Research Article Pantoea sp. Isolated from Tropical Fresh ...

Research ArticlePantoea sp Isolated from Tropical Fresh Water ExhibitingN-Acyl Homoserine Lactone Production

Wen-Si Tan Nina Yusrina Muhamad Yunos Pui-Wan Tan Nur Izzati MohamadTan-Guan-Sheng Adrian Wai-Fong Yin and Kok-Gan Chan

Division of Genetics and Molecular Biology Institute of Biological Sciences Faculty of Science University of Malaya50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Kok-Gan Chan kokganumedumy

Received 27 March 2014 Accepted 22 July 2014 Published 12 August 2014

Academic Editor Amparo Latorre

Copyright copy 2014 Wen-Si Tan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulatevarious physiological activities including pathogenicityWith the aim of isolating freshwater-borne bacteria that can cause outbreakof disease in plants and portrayed QS properties environmental water sampling was conducted Here we report the preliminaryscreening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors The 16SrDNA gene sequence of isolateM009 showed the highest sequence similarity to Pantoea stewartii S9-116 which is a plant pathogenThe isolated Pantoea sp was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolutionmass tandem mass spectrometry

1 Introduction

Quorum sensing (QS) expresses the mechanism in whichgene expression is regulated in relation to cell-density depen-dent manner to induce concerted physiological process inbacterial community [1 2] The concerted action by bacterialcommunication in relation to population density is coupledwith the production of signaling molecules which functionas ldquoautoinducersrdquo [1] There are two classified autoinduc-ers namely autoinducer-1 and autoinducer-2 [1 3] Gram-negative bacteria utilize the autoinducer-1 involving N-acylhomoserine lactone (AHL) and Gram-positive bacteria uti-lize the posttranslationally modified oligopeptide signalingmolecules classified as autoinducer-2 [1 4] To date the onlyshared QS mechanism for both Gram-positive and Gram-negative bacteria involves the autoinducer-2 production bythe enzyme LuxS [5 6]

Autoinduction of bacterial bioluminescence was firstdiscovered in the early 1970s when this discovery becamethe focus of the QS mechanism [7 8] The marine symbioticVibrio fischeri and its free living relative V harveyi are able toinduce bioluminescence in a cell density-dependent mannerThe AHL molecules which have been studied intensely are

utilized by a vastmajority of Gram-negative bacteria typicallydependent on a ldquoLuxIrdquo autoinducer synthase and a cognateldquoLuxRrdquo transcriptional activator protein [9] When AHLthreshold level is achieved the signaling molecules will bindto the transcriptional activator (LuxR protein) which willthen form a AHLLuxR complex and regulate expression oftarget genes [10 11]

Since QS play an important role in bacteria cell-to-cellcommunication it is therefore important for us to study thefresh water inhabiting bacteria which exhibits QS properties[12 13]Therefore it raises significant interest on the presenceof QS phytopathogen in aquatic environment as river wateris commonly used for irrigation Presence of phytopathogenmay have implication in the agriculture if the QS phyto-pathogens-contaminated fresh water is used for crop irri-gation Waterfall is chosen as sampling source for bacteriaisolation There are many waterfalls that are located insidetropical rainforest in Malaysia and Sungai Tua waterfall is theinterest in our study as it is a touristic site for locals In viewof this we investigated the presence of QS bacteria inMalaysia tropical rainforest waterfall sample and we reportthe isolation of plant pathogen that portrayed QS propertiesnamely Pantoea sp Plant pathogen that causes diseases tomajor crops could be water-borne [14]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 828971 5 pageshttpdxdoiorg1011552014828971

2 The Scientific World Journal

2 Experimental Section

21 Sample Collection and Bacterial Isolation Water samplewas collected from the top of Sungai Tua waterfall duringOctober 2013 The waterfall was located 10 km from Selayangand Ulu Yam with GPS coordinates of N03 19911015840 E101 42151015840The sample was collected below water surface at the depthof 12 cm Sample was collected in sterilized plastic tubes andkept at 4∘C till further processing [15] The water samplewas then serially diluted with sterile saline and spread ontoReasonerrsquos 2A agar (05 gL proteose 05 gL casamino acids05 gL yeast extract 05 gL dextrose 05 gL soluble starch03 gL dipotassium phosphate 005 gL magnesium sulfateand 03 gL sodium pyruvate) and incubated overnight (24 h)at 28∘CTheobservable differentmorphologies of the bacteriacolonies were isolated Pure colony was obtained with severalpassages on Trypticase Soy (TS) medium (10 gL tryptone5 gL soytone 5 gL NaCl and 15 gL Bacto agar)

