimportantnosocomialpathogens[1–3].The development in A. baumannii is a highly regulated process pathogen has become a universal challenge to treatment, and could be the interplay of several genetic determinants owingtoitsmultidrugresistant(MDR)natureandaplethora [13].Theextracellularmatricesofbacterialbiofilmcomprise of virulence attributes [2, 4, 5]. Associated mortality up ofproteins,nucleicacids,andpolysaccharides[14]whichare to 30% is seen with A. baumannii infections [6], such as often considered as ideal start-points to further investiga- ventilator-associatedpneumonia,bacteraemia,urinarytract tion towards effective treatment measures against biofilm- infections, burn wound infections, endocarditis, secondary associatedpathogens,suchasMDRA.baumannii. meningitis,andsepticemiaespeciallyinintensivecareunits Acinetobacterspp.andPseudomonasaeruginosatogether [1]. A. baumannii infection and colonization often involve are known to be responsible for a significant proportion of 2 TheScientificWorldJournal nosocomialinfections[15]withcrudemortalityratesof30% biofilmdevelopment.Moleculardynamics(MD)simulation to75%incaseofnosocomialpneumoniaonly[16].Inpatients wasperformedonthethreedimensional(3D)structureofthe with cystic fibrosis, alginate production by P. aeruginosa is geneproduct(enzymePMM/PGM)toconfirmthestability foundtobeassociatedwithhighmorbidityandmortality[17, and to compare with that of P. aeruginosa, besides phylo- 18].Productionofalginate,anexopolysaccharide,isrespon- geneticanalysis.Subsequently,biofilmaugmentationdueto siblefordevelopmentofmucoidbacterialphenotype[17,19] algC gene was evaluated using cloning and heterologous which is associated with biofilm formation in P. aeruginosa expressionstudiesfollowedbyscanningelectronmicroscopy. underironlimitingconditions[20];althoughproportionsof alginate in the extracellular polysaccharide/polymeric sub- 2.MaterialsandMethods stances(EPS)ofbiofilmcouldvarysignificantlyinstrainslike PA14andPA01ofP.aeruginosa[21].Itactsasanintercellular 2.1. Bacterial Strains and Culture Conditions. Multidrug material in complex biofilm structures and facilitates non- resistant clinical isolate of Acinetobacter baumannii (strain specific attachment of bacteria to surfaces, thus increasing AIIMS 7) was used in this study, as described previously cohesion [22]. Biosynthesis of alginate is well characterized [9]. The bacterium was grown on CLED (cysteine-lactose in P. aeruginosa [23, 24] and Escherichia coli [25]. The gene electrolyte-deficient) agar and Luria Bertani (LB) broth algCinP.aeruginosacodesforacrucialbifunctionalenzyme (HiMedia,India).E.coliDH5𝛼wasusedforthecloningand phosphomannomutase/phosphoglucomutase (PMM/PGM), heterologous expression experiments. Clones were main- belongingtothe𝛼-D-hexomutasesuperfamily,synthesizing tainedonLuriaagarplatescontaining100mg/Lampicillin. alginateandlipopolysaccharide(LPS)core,respectively[24, 26,27].ThegeneticregulationofalgCgeneinP.aeruginosa 2.2.NucleicAcidsPurificationandPCR. GenomicDNAwas [23, 24, 28] could be dependent on surface attachment and purified using a DNA isolation kit (Sigma Aldrich, USA). otherimportantfactors. Total RNA and plasmid purification was done using Trizol Earlierstudyshowsbiofilmformationbyclinicalstrains reagent(Invitrogen,USA)andGenEluteplasmidextraction A.baumanniionabioticsurface(urinarycatheters)asamajor kit (Sigma Aldrich, USA) respectively, according to manu- reasonfordevice-relatedinfections[29].Toexplorethebasis facturer’s instructions. Concentration and purity criteria of ofpersistenceonsuchclinicallyimportant(abiotic)surfaces, the nucleic acids were quantified in a BioPhotometer Plus wecharacterizedtheroleofextracellularmacromoleculeslike (Eppendorf, Germany). Primers used for amplification of extracellularDNA(eDNA),amajorcomponentofthebiofilm directPCRforalgCandRT-PCRofinternalregionsofalgC EPS matrix and evaluated its role in biofilm formation [9]. are listed in Table1. Primers were designed using Primer Besides eDNA, other extracellular macromolecules such as Quest (IDT, USA) and synthesized (Sigma Aldrich, USA). exopolysaccharides also play significant role in the consti- Foramplificationofthe1781bpfragmentalgCfromgenomic tutionofthebiofilmEPS.RecentfindingsontheEPSof A. DNA,PCRconditionsusedwereinitialdenaturationof5min baumanniiindicateitasauniversalprotectorfromantibiotics ∘ ∘ at94 C,followedby30cycles of30secat94 C,20seconds liketobramycin[30]andoneofitscapsularpolysaccharides ∘ ∘ at 54.5 C, and 45 seconds at 72 C with final extension of 5 (K1)beinghighlyimmunogenicandapotentialtherapeutic ∘ minutesat72 C.AmplificationofalgCwasalsoverifiedwith target via passive immunization [31]. The synthesis of spe- nativeplasmidsasPCRtemplate.PCRassayswereperformed cific exopolysaccharides like alginate and LPS core is well inanep-gradientPCR(Eppendorf,Germany);productswere characterized in P. aeruginosa along with their involvement separatedonagarosegelsstainedwithethidiumbromideand inbiofilmformationasmentionedearlier;however,remains documented in a gel documentation system (Alpha Imager unclear in A. baumannii, which is a close relative and a HP, Alpha Innotech, USA). DNase-, RNase-, Protease-free globally important nosocomial pathogen. Presumably, the water was used as negative control. All PCR chemicals and geneticbasisoftheassociationofexopolysaccharidessuchas