22 Bacterial Strains Culture Conditions AHL BiosensorAssay and Controls for Short Chain AHLs Bacterial isolateM009 isolated from waterfall sample was selected for fur-ther Analysis and cultured on TS medium Two differentAHL biosensors (Chromobacterium violaceum CV026 andEscherichia coli [pSB401]) were used for the preliminaryscreening of QS signaling molecules Both the biosensorsrespond to the presence of short chain AHL molecules inwhich CV026 produced purple violacein pigmentation whileE coli [pSB401] respond by induction of luminescence [1617] In the AHL preliminary screening Erwinia carotovoraGS101 and E carotovora PNP22 were used as positive andnegative controls respectively [18] Lysogeny broth (LB)medium (10 gL tryptone 5 gL yeast extract 5 gL NaCl and15 gL Bacto agar) was used as growth medium for routineculture ofC violaceumCV026 E coli [pSB401] E carotovoraGS101 and E carotovora PNP22

23 Preliminary Screening of AHLs Using Bacterial BiosensorsIsolate M009 was screened for AHL production by crossstreaking the bacterial isolates with CV026 on a LB agar plate(24 h at 28∘C) Secondly E coli [pSB401] was used as an AHLbiosensor to screen for the production of AHLAfter 24 hoursincubation at 28∘C the photon camera with 60 s of exposurewas used to observe the bioluminescence induced [19]

24 Strains Identification Molecular identification of isolateM009 was done by analyzing the PCR-amplified bacterial 16SrDNA gene The primer pair 27F-1525R [20 21] was used forPCR amplification using PCR mix from Promega (PromegaKit Madison WI USA) Prior to that genomic DNA ofisolateM009 was extracted usingMasterPureTM DNAPurifi-cation Kit (Epicentre Inc Madison WI USA) PCR ampli-fication and amplicons purification were conducted asdescribed previously and the PCR product sequence align-ment was done using GenBank BLASTN program followedby phylogenetic tree reconstruction using theMolecular Evo-lutionary Genetic Analysis version 60 [22 23]

25 AHL Extraction Isolate M009 was cultured in LB brothbuffered to pH 55 with 50mM of 3-(N-morpholino) pro-panesulfonic acid (MOPS) in an incubator shaker (200 rpm28∘C 18 h) [24] The cultured supernatant was extractedtwice with equal volume of acidified (01 vv glacial aceticacid) ethyl acetate as described previously [24] The organicsolvent was dried in fume hood and the dried extracts wereresuspended in 1mL of acidified ethyl acetate and desiccatedcompletely Finally 200 120583L of acetonitrile (HPLC grade) wasadded and vortexed to dissolve the dried extractsThe extractwas then centrifuged at 12000 rpm for 5min to remove anyinsoluble residue The dissolved sample with 75120583L aliquotwas withdrawn from the upper layer and inserted in samplevials for mass spectrometry Analysis

26 AHL Profiling by Mass Spectrometry (MS) AHL profilewas analyzed byMS as described previously [24]The AgilentRRLC 1200 system was utilized as the liquid chromatography(LC) delivery system with the use of Agilent ZORBAX RapidResolution HT column (21mm times 100mm 18 120583m particlesize) for separation of AHL molecules The flow rate for theAnalysis was set to 03mLmin at 60∘C and injection volumewas adjusted to 20 120583L The mobile phases A (LCMS gradewater with 01 vv formic acid) and B (HPLC grade acetoni-trile with 01 vv formic acid) were prepared and set a ratioof 80 20 respectively The high resolution electrospray ion-ization mass spectrometry (ESI-MS) was performed with theAgilent 6490 Triple Quadrupole LCMS system and wascarried out in the ESI-positive mode (probe capillary voltage3 kV nebulizer pressure at 20 psi sheath gas at 11mLh des-olvation temperature 200∘C collision energy and fragmen-tation at both 5 eV and 380 eV resp) The precursor ion scanmode targeting at the production ion with mz 102 indicatesthat [M+H]+ ion of the core lactone ring moiety The mzvalue range to detect the precursor ions was set at mz 150ndash400 To analyse the MS spectra we used Agilent MassHuntersoftware