reagentswerepurchasedfromSigmaunlessstatedotherwise. alginateandLPScorewithbiofilmformationmayexacerbate A. baumannii infections on clinically important surface, which can multiply the intrinsic antibiotic resistance and 2.3. Confirmation of Internal Regions of cDNA and DNA worsenthetreatmentscenario.However,therearenoreports Sequencing. ToconfirmthetranscriptionofalgC,twointer- as yet, which describe the genetic association of algC with nalregionsofalgCcDNA(1360bpand463bp)wereampli- biofilmformationinA.baumannii.Thereforeinthisstudy,we fied using upstream primer 363F and CD1701R and inter- setouttocharacterizetheA.baumanniialgCgeneexpression nal nested primers 1260 nest1F and CD1701R, respectively patternduringbiofilmformationanditsassociationwiththe (Table1).TotalRNAsamplesweretreatedwithDNaseIand biofilmdevelopment,whichcouldfurtherpavewayforfuture thenusedinRT-PCR,whichwasperformedusingasingle- research on potential drug target(s) for biofilm-associated stepReverseTranscriptionkit(Promega,USA).Totestpossi- infectionscausedbyMDRA.baumannii. bleDNAcontaminationinRNAsamples,directPCRoftotal In the current study, we identified the algC gene in RNA without reverse transcription was performed. DNase- the MDR strain of A. baumannii AIIMS 7 genome and , RNase-, Protease-free water was used as negative control. characterized its quantitative gene expression patterns in RT-PCRwasperformedwithone𝜇LofcDNAtemplateand growingbiofilmcellscomparedtotheplanktoniccellsusing 100pm of primers. cDNA from E. coli DH5𝛼 with pGEM- therelativequantification(ΔΔCtmethod)inreal-timePCR. 3Zf (+) plasmid as emptyvector(AppliedBiosystem, USA) Geneexpressionpatternwascorrelatedwithspecificstagesof servedasnegativecontrol.DNAsequencingwasperformed TheScientificWorldJournal 3 Table1:Oligos/probesusedforPCR,RT-PCR,andreal-timePCRanalysis. 󸀠 󸀠 Gene Primer 5-3 Nucleotidesequence 118 F TTAGAACCGGGTGAGCGTTTAGCA algC 1897 R AGATGCTGATCTTGTGGCATTGCG ALGC1 F GCTGTGAAGTCATTGCTTTACGT 𝑎𝑙𝑔𝐶𝜓 ALGC1 R TGAAGGGTCTGGTGCATGATC ALGC1 MFAM 6FAM-CAAATGGTGAGTTCCC-TAMRA CD363F CCGTGCTTACGATATTAGAGGCAA algCcDNAint region1 CD1701R AATTTGCGGATAACGCTCTTGC 1260nest1 CTTCCTCCGCGCGTATTTATC algCcDNAint region2 CD1701R AATTTGCGGATAACGCTCTTGC 16B-F TGGCTCAGATTGAACGCTGGCGGC 16SrRNA 16B-R TACCTTGTTACGACTTCACCCCA 16SRR F TGTCGTGAGATGTTGGGTTAAGTC 16𝑆𝑟𝑅𝑁𝐴𝜓 16SRR R CCGAAATGCTGGCAAGTAAGGA 16SRR MFAM 6FAM-ACGAGCGCAACCCTTT-TAMRA 𝜓 Primersforreal-timePCRassays. in the Applied Biosystems 3730 DNA Analyzer platform. 2.5.RelativeQuantificationofalgCExpressionPatternbyReal- Sequenceswereanalyzedbysequenceanalysissoftwarev5.1.1 TimePCR. SpecificprimersandprobesforalgCgenealong (Applied Biosystems, USA) and assembled using software with 16SrRNA gene (endogenous control) were designed ContigExpress (Vector NTI Advance v11.5.0, Invitrogen). and synthesized as “assay-by-design” from Applied Biosys- Promoter and the ribosome binding sites of algC were pre- tems as listed in Table1. Prior to proceeding with relative dictedusingonlinetoolNeuralNetworkPromoterPrediction quantification, the cDNA template of 24 hour old biofilm program(http://www.fruitfly.org/seq tools/promoter.html). sampleswith10-foldserialdilutionswasusedtoanalyzethe standard curves of both algC and 16srRNA gene. Real-time 2.4. Total RNA Extraction and cDNA Synthesis from Biofilm PCRwasperformedin10𝜇Lreactionsin96-wellPCRplates andPlanktonicA.baumanniiCells. Tocomparegeneexpres- using100ngcDNA,2XTaqMangeneexpressionmastermix sionpatternofalgCinbiofilmversusplanktonicA.bauman- (Applied Biosystems, USA) and 20X algC assay mix (20X niiAIIMS7,totalRNAwasextractedfromcellsgrowingin 16SrRNA assay mix in case of controls) in a 7500 fast real- biofilmandplanktonicmodeandthenreal-timequantitative time PCR system (Applied Biosystems, USA). The relative RT-PCRwasperformed.Biofilmswereallowedtoformin6- numberofalgCmRNAwasdeterminedusingΔΔCt-method wellpolystyreneplates(Tarsons,India)beginningat3hours (comparative threshold cycle) by normalizing Ct values of 6 upto96hours.A.baumanniiAIIMS7cultures(10 CFU/mL, algCmRNAwiththatof16srRNAinbiofilmandplanktonic overnightgrown)wereinoculatedontothewellsanddiluted samples at various time points from 3 to 96 hours. Relative initially with sterile distilled water (without LB broth) at a quantification (RQ) represented in “fold over increase” was ∘ dilutionof1:40.Afterincubationfor3hoursat37 Cunder determined according to methods described elsewhere [32] static conditions, the supernatant was discarded and fresh andtheformulausedforcalculationwasRQ=2−ΔΔCt.Cutoff sterile nutrient medium (Luria broth) was added at a final Ctwaskept35,withanautomaticthresholdof0.2andbase- dilutionof1:40andthenincubatedundersimilarconditions linefromcycle3–15with95%confidencelevel.The24-hour toallowbiofilmformation.Afterappropriateincubation(s), biofilmsamplewassetasthecalibratorforthiscomparative platesweretakenout;nonadherentcellswereaspiratedand geneexpressionanalysis.StatisticalcomparisonofRQvalues discarded after brief sonication. Plates containing biofilms (between biofilm and planktonic samples) was performed were washed twice with phosphate buffer saline (PBS). usingStudent’s𝑡-testand𝑃value<0.05wasconsideredtobe Surface-attachedbacteria(biofilmforming)werescrapedoff statisticallysignificant. andtotalRNAwaspurifiedusingTrizolreagent(Invitrogen, USA)aspermanufacturer’sinstructions.Similarly,totalRNA 2.6.BiofilmDevelopmentAssay. Biofilmdevelopmentassays waspurifiedfromplanktoniccellcultureatthecorrespond- wereperformedtoassessthepatternofbiofilmformationof ing time points (3–96h). RNA samples were treated with A.baumanniiAIIMS7onpolystyrenesurfaceandforfurther DNaseI(Sigma,USA).TotestpossibleDNAcontamination in RNA samples, direct PCR of total RNA without reverse correlationwithgeneexpressionplotoverthesametimeline. transcriptionwasdone.cDNAwassynthesizedusingVerso Beginning at 3h up to 96h, quantitative biofilm assay was cDNA synthesis kit (Thermo, USA) as per manufacturer’s performedon96-wellmicrotitreplates,accordingtoearlier instructions. cDNA concentrations were determined using methods [10] with appropriate modifications. Briefly, A. 6 BioPhotometerPlus(Eppendorf,Germany). baumanniiAIIMS7cultures(10 CFU/mL,overnightgrown) 4 TheScientificWorldJournal wereinoculatedontopolystyrenemicrotitrewellsofa96-well USA).AminoacidsequencesweretranslatedusingTranslate plate(Tarsons,India)anddilutedinitiallywithsteriledistilled tool of the Expert Protein Analysis System (ExPASy, Swiss water(nonutrientbroth)atadilutionof1:40.Afterastatic InstituteofBioinformatics).Proteinsequencesretrievedfrom ∘ incubationof3hoursat37 C,thesupernatantswereaspirated NCBIwerealignedusingCLUSTALW[33]andphylogenetic andthensterileLBbrothwasaddedtothewellswithafinal tree was constructed with the help of analysis software dilutionof1:40withsterileLBbroth.Foralltimepoints(3– MEGA v5.0 [34]. Bootstrapping was performed with 1,000 96h),thiswasrepeatedinordertoallowA.baumanniicells replicates for checking relative support for the branches toattachontopolystyreneinitiallybutnottogrowandthen in the phylogenetic tree. Comparison was restricted to tosubstituteitwithadditionoffreshLuriabrothtofacilitate sequences of close organisms, for example, Pseudomonas adherentandmicro-aggregatedcellstodevelopbiofilms.All aeruginosa,Pseudomonasputida,Pseudomonassyringae,and ∘ plates were incubated at 37 C under static conditions for previously predicted PMM sequence from whole genome biofilm development, including negative controls (no cells, sequence of Acinetobacter baumannii strain AYE, Acineto- onlynutrientmedium)foreachtestsamplesintheplate.After bacterhaemolyticus,andAcinetobactercalcoaceticus.Higher appropriateincubationperiod(s),platesweretakenoutand eukaryote sequences, for example, Oryctolagus cuniculus nonadherentanddeadcellswereremovedbybriefsonication PGM isoform 1 and PMM from Homo sapiens, were also and aspiration carefully. The wells were washed thrice with includedforcomparison. sterile PBS, dried, and stained with 0.1% Gentian Violet (HiMedia, India). Excessive stain was removed by sub- 2.10.CloningandHeterologousExpression. Upstreamprimer merging the plate in a water trough and then dried in (alg 118F)anddownstreamprimer(alg 1897R)wereusedin laminarairflowfollowedbyaddition200𝜇Lof100%ethanol aPCRprogramwithanadditional15minfinalextensionfor for solubilizing the stained biofilm matrices. To measure producingpoly-AtailsinthePCRproductstoaidwhileTA the absorbance, plates were read in a Multi-Plate Reader cloning. Amplified algC was purified using gel purification (MolecularDevices,USA)at570nmbeforebeingshakenat kit (Bangalore GeNei, India) and ligated into pGEMT-Easy 1020rpmfor10seconds.Allbiofilmdevelopmentassayswere vector (Promega, USA) to produce resultant recombinant performedinreplicateof12andrepeated.Absorbancevalues plasmid pGEalgCA7. The plasmid was transformed into weretermedasbiofilmgrowthindexafternormalizationwith chemically competent E. coli DH5𝛼. Colonies were picked LB control, and values were plotted in Excel spreadsheets anddirectPCRwasperformedtoconfirmpresenceofalgC (Microsoft,USA)forfurtheranalysis. gene. 2.7. Statistical Correlation of Biofilm Formation with algC 2.11. Assessment of AlgC (PMM/PGM) Protein Expression. Gene Expression. To find the concurrence of algC gene Mid-log phase grown (with 100mg/L ampicillin in Luria expression pattern with biofilm formation, RQ values (fold broth,undershakingconditions)E.coliDH5𝛼-pGEalgCA7 overincreaseinalgCgeneexpression)inbiofilmmodewere andE.coliDH5𝛼-pGEM-3Zf(+)atOD600=0.8wereused correlatedlinearlywiththebiofilmindicesatcorresponding topreparewholecellextracts.Thecellextractswereanalyzed timepointsandcorrelationcoefficientsweredeterminedusing bySDS-PAGE(12.5%)andproteinbandsweredocumentedin Excel spreadsheet (Microsoft, USA). Correlation statistics ageldocumentationsystem(AlphaInnotech)afterstaining wasrestrictedtothreemajorstages,thatis,initialattachment with0.2%Coomassiebrilliantblue(Himedia). (3–9 hours), consolidation of the surface-attached micro- aggregation (12–24 hours), and maturation of biofilm (36– 2.12.AssessmentofBiofilmAugmentation. Biofilmaugmenta- 48 hours). 24-hour biofilm gene expression data (calibrator tionwasvisualizedbyscanningelectronmicroscopy.Briefly, sample)wasexcludedfromthecorrelation.𝑃valueof<0.05 overnightgrowncells(3×109 cells)wereinoculatedon(1× wasconsideredtobestatisticallysignificant. 