3 Results and Discussion

31 Strains Isolation and Identification This study aimed toisolate AHL producing bacterial isolate from the tropicalrainforest waterfall Irrigation systems carry water directlyfrom river to farmswhereby the usual source is fromwaterfall[25] The temperature of the water was 25∘C at daytime andthe pH gave a reading value of 7 at the point of the samplingThewater samplewas collected at themost top of thewaterfallwhere human activities were less common in order to reducethe faecal contamination during water collection [25 26]

The number of QS bacteria identified has been increaseddue to the availability of AHL biosensors which are able todetect the presence of AHLs [6 27] The AHL biosensorspractically rely on LuxR proteins and display specificitytowards the cognate AHL and positively regulate the tran-scription of a reporter gene [27] The biosensor C violaceumCV026 responds to AHL with N-acyl chain length of C4 toC8 that will induce purple violacein pigmentation [16] It is acommonly used bacterial biosensor due to rapidness and

The Scientific World Journal 3

M009

Positive control

Negative control

(a)

M009

Positive control

Negative control

(b)

Figure 1 (a) AHL screening of strain M009 with CV026 E carotovora PNP22 (Negative control) devoid of QS activity was included andE carotovora GS101 (Positive control) that can activate CV026 was included for comparison Both positive control and strain M009 inducethe purple violacein pigmentation (b) AHL screening of strain M009 with E coli [pSB401] E carotovora PNP22 (negative control) andE carotovora GS101 (positive control) served as negative and positive controls respectively The positive control and strain M009 exhibitbioluminescence

M009

0005

gi|587023135|gb|KF8310241|

gi|157780309|gb|EF6880061| LMG 22050 (EF699006)gi|645320578|ref|NR 1177511| (HQ992945)

gi|530758821|gb|KF5272171| (KF527217)gi|164710484|gb|EU2391461| KNUC358 (EU239416)

Pantoea stewartii strain L-2 (KF831024)gi|381217925|gb|JQ6602861|Pantoea stewartii strain S9-116 (JQ660286)

gi|586962683|gb|KF9138281|Pantoea vagans strain iHA8 (KF913828)

Citrobacter koseri strain LMG 5519

Pantoea dispersa strain XSPantoea dispersa strain

7

Pantoea punctate strain

Figure 2 Phylogenetic tree reconstructed using maximum likelihood algorithm showing phylogenetic relationships of isolate M009 tomembers of other species of the genus Pantoea There are a total of 1220 positions in the final dataset Bar 5 substitutions per 1000 nucleotidepositions

accuracy of AHL detection On the other hand E coli[pSB401] biosensor will exhibit luminescence due to the pres-ence of short chain AHLs and it is most sensitive to cognate3-oxo-C6-HSL [17] In this study both CV026 and E coli[pSB401] were employed for preliminary screening of AHLsproduced by strain M009 (Figure 1)

Preliminary screening of the AHL production withCV026 (Figure 1(a)) and E coli [pSB401] (Figure 1(b)) indi-cated that isolate M009 strain produced short chain AHL[16 17]This strain was then sent for molecular identification

32 Molecular Bacterial Identification Isolate M009 identitywas confirmed by analysis of its 16S rDNA gene nucleotidessequences showing that it clusters closely with the Pantoeagenus showing 98-99 similarity in the BLAST searchThe nucleotide sequences were subsequently deposited intoGenBank with the accession number KJ830125 Accord-ing to the phylogenetic tree constructed (Figure 2) withthe evolutionary history which was inferred by using theMaximum Likelihood algorithm based on the Tamura-Nei

model [1] strain M009 is identified as Pantoea stewartiia plant pathogen The tree with the highest log likelihood(minus23887284) is shown Initial tree(s) for the heuristic searchwere obtained automatically as follows When the number ofcommon sites was lt 100 or less than one fourth of the totalnumber of sites the maximum parsimony method was usedotherwise BIONJ method with MCL distance matrix wasused There were a total of 1221 positions in the final datasetEvolutionary analyses were conducted in MEGA6