1cm)sterileglassslidesinside12-wellcultureplates(Tarsons, ∘ India)andincubatedat37 Cstatic.After24hofincubation, 2.8.MicroscopicExaminationofA.baumanniiBiofilmDevel- culture supernatant was removed; slides were immediately opment. To localize the stages of biofilm growth over a flooded with 2.5% glutaraldehyde in PBS and incubated at temporal scale (3–96h), biofilms were formed on sterile room temperature for 2 hours, followed by rinsing with polystyrene culture dish, 55mm × 15mm (Tarsons, India), steriledistilledwater,andseriallydehydratedwithanethanol ∘ and incubated at 37 C under similar culture conditions as gradient (25–100%), CO2-critical point dried and coated describedabove.Afterappropriateincubationperiods,plates with platinum in an Auto Fine Coater (JFC-1600, JEOL, were taken out; supernatants were aspirated followed by Japan).Theslideswerethenobservedinascanningelectron washingthreetimeswithphosphatebuffersalineforremoval microscope (Vega, Tescan, USA) with 30KV input voltage. of nonadherent cells and fixed with 1mL of methanol, air- For quantitative biofilm augmentation in the algC clones, dried,andobservedwithoutstainingunderamodularbright biofilms were formed in microtitre plates and quantified as fieldmicroscope(AxioscopeA1,Zeiss,Germany)withbright permethodsdescribedearlier[10]. fieldsettingsatmagnificationof100x. 2.13.SequenceAlignmentandBuildingModelforPMM/PGM. 2.9. Bioinformatics and Phylogenetic Analysis. Nucleotide PMM/PGMsequenceofA.baumanniiAIIMS7(AEC46864) and protein sequence comparisons were performed using wasusedasatargetsequenceinBLASTpprogramtoidentify BLASTn (National Center for Biotechnology Information, possibletemplatestructuresavailableinProteinDataBank. TheScientificWorldJournal 5 Theselectedtemplateshavinggoodalignmentscorewiththe 90 target sequence were aligned using CLUSTALW. Template 80 proteinstructure1K2Y.pdb(P.aeruginosaPMM/PGMS108A mutant),havingmaximumsequencealignmentscore(32%) ase) 70 e withtargetsequence,wasusedasthefinaltemplatetobuild cr 60 n homologymodel of PMM/PGM. Three-dimensionalmodel er i 50 v ofPMM/PGMwasconstructedusingMODELLER9v7[35] d o 40 boyf tchoensmidoedreinlgw1aKs2tYe.spteddbbasyautseimngpl1a0tensstrmuoctleucrue.laThr deysntaabmiliictys Q (fol 30 R 20 (MD)simulation. 10 2.14.MolecularDynamics(MD)Simulation. MDsimulations 0 3 6 9 12 18 24 36 48 72 96 were performed on the homology model of PMM/PGM Time (hours) from A. baumannii AIIMS 7 in a HP workstation with the help of GROMACS 4.0.4 program using the GROMOS96 Planktonic 45a3 force field [36]. The structure was fully solvated Biofilm + with water (SPC) and system was neutralized with 9Na Figure1:Comparisonof algC geneexpressionpatterninbiofilm ions. The solvated structure was minimized by steepest andplanktonicA.baumanniiAIIMS7.Real-timequantitativePCR descent method for 1000 steps at 300K temperature and resultsshowingrelativequantificationofalgCgeneexpressionlevels constant pressure. The LINCS algorithm with 8.0A˚ cutoffs (calculated according to ΔΔCt-method and represented as “fold was used for energy minimization of PMM/PGM whereas overincrease”)atcorrespondingtimepoints.Expressionlevelswere PME algorithm with 8.0A˚ nonbonded cutoffs was utilized normalized with indigenous control (16SrRNA gene expression) similarly as used in an earlier MD simulation study [37]. in both biofilm and planktonic cells (calibrator: 24-hour biofilm AfterequilibrationperiodproductionMDwasrunfor10ns sample,foldoverincrease=1). at300Ktemperature,pressure,andconstantvolumeensem- ble. Solvent accessible surface (SA, Richards’ surface) and molecular surface (MS, Connolly’s surface) areas were cal- showedsignificantvariationinthethresholdcycle(Ct)values culated using CASTp analysis [38]. Structural comparison inbiofilmandplanktonicmodeofgrowthofA.baumannii, aftermoleculardynamicssimulationofinitialstructurewith whichdescribeddifferentialexpressionalpatternsofthetwo final structure was done using PDBeFOLD software [39]. modesofgrowth(Figure1).NumberofcopiesofalgCmRNA The MD simulation trajectory was then visualized using were calculated using 16SrRNA gene as internal control at theVMD(visualmoleculardynamics)package[40].Three- eachtimepoint(ΔCt).Toevaluatetherelativegeneexpres- dimensionalimagesofenzymePMM/PGMafterMDsimu- sion in biofilm and planktonic modes of growth over the lationrunweregeneratedusingtheUCSF-Chimeratool[41] temporalscaleof3to96hours,the24-hourbiofilmsample andPymol(http://pymol.sourceforge.net/). was used as calibrator (ΔΔCt). Relative quantification (RQ) plot for gene expression at various time points from 3 to 2.15.NucleotideSequenceAccession. TheGenBankaccession 96 hours showed low basal (almost linear) expression of number for A. baumannii AIIMS 7 algC gene sequence algCinplanktonicmode(maximumof3.24-fold,48hours) is JF701279 and AEC46864 for the protein (PMM/PGM) whereas highly variable and elevated algC expression was sequence. seen in biofilm mode (maximum 81.59-fold, 48 hours) and displayingadefinitepattern.Theinitialattachmentstage(9 3.Results hours) and maturation stage (48 hours) of biofilm showed maximalexpressionofalgC,whichwasincontrastandcould 3.1.IdentificationofalgCGeneinA.baumanniiAIIMS7. PCR be seen as steadily low expression at almost all time points with genomic DNA yielded a fragment of 1781bp (Supple- in planktonic mode of growth (Figure1). This observation mentary Figure S1 in the Supplementary Material available affirmsthatthealgCgeneishighlyup-regulated(atthetran- onlineathttp://dx.doi.org/10.1155/2014/593546).PCRampli- scriptionlevel)whiletheA.baumanniicellsgrowinabiofilm fications with plasmids were