33 Assay of AHL from Supernatant Pantoea stewartii iscausative agent of Stewartrsquos wilt a bacterial disease transmit-ted by the corn flea beetle mainly sweet corn (Zea mays) Ithas been classified as a quarantine microorganism and mustbe differentiated fromother yellow enteric bacteria that occurfrequently with corn [28] Stewartrsquos wilt is also a problem oncertain elite inbred maize lines used for producing hybridfield corn seed and more than 60 countries place importregulations onmaize seed imports from affected areas to pre-vent the distribution of pathogen and to reduce the influence


2143000

317100017220000

04081216

150 170 190 210 230 250 270 290 310 330 350 370 390

times103

Counts () versus mass-to-charge (mz)

+ESI precursor ion 47min) Frag = 3797V CID50 (lowastlowast 1020000) 16d1 (09

Figure 3 Mass spectrometry analysis of AHL extracts from Pantoea sp strain M009 Mass spectra of 3-oxo-C6 HSL (mz 2143000) wasdetected

towards agriculture economy [28] Thus the availability todetect AHL profile of Pantoea sp could be a milestone inunderstanding this potential plant pathogen

The spent culture supernatants of M009 strain wereanalyzed using the Agilent 6500 Q-TOF LCMS system andmass spectrometry analysis The presence of 3-oxo-C6-HSLis confirmed (Figure 3) and this is the type of AHL which isidentical to the V fischeri autoinducer [11ndash13] Our result isalso in agreement with other report work that showed P stew-artii primarily produces 3-oxo-C6-HSL molecules [29 30]The AHL synthase and cognate AHL receptor for 3-oxo-C6-HSL in the reported P stewartii are EsaI and EsaR which arethe homologues of luxI and luxR [30] The similarity of theseresults further confirmed that bacterial communicationoccurs on plant surfaces and participants likely include thesignal producers community signal eavesdroppers and planthost [4] In addition our group is currently expanding thiswork to the whole genome sequence research to gain moreinsights into this strain particularly to isolate the luxI andluxR homologues of our isolate

QS regulates a battery of bacterial pathogenicity factors[30] and hence this work facilitates the evidence to illustratethe importance to emphasize the research onAHL-producingplant bacteria that are present in the environmentEsaI whichproduces 3-oxo-C6-HSL in P stewartii has been shown toregulate extracellular polysaccharide capsule production andpathogenicity [30] This work suggests that fresh water maybe a potential reservoir for QS pathogens that should be givenattention by more intense research

4 Conclusion

We reported here the production of 3-oxo-C6-HSL in Pan-toea sp M009 strain isolated from fresh water sampleThis discovery could form the basis for understanding thecommunication system of this isolate and relate it to QS-modulated virulent determinants

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

Thisworkwas supported by theUniversity ofMalaya throughthe award ofHIRGrant (UMC6251HIRMOHECHAN01Grant no A-000001-50001) to KG Chan

References

[1] P Williams K Winzer W C Chan and M Camara ldquoLookwhorsquos talking communication and quorum sensing in the bac-terial worldrdquo Philosophical Transactions of the Royal Society BBiological Sciences vol 362 no 1483 pp 1119ndash1134 2007

[2] W Yin K Purmal S Chin et al ldquoN-Acyl homoserine lactoneproduction by Klebsiella pneumonia isolated from humantongue surfacerdquo Sensors vol 12 no 3 pp 3472ndash3483 2012

[3] J W Chen H M Gan W F Yin and K G Chan ldquoGenomesequence of Roseomonas sp strain B5 a quorum-quenchingN-acylhomoserine lactone-degrading bacterium isolated fromMalaysian tropical soilrdquo Journal of Bacteriology vol 194 no 23pp 6681ndash6682 2012

[4] B L Bassler ldquoHow bacteria talk to each other regulation ofgene expression by quorum sensingrdquoCurrent Opinion inMicro-biology vol 2 no 6 pp 582ndash587 1999

[5] L Y TanW F Yin and K G Chan ldquoSilencing quorum sensingthrough extracts ofMelicope lunu-ankendardquo Sensors vol 12 no4 pp 4339ndash4351 2012

[6] K Winzer K R Hardie and P Williams ldquoBacterial cell-to-cellcommunication sorry canrsquot talk now Gone to lunchrdquo CurrentOpinion in Microbiology vol 5 no 2 pp 216ndash222 2002

[7] H M H N Bandara O L T Lam L J Jin and L Sama-ranayake ldquoMicrobial chemical signaling a current perspectiverdquoCritical Reviews inMicrobiology vol 38 no 3 pp 217ndash249 2012

[8] J Loh E A Pierson L S Pierson III G Stacey and A Chat-terjee ldquoQuorum sensing in plant-associated bacteriardquo CurrentOpinion in Plant Biology vol 5 no 4 pp 285ndash290 2002

[9] S Schauder K Shokat M G Surette and B L Bassler ldquoTheLuxS family of bacterial autoinducers biosynthesis of a novelquorum-sensing signal moleculerdquoMolecular Microbiology vol41 no 2 pp 463ndash476 2001

[10] A Deep U Chaudhary and V Gupta ldquoQuorum sensing andbacterial pathogenicity from molecules to diseaserdquo Journal ofLaboratory Physicians vol 3 no 1 pp 4ndash11 2011

[11] S Atkinson and P Williams ldquoQuorum sensing and socialnetworking in the microbial worldrdquo Journal of the Royal SocietyInterface vol 6 no 40 pp 959ndash978 2009

[12] A M Duda and R J Johnson ldquoCost-effective targeting of agri-cultural nonpoint-source pollution controlsrdquo Journal of Soil ampWater Conservation vol 40 no 1 pp 108ndash111 1985

[13] V Koiv and A Mae ldquoQuorum sensing controls the synthesisvirulence factors by modulating rsmA gene expression inErwinia carotovora subsp carotovorardquo Molecular and GeneralGenetics vol 265 no 2 pp 287ndash292 2001

[14] C X Hong and GW Moorman ldquoPlant pathogens in irrigationwater challenges and opportunitiesrdquo Critical Reviews in PlantSciences vol 24 no 3 pp 189ndash208 2005


[15] J R Firth and C Edwards ldquoDenitrification by indigenousmicrobial populations of river watermeasured usingmembraneinlet mass spectrometryrdquo Journal of Applied Microbiology vol89 no 1 pp 123ndash129 2000

[16] K H McClean M K Winson L Fish et al ldquoQuorum sensingand Chromobacterium violaceum exploitation of violacein pro-duction and inhibition for the detection of N-acylhomoserinelactonesrdquoMicrobiology vol 143 no 12 pp 3703ndash3711 1997

[17] M KWinson S Swift L Fish et al ldquoConstruction and analysisof luxCDABE-based plasmid sensors for investigating N-acylhomoserine lactone-mediated quorum sensingrdquo FEMS Micro-biology Letters vol 163 no 2 pp 185ndash192 1998

[18] E H J Robson W F Yin and K G Chan ldquoPandoraea sp RB-44 a novel quorum sensing soil bacteriumrdquo Sensors vol 13 no10 pp 14121ndash14132 2013

[19] L Y Tan W Yin and K Chan ldquoPiper nigrum Piper betleand Gnetum gnemonmdashnatural food sources with anti-quorumsensing propertiesrdquo Sensors vol 13 no 3 pp 3975ndash3985 2013

[20] J A Frank C I Reich S Sharma J S Weisbaum B AWilsonand G J Olsen ldquoCritical evaluation of two primers commonlyused for amplification of bacterial 16S rRNA genesrdquoApplied andEnvironmental Microbiology vol 74 no 8 pp 2461ndash2470 2008

[21] F E Dewhirst C C Chien B J Paster et al ldquoPhylogeny of thedefined murine microbiota altered Schaedler florardquo Appliedand Environmental Microbiology vol 65 no 8 pp 3287ndash32921999