unsuccessful, confirming that mode (i.e., attached to an abiotic surface) and follows a the gene is not plasmid-borne. Reverse transcription PCR pattern as depicted (Figure1); however in planktonic (free using total RNA showed amplification of internal coding form,inasuspension,unattachedtosurface)thealgC gene regions1(1360bp)and2(463bp),whichconfirmedtheactive istranscribedatabasalrate. transcription of the algC ORF (Supplementary Figure S2). DNAsequenceofthegenewasanalyzedtopredictregulatory 3.3.BiofilmGrowthPatternCorrelateswithQuantitativealgC regions such as promoter, ribosomal binding site, transla- Gene Expression. Biofilm growth pattern A. baumannii on tion start, and end, which were identified and annotated polystyrene surface can be seen in Figure2, which can be (SupplementaryFigureS3). explained in three distinct stages. First, an exponential rate of increase was observed at 3 to 9 hours, which defined 3.2.ElevatedLevelsofalgCGeneTranscriptionduringBiofilm the initial attachment and micro-colony aggregation stage Mode of Growth. Quantitative real-time PCR experiment (Figure2).After9hourtill36hoursitshowedsteadylevelof 6 TheScientificWorldJournal 3 followedbyappearanceofrobustthree-dimensionalbiofilm structures, which were found persistent till 72 and 96h 2.5 (Figures 3(i) and 3(j)). Overall, visualization of biofilm x 2 growthatdevelopingstageswasintandemwiththepatterns e d ofbiofilmgrowthaswellasalgCgeneexpression. n m i 1.5 ofil 3.5. In Silico Analysis of Protein Reveals Highly Conserved Bi 1 Identity. TocompareandevaluatetherelatednessofA.bau- 0.5 manniiAIIMS7algCencodedPMM/PGM,insilicoanalyses were performed. BLASTn results showed 100% similarity 0 with the predicted PMM/PGM sequence in whole genome 3 6 9 12 18 24 36 48 72 96 sequences of A. baumannii available in NCBI database. Time (hours) Alignment results also displayed significant similarity with Figure 2: Pattern of biofilm growth of A. baumannii AIIMS 7. PMM/PGMnucleotidesequencesofothergenera.472amino Graphshowingcorrespondingbiofilmindicesofbiofilmsformedon acid residues could be theoretically translated in one frame 󸀠 󸀠 polystyrenemicrotitresurface,calculatedafternormalizationwith (5 –3 )oftheDNAsequence.BLASTpshowedsimilarresults controlandplottedversusrepresentativetimepoints(threeto96h). asinBLASTnalignment.Themolecularweightoftheprotein was predicted to be 52.94kDa with isoelectric point (pI) of 5.67. CLUSTALW alignment with selected bacterial and increaseinbiofilmindices,indicatingtheconsolidationstage eukaryoticPMM/PGMsequencesshowedconservedregions oftheattachedmicro-coloniesandapproachingmaturation in the protein as shown in Figure4(a) (active site, Mg2+ of biofilm. Third, maximum production of biofilm matrix binding site, and sugar binding site marked as colored could be seen at 36 to 48 hours, marking the maturation rectangles) in the protein as shown. Figure4(b) shows the stage. Lastly, as like the plateau phase in the planktonic phylogenetictreeindicatingrelatednessbetweenPMM/PGM growth curve of A. baumannii AIIMS 7 (data not shown), enzymesfrompseudomonadsandhighereukaryotes(rabbit the biofilm growth pattern also exhibited a steady state andhuman).PMM/PGMsequencefromA.baumanniihad and persistent nature after maturation (after 48 hour till less than 25% identity with rabbit muscle PGM isoform 96 hours). To find the association of algC gene expression 1, whereas it had a mere 10% identity with human PMM patternattranscriptionlevel(asrevealedfromreal-timePCR (Figure4(b)). analysis)withthatofbiofilmgrowth,correlationcoefficients weredeterminedusingtheRQvaluesinbiofilmsamplesand 3.6. Assessment of Protein. To demonstrate the synthesis of biofilm indices at corresponding time points. High corre- PMM/PGM protein, whole cell extracts of cloned E. coli lation was observed at all three major stages of biofilm. DH5𝛼withresultantrecombinantplasmidpGEalgCA7cells Correlation coefficients were 0.902, 0.925, and 0.983 (𝑃 < were used in SDS-PAGE analysis, which showed a clearly 0.05) corresponding to initial attachment, consolidation of overexpressedproteinof∼53kDasuggestingthePMM/PGM the surface-attached micro-aggregation, and maturation of protein in question (Figure5). Control E. coli DH5𝛼 (with biofilmstages.Correlationwasfoundtobemaximum(0.983, emptyvectorpGEM-3Zf+lackinganalgCgene)didnotshow 𝑃<0.05)atstageswherebiofilmsweremature.Thisindicated anyPMM/PGMproteinband. astrongpossibilitythattranscriptionofalgCcouldbeinclose associationwithbiofilmformation,especiallythematuration 3.7.BiofilmAugmentationbyalgCafter24h. Electronmicro- stage(36–48hours)andalsotheinitialattachmentstage(3–9 graphs of biofilm formed by the algC clones (Figure6) hours). showed significant increase in biofilm formation compared tocontrols,probablyindicativeoftheoverexpressionofthe 3.4. Microscopic Visualization of Biofilm Development. PMM/PGM protein and the resultant exopolysaccharides. To visualize the growth pattern of biofilm, bright- Thicker biofilms could be seen in the algC clones having field microscopy was performed on biofilms growing clear dense matrices (Figures 6(a), 6(c), and 6(e)) with a on polystyrene surface, which showed varied biofilm intracellularcementingmaterialclearlyvisible(Figures6(a) morphologyatgradualtimepoints(Figure3)withastrong and6(e))indicativeofthebiofilmEPS.OverallSEManalysis concurrence with