[22] K Tamura andMNei ldquoEstimation of the number of nucleotidesubstitutions in the control region of mitochondrial DNA inhumans and chimpanzeesrdquo Molecular Biology and Evolutionvol 10 no 3 pp 512ndash526 1993

[23] K Tamura D Peterson N Peterson G Stecher M Nei and SKumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[24] C A Ortori J Dubern S R Chhabra et al ldquoSimultaneousquantitative profiling of N-acyl-l-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signalingmolecules using LC-MSMSrdquo Analytical and BioanalyticalChemistry vol 399 no 2 pp 839ndash850 2011

[25] M J Brown W D Kemper T J Trout and A S HumpherysldquoSediment erosion and water intake in furrowsrdquo IrrigationScience vol 9 no 1 pp 45ndash55 1988

[26] Y Madrid and Z P Zayas ldquoWater sampling traditional meth-ods and new approaches in water sampling strategyrdquo Trends inAnalytical Chemistry vol 26 no 4 pp 293ndash299 2007

[27] L Steindler and V Venturi ldquoDetection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosen-sorsrdquo FEMS Microbiology Letters vol 266 no 1 pp 1ndash9 2007

[28] A Wensing S Zimmermann and K Geider ldquoIdentification ofthe corn pathogen Pantoea stewartii by mass spectrometry ofwhole-cell extracts and its detection with novel PCR primersrdquoApplied and Environmental Microbiology vol 76 no 18 pp6248ndash6256 2010

[29] S B von Bodman D R Majerczak and D L Coplin ldquoA nega-tive regulator mediates quorum-sensing control of exopolysac-charide production in Pantoea stewartii subsp Stewartiirdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 95 no 13 pp 7687ndash7692 1998

[30] S B von Bodman and S K Farrand ldquoCapsular polysaccharidebiosynthesis and pathogenicity in Erwinia stewartii requireinduction by an N-acylhomoserine lactone autoinducerrdquo Jour-nal of Bacteriology vol 177 no 17 pp 5000ndash5008 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


2 Experimental Section

21 Sample Collection and Bacterial Isolation Water samplewas collected from the top of Sungai Tua waterfall duringOctober 2013 The waterfall was located 10 km from Selayangand Ulu Yam with GPS coordinates of N03 19911015840 E101 42151015840The sample was collected below water surface at the depthof 12 cm Sample was collected in sterilized plastic tubes andkept at 4∘C till further processing [15] The water samplewas then serially diluted with sterile saline and spread ontoReasonerrsquos 2A agar (05 gL proteose 05 gL casamino acids05 gL yeast extract 05 gL dextrose 05 gL soluble starch03 gL dipotassium phosphate 005 gL magnesium sulfateand 03 gL sodium pyruvate) and incubated overnight (24 h)at 28∘CTheobservable differentmorphologies of the bacteriacolonies were isolated Pure colony was obtained with severalpassages on Trypticase Soy (TS) medium (10 gL tryptone5 gL soytone 5 gL NaCl and 15 gL Bacto agar)

22 Bacterial Strains Culture Conditions AHL BiosensorAssay and Controls for Short Chain AHLs Bacterial isolateM009 isolated from waterfall sample was selected for fur-ther Analysis and cultured on TS medium Two differentAHL biosensors (Chromobacterium violaceum CV026 andEscherichia coli [pSB401]) were used for the preliminaryscreening of QS signaling molecules Both the biosensorsrespond to the presence of short chain AHL molecules inwhich CV026 produced purple violacein pigmentation whileE coli [pSB401] respond by induction of luminescence [1617] In the AHL preliminary screening Erwinia carotovoraGS101 and E carotovora PNP22 were used as positive andnegative controls respectively [18] Lysogeny broth (LB)medium (10 gL tryptone 5 gL yeast extract 5 gL NaCl and15 gL Bacto agar) was used as growth medium for routineculture ofC violaceumCV026 E coli [pSB401] E carotovoraGS101 and E carotovora PNP22