quantitative evaluation of biofilm growth pattern(Figure2,asdescribedabove).Atinitialattachment atagradientofmagnificationsandconcurrentcomparisons stages, planktonic cells were seen scantily aggregated with control clones (lacking functional copy of algC) sug- (Figures3(a)and3(b))butgraduallythemicro-aggregation gestedsubstantiallythatthereissignificantfacilitationofthe on the surface increased, subsequently making completely overallbiofilmformation,emphasizingtheenrichmentofthe attached micro-colonies at 9h which could be seen clearly biofilmmatrices,whichcouldbeduetothecohesiveactivity (Figure3(c)). Further stages of growth (12, 18, and 24h) of EPS including alginate and LPS cores. The quantitative showed consolidation of micro-colonies leading to stable biofilmassayconcurredwiththemicroscopicanalysis,with structures of biofilm (Figures 3(d)–3(f)). At 36–48h, fully augmentation of biofilm up to 3.87-fold in the algC clones mature biofilm communities of A. baumannii were seen, compared to the control cells lacking algC gene (𝑃 < 0.02, enriched with thick EPS matrices (Figures 3(g) and 3(h)), datanotshown). TheScientificWorldJournal 7 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) Figure 3: (a)–(j) Microscopic visualization of A. baumannii AIIMS 7 biofilms. Representative bright field micrographs depicting static biofilmsformedonpolystyrenemicrotitresurfaceatrespectivetimepoints(3,6,9,12,18,24,36,48,72,96h)(magnification,100x). 3.8. Molecular Dynamics Simulation Study of PMM/PGM. eachdomainsequentially(Figure7(a))formingheartshaped To predict the functional association between PMM/PGM geometry. Sequence alignments (Supplementary Figure S4) enzyme (encoded by algC gene) resulting in alginate/LPS and model building study showed that the active site was core mediated biofilm formation in A. baumannii, molec- located at the centre of the two domains (domains 1 and ular dynamics (MD) simulations up to 10ns were carried 2) in a deep cleft formed by Ser104, Asp244, Asp246, and out on homology model of PMM/PGM of A. baumannii Asp248. PMM/PGM sequence of A. baumannii AIIMS 7 AIIMS 7 using GROMACS v4.0.4 program. The generated showedconservedsequencemotifindomain3fromresidues PMM/PGMmodelofA.baumanniiAIIMS7containedfour 327-331(GEYAGH)whichwouldactasasugarbindingsite. domains of equal size arranged in “heart shaped” manner A cluster of positively charged conserved residues found in (Figure7(a)) which form a compact structure, similar to P. domain 3 (Lys287) and domain 4 (Arg427, Arg438) could aeruginosa [42]. The polypeptide chain proceeds through be involved in phosphate binding. The cleft showed solvent 8 TheScientificWorldJournal H.sapiens ----------mavtaqAarrrerVlclfDvdGtL---------------------tpaRQ O.cuniculus ----mEegPlplltirtapYhdqkpgtsglRkKt-yyfedkpcylenFiqsiffsidlKd A.haemolyticus -------------mnIkhkFPlNIFRAYDIRGKL-TnLTptiiRsIavA----LAAQYtE A.calcoaceticus -------------mnVrhsFPkSIFRAYDIRGKL-SyLTtDvvRsIaYg----LAqQYKQ AB AIIMS 7 -------------mnVrhsFPkNIFRAYDIRGKL-SyLTtDvvRsIaYg----LAqQYKQ AB AYE -------------mnVrhsFPkNIFRAYDIRGKL-SyLTtDvvRsIaYg----LAqQYKQ P.aeruginosa -----------mstakAptlPaSIFRAYDIRGvvGdtLTAETAywIGrA----igSEsla P.syringae ---------mnspasVApnlPeTIFRAYDIRGvvGdtLnAETAywIGrA----igSEsla P.putida --------mndmahlVpaalPdSIFRAYDIRGvvGktLhAETAywIGrA----igAQsla H.sapiens kidPEva--------------aflQkLr----SrvqI---GvVggsdyckIAeqLGdGDe O.cuniculus rqgssLVvGgDGRyfnkSaietIlQmaaanGigrlvIgqnGilsTPavscIiRkIkaigG A.haemolyticus agQkQiVIGYDaRLtSPtyAniIqQifehqGleVinI---GCcsTPMMYYIARdya-GNG A.calcoaceticus vkQtQLVIGYDaRLtSPAyAylIeeiLiEqGlNVTnI---GCcssPMMYYIARdfG-GNG AB AIIMS 7 aeQtQLIIGYDaRLtSPAyAhlIeeiLvEqGlNVTnI---GCcsTPMMYYIARefG-GNG AB AYE aeQtQLIIGYDaRLtSPAyAhlIeeiLvEqGlNVTnI---GCcsTPMMYYIARefG-GNG P.aeruginosa rgEPcvavGrDGRLSgPeLvkqliQgLvDcGCqVSDv---GmVPTPVLYYaAnvLegkSG P.syringae qnEPnvsvGrDGRLSgPeLvqqliQgLhDsGCHVSDv---GlVPTPaLYYaAnvLagktG P.putida qgEPQvsvGrDGRLSgPmLveqlikgLvDsGCHVSDv---GlVPTPaLYYaAnvLagkSG H.sapiens Viekfdyvfaen----GtvqykhGrlLSkqtIQnh-------LgEellq--d-------- O.cuniculus iilTASHNPggpNgdfGiKFnIsnggpaPEAItdkifqisktieEyAic--PDlkvdlgv A.haemolyticus iMVTASHNPKSdN---GiKWILkGEPpSPEAIQQ--------vglyAegftdqiieiqnl A.calcoaceticus iMVTASHNPKSdN---GiKWILkGEPpSPEmIQQ--------vgEeAqtYVPNhsisvle AB AIIMS 7 iMVTASHNPKSdN---GiKWILrGEPpSPEmIQQ--------vgEfAqtYVPthtislle AB AYE iMVTASHNPKSdN---GiKWILrGEPpSPEmIQQ--------vgEfAqtYVPthtislle P.aeruginosa VMlTgSHNPpdYN---GFKiVvaGEtLanEqIQa--------LrEriek--nDl--asgv P.syringae VMlTgSHNPKdYN---GFKiVIaGDtLanEqIQa--------LhErikt--nNl--tsqk P.putida VMlTgSHNPsdYN---GFKiVIaGDtLanEqIQa--------Lltrlkt--nDl--tlaq H.sapiens ----li--nFClsY---------------------------mallRLpkkrgtfIefrNG O.cuniculus lgkqQF--DlenKfkpftveivdsveayatmlrnifdfnAlkellsgpnrLKIrIDamhG A.haemolyticus dqlhkiipqYClQY-----------------------qQAllSDIhLskPLKIVLDglhG A.calcoaceticus lTlpQFkAeFCqQY-----------------------qQAIfkDIQLkRPLKVVLDglhG AB AIIMS 7 lStpQFnSeFCkKY-----------------------qQAIfNDIQLkRPLKVVLDglhG AB AYE lStpQFnSeFCkKY-----------------------qQAIfNDIQLkRPLKVVLDglhG P.aeruginosa gSveQv--DilpRY-----------------------fkqIrDDIamakPmKVVvDCgNG P.syringae gSitQv--DileRY-----------------------fQqIkNDIvmaRkLKVVvDCgNG P.putida grvekv--DilgRY-----------------------fQqIvgDvKLakkLKVVvDCgNG H.sapiens mlni-------spiGrs------CtLEeri-----efSeldkkEkireKfvE---Dlkte O.cuniculus vvGpyvKKiLcEeLGapanSavnCvpledFggHHPDP-nltyaaDLvetmKsgehDfGaA A.haemolyticus SAGrCAKsvL-EkLGCdVIALr-CEanGhFPdHaPDPSHAehLKqLqqaIisenADlGIA A.calcoaceticus SAGhCsKliL-EkmGCEVIALr-tnpnGeFPdHaPDPSHAAhLisLrkaVvEqqADIGIA AB AIIMS 7 SAGhCsKlvL-EkmGCEVIALr-ttpnGeFPdHaPDPSHAAhLKeLrktIiEqGADIGIA AB AYE SAGhCsKlvL-EkmGCEVIALr-ttpnGeFPdHaPDPSHAAhLKeLrktIiEqGADIGIA P.aeruginosa vAGviApQLi-EALGCsVIpLy-CEVDGNFPNHHPDPgkpeNLKDLiAKVKaenADlGLA P.syringae AAGviApQLi-EALGCEVISLf-aEVDGNFPNHHPDPgkleNLQDLiAKVKEtGADlGLA P.putida AAGvvApQLi-EALGCEVIpLf-CEVDGNFPNHHPDPgkpeNLEDLiAKVKEtGADIGLA H.sapiens FaGkG--------------------------LrfSrGgmIsFDV---------------- O.cuniculus FDGDGDRnmIlgkhGffVnPsdsvaviAaNIfsip-----yFqqtgvRgfarsmptsGal A.haemolyticus lDGDGDRVvlldEqahIIsPDRLLsLFAQmcLQQHPhkEIVFDVKCSRmVadtVEQlnGQ A.calcoaceticus lDGDGDRVvlVdEkaqIltaDRLLsLFAQmcLEQHPeqEIVFDVKCSRmVqetVEKlGGK AB AIIMS 7 lDGDGDRVvlldEkanIltaDRLLsLFAQmcLEQqPdkEIVFDVKCSlmVqrtVERlGGK AB AYE lDGDGDRVvlldEkanIltaDRLLsLFAQmcLEQqPdkEIVFDVKCSlmVqrtVERlGGK P.aeruginosa FDGDGDRVGVVTntGtIIYPDRLLMLFAKDVvsRNPGADIIFDVKCtRrlialIsgyGGR P.syringae FDGDGDRVGVVTnaGnVVYPDRLLMLFAlDVLKRNPGADIIFDVKCtRrltplIsehGGR P.putida FDGDGDRVGVVTntGsIVYPDRLLMLFAQDVLsRNPGAEIIFDVKCtRrltplIEQhGGR -------------fpeGwd-----------------------krYclds-----lDqdsf H.sapiens O.cuniculus drvanatkialyetpTGwkFfgnlmdaSk--lsLcGEeSfgtGsdhiRE------kDGLw A.haemolyticus akM----------iRTGsSFLrSyLSQSnGNAvfgGEya---GHYvFndGRgFGyDDGLY A.calcoaceticus akM----------iRTGsSFLrAyLSQSnGrAifgGEya---GHYvFndGRgFGyDDGLY AB AIIMS 7 PkM----------iRTGsSFLrAyLSQSnGNAifgGEya---GHYvFndGRgFGyDDGLY AB AYE PkM----------iRTGsSFLrAyLSQSnGNAifgGEya---GHYvFndGRgFGyDDGLY P.aeruginosa PvM----------WKTGHSlIKkKmketG--AlLAGEMS---GHvFFKE-RWFGFDDGiY P.syringae PvM----------WKTGHSlIKkEmkkSG--AlLAGEMS---GHiFFKE-RWFGFDDGiY P.putida alM----------WKTGHSlIKkKmkQtG--slLAGEMS---GHiFiKE-RWyGFDDGiY dtihffgneTspggnd-fEiFAd-----Prtv---------------------------- H.sapiens O.cuniculus AvlawLsILatrKqsv-eDilkdhwhkFgrnffTrydYeeVeaegatkmmKdLeAlmfdr A.haemolyticus AAlRvmEyLgQsEArclSELlAa----FPEryCTedIYIsthnasPqqVlnni-----Ei A.calcoaceticus AAlRvmEyfTQssATtISELFAp----YPErCCTedtYIgtyQsDPkyVlQdi-----Ei AB AIIMS 7 AAlRvmEyfTEssATtISDLFSn----YPErCCTedtYIgthQsDPkhVlQdi-----Ei AB AYE AAlRvmEyfTEssATtISDLFSn----YPErCCTedtYIgthQsDPkhVlQdi-----Ei P.aeruginosa sAARLLEILsQdQrds-ehvFSa----FPsdisTPEInItVtEdskfaIIEaL-----qr P.syringae sAARLLEILsQesAna-eDLFet----FPndisTPEInIkVtDvtkfsIIKaL-----Et P.putida sAARLLEILsktEqsa-enLFAa----FPndisTPEInIdVtDegkfsIIdaL-----qr ---------------------------------------------GhsVV---------- H.sapiens O.cuniculus sfvgkqfsandkvytvekadnfeyhdpvdg-sVSKnqGlRLiFaDGsrIIfrlsgtgsAg A.haemolyticus i-----------------------SHrlaA-RlSKIDGVRLDFDDGFGII-------RAS A.calcoaceticus l-----------------------SHrlGA-RISKIDGVRLDFDDGFGII-------RAS AB AIIMS 7 l-----------------------SHrlGA-RISKIDGVRLDFDDGFGII-------RAS AB AYE l-----------------------SHrlGA-RISKIDGVRLDFDDGFGII-------RAS P.aeruginosa d-----------------------Aqwgeg-nIttlDGVRvDypkGwGlV-------RAS P.syringae d-----------------------AqwgdA-KlttIDGVRvDypkGwGlV-------RAS P.putida d-----------------------AdwgeA-nlttIDGVRvDyanGwGlV-------RAS ------------spqdtvQRcrEiF----------------fPEtAhEa--- H.sapiens aTirlYIdsyEkDNakinQdpQvmlaplisialkvsqLQErtgrtAptvit- O.cuniculus NTgeyFtvRFdADhPdrLveIrQKF---------isMLQDqyPQIAqElaQS A.haemolyticus NTgeyFtvRFdADNPlrLkeIQQKF---------vDMLQEhyPQIAqElsEA A.calcoaceticus AB AIIMS 7 NTgeyFtvRFdADNPlrLkeIQQKF---------iDMLQEryPQIAqElmsl AB AYE NTgeyFtvRFdADNPlrLkeIQQKF---------iDMLQEryPQIAqElsEA NTTPVlVLRFEADteeeLERIktvF---------rNqLkavdssLpvpF--- P.aeruginosa NTTPVlVLRFEAeteaeLQRIkdvF---------haeLkKvaPdLdlpF--- P.syringae P.putida NTTPVlVLRFEADsdaeLQRIkdvF---------rtqLlrvePELqlpF--- (a) Figure4:Continued. TheScientificWorldJournal 9 92 A. baumanniiAIIMS7AlgC PMM 94 A. baumanniiAYE PMM 92 A. calcoaceticusPMM 76 A. haemolyticusPMM 36 O. cuniculusPGM isoform1 39 P. aeruginosaPAO1PMM P. putidaPMM/PGM P. syringaePMM H. sapiensPMM 0.5 (b) Figure4:(a)-(b)MultiplesequencealignmentandphylogeneticrelationshipofPMM/PGM.(a)CLUSTALWanalysesofPMM/PGMprotein 2+ sequencesdisplayingrepresentativecolorofboxwhichhighlightsconservedregions,thatis,activesite(red),Mg bindingsite(green),and substratebindingsite(blue).(b)BootstrapconsensustreeforPMM/PGMenzymes,constructedbyMEGAv5.0usingtheneighbor-joining (NJ)method(basedon1,000replicates).Numbersonthenodesarebootstrapvaluesandthebarrepresentsscaleofestimatedevolutionary distance(20substitutionsatanyaminoacidpositionper100aminoacidpositions). 1 2 M residues (Figure7(d); Supplementary Table S1). The metal 2+ ion Mg is required for enzymatic activity of PMM/PGM 2+ of A. baumannii AIIMS 7 (Figure7(d)), rather than Mn 2+ 2+ andZn .Specificinteractionsbetweenmetalion(Mg )and 94.3 protein, water-mediated H-bonds (so-called water bridges), and hydrophobic (Lennard-Jones) interactions have been identifiedandarelisted(SupplementaryTableS1). 66.0 3.9.StabilityofPhosphomannomutaseduringSimulation. To verifythesimulationstabilityandtomeasurestructureand dynamics of PMM/PGM model of A. baumannii AIIMS 7, AlgC standardstructuralparameterslikeRMSD,rootmeansquare 44.0 fluctuation(RMSF),andradiusofgyration(RG)werecalcu- latedandrepresentedinFigures8(a)–8(c).Duringmolecular dynamicssimulationperiodof10ns,theaverageRMSDvalue was 0.3nm as depicted (Figure8(a)). Steady RMSD result showed for the atoms in the four domains of PMM/PGM 29.0 modelindicatedthatthecatalyticactivityisrelativelystable duringthesimulation.Theresultforradiusofgyration(RG) alsoindicatedthestabilityofmodeloverthewholesimulation period (Figure8(b)). Minor fluctuations in C-alpha atoms Figure5:SDS-PAGEanalysisofAlgC(PMM/PGM)protein.Lane1: wholecellextractofcontrolE.coliDH5𝛼(lackingalgCgene);lane2: (RMSF) of helical and sheet structural elements could be wholecellextractofE.coliDH5𝛼withalgCgeneshowingtheAlgC seenwhereasloopregionshowedvariabilitytosomeextent proteinband∼53kDa;laneM:standardproteinmolecularweight (Figure8(c)). Overall, these results of RMSD, RMSF, and marker(kDa). RGstronglyindicatedthestabilityofsystemovertheentire simulationperiod. accessible surface (SA, Richards’ surface) and molecular 4.Discussion surface(MS,Connolly’ssurface)areasascalculatedbyalpha shapemethod.Theenzymeshowedatotalcavityof3641.5A˚ Biofilm formation substantially aids to the spectrum of andsphericalcentralcavityof1878.13A˚ wheresubstratecould multidrugresistancedisplayedbyA.baumanniiandisoften bind as shown in Figure7(b). To compare the 3D struc- attributed as the major cause for antibiotic treatment fail- tures obtained before and after MD simulations, structural ure [3, 5]. The process of bacterial biofilm development is superpositionsweremadeusingPDBeFOLD,resultsofwhich believedtobeacomplexinterplayofthebiofilmEPSmatrix showedrootmeansquaredeviation(RMSD)oftwoaligned components,thatis,aplethoraofproteinsandextracellular structureswithintherangeof2.51A˚ (Figure7(c)).Metalion macromolecules,forexample,DNAandpolysaccharides.The 2+ Mg interacts with Asp244, Asp246, Asp248, and Ser104 synthesisofspecificexopolysaccharidessuchasalginateand 10 TheScientificWorldJournal (a) (c) (e) (g) (b) (d) (f) (h) Figure6:(a)–(h)Visualizationofbiofilmaugmentationbyscanningelectronmicroscopy.Electronmicrographs(a,c,e,g)showingbiofilms formedbyalgCclonesafter24h,atagradientofmagnifications(Bar=2𝜇m,5𝜇m,10𝜇m,and20𝜇m;absolutemagnificationsindicatedas Kxintheverypicture)asobservedunderascanningelectronmicroscope.algCclonescanbeseenclearlyproducingdenseandrobustbiofilm assuggestedbythethicknessandintegrityofthebiofilmmatrices,comparedtothecontrolbiofilmsbyE.coliDH5𝛼(lackingA.baumannii algCgene)atcorrespondingcomparablemagnifications(b,d,f,h). LPS core has been described in various bacterial systems reportergeneactivityincontinuousbiofilmculturecellstobe [25,43,44],inparticularP.aeruginosa,withitscontribution 19-foldincreasedthanplanktoniccells.Earlier,Daviesetal. in biofilm formation [20, 22]. However, algC gene function [46] have shown that expression of P. aeruginosa algC is and its expression pattern have not been elucidated in A. upregulated after initial attachment of cells to teflon or baumannii in order to understand the genetic basis of its glasssubstrata,wherecellsthatfailedtoupregulatealginate associationwithbiofilmformation.Herein,aninitialgenetic biosynthesistypicallydetachedfromthesurface.Asreviewed characterization of the algC gene and its association with earlierbyGacesa[47],itispresumedthatexopolysaccharide biofilmformationisreportedintheMDRstrainAIIMS7of alginate plays a role in consolidation of the biofilm rather A. baumannii, which depicts differential expression of algC than in the initial adhesion event and hence higher rate of gene during the biofilm development. Besides, MD simula- algC transcription was not a prerequisite to surface attach- tion was performed on the three-dimensional (3D) struc- ment by P. aeruginosa, indicating alginate biosynthesis not ture of the gene product (enzyme PMM/PGM) to compare necessaryforitsattachmenttoglasssurface.Incontrast,our with that of P. aeruginosa, followed by its cloning, heterol- data in support of the relative quantification of algC tran- ogousexpression,andphylogeneticanalysis.Finally,biofilm scription by real-time PCR and biofilm augmentation by augmentationbyalgCgenewasevaluatedquantitativelyand algC as visualized by SEM is indicative of the possible confirmedbySEM. involvementofalgCexpressioninprobablyboththeevents, Analysis of the algC gene expression pattern in the that is, initial attachment and consolidation of biofilms temporalscale(upto96hours)revealsthatsurface-attached formed by A. baumannii. Furthermore, the upregulation of bacteriahavesignificantlyhigherrateoftranscriptionlevels algC transcription at the maturation stages (36–48 hours) than nonadherent or shaking cultures. The attachment or explains that it is strongly associated with maturation of “surface sensing” could be the trigger for the activation of biofilms, as the correlation of algC expression with biofilm algCpromoter,asaresultofwhichthealgCexpressioncould formationduringtheverystagewassignificantlyhigherthan beseenconsistentlyhigher(maximum81.59-fold,𝑃 < 0.05) theinitialevents(0.983comparedto0.902,𝑃<0.05).During inbiofilmformingA.baumanniiculturesthantheplanktonic planktonicmodeofgrowth,lowlevelsofalgCtranscription counterparts (maximum 3.24-fold) as revealed by real time wereobservedascomparedtobiofilmformingA.baumannii, PCR analysis (Figure1). This observation is in accordance whichshouldbeideallyduetolackofsurfaceattachment(less withanearlierstudyinP.aeruginosa[45]whichshowsalgC promoter activity) and variations in the oxygen tension in