23 Preliminary Screening of AHLs Using Bacterial BiosensorsIsolate M009 was screened for AHL production by crossstreaking the bacterial isolates with CV026 on a LB agar plate(24 h at 28∘C) Secondly E coli [pSB401] was used as an AHLbiosensor to screen for the production of AHLAfter 24 hoursincubation at 28∘C the photon camera with 60 s of exposurewas used to observe the bioluminescence induced [19]

24 Strains Identification Molecular identification of isolateM009 was done by analyzing the PCR-amplified bacterial 16SrDNA gene The primer pair 27F-1525R [20 21] was used forPCR amplification using PCR mix from Promega (PromegaKit Madison WI USA) Prior to that genomic DNA ofisolateM009 was extracted usingMasterPureTM DNAPurifi-cation Kit (Epicentre Inc Madison WI USA) PCR ampli-fication and amplicons purification were conducted asdescribed previously and the PCR product sequence align-ment was done using GenBank BLASTN program followedby phylogenetic tree reconstruction using theMolecular Evo-lutionary Genetic Analysis version 60 [22 23]

25 AHL Extraction Isolate M009 was cultured in LB brothbuffered to pH 55 with 50mM of 3-(N-morpholino) pro-panesulfonic acid (MOPS) in an incubator shaker (200 rpm28∘C 18 h) [24] The cultured supernatant was extractedtwice with equal volume of acidified (01 vv glacial aceticacid) ethyl acetate as described previously [24] The organicsolvent was dried in fume hood and the dried extracts wereresuspended in 1mL of acidified ethyl acetate and desiccatedcompletely Finally 200 120583L of acetonitrile (HPLC grade) wasadded and vortexed to dissolve the dried extractsThe extractwas then centrifuged at 12000 rpm for 5min to remove anyinsoluble residue The dissolved sample with 75120583L aliquotwas withdrawn from the upper layer and inserted in samplevials for mass spectrometry Analysis

26 AHL Profiling by Mass Spectrometry (MS) AHL profilewas analyzed byMS as described previously [24]The AgilentRRLC 1200 system was utilized as the liquid chromatography(LC) delivery system with the use of Agilent ZORBAX RapidResolution HT column (21mm times 100mm 18 120583m particlesize) for separation of AHL molecules The flow rate for theAnalysis was set to 03mLmin at 60∘C and injection volumewas adjusted to 20 120583L The mobile phases A (LCMS gradewater with 01 vv formic acid) and B (HPLC grade acetoni-trile with 01 vv formic acid) were prepared and set a ratioof 80 20 respectively The high resolution electrospray ion-ization mass spectrometry (ESI-MS) was performed with theAgilent 6490 Triple Quadrupole LCMS system and wascarried out in the ESI-positive mode (probe capillary voltage3 kV nebulizer pressure at 20 psi sheath gas at 11mLh des-olvation temperature 200∘C collision energy and fragmen-tation at both 5 eV and 380 eV resp) The precursor ion scanmode targeting at the production ion with mz 102 indicatesthat [M+H]+ ion of the core lactone ring moiety The mzvalue range to detect the precursor ions was set at mz 150ndash400 To analyse the MS spectra we used Agilent MassHuntersoftware

3 Results and Discussion

31 Strains Isolation and Identification This study aimed toisolate AHL producing bacterial isolate from the tropicalrainforest waterfall Irrigation systems carry water directlyfrom river to farmswhereby the usual source is fromwaterfall[25] The temperature of the water was 25∘C at daytime andthe pH gave a reading value of 7 at the point of the samplingThewater samplewas collected at themost top of thewaterfallwhere human activities were less common in order to reducethe faecal contamination during water collection [25 26]

The number of QS bacteria identified has been increaseddue to the availability of AHL biosensors which are able todetect the presence of AHLs [6 27] The AHL biosensorspractically rely on LuxR proteins and display specificitytowards the cognate AHL and positively regulate the tran-scription of a reporter gene [27] The biosensor C violaceumCV026 responds to AHL with N-acyl chain length of C4 toC8 that will induce purple violacein pigmentation [16] It is acommonly used bacterial biosensor due to rapidness and


M009

Positive control

Negative control

(a)

M009

Positive control

Negative control

(b)


M009

0005

gi|587023135|gb|KF8310241|







7









2143000

317100017220000

04081216

150 170 190 210 230 250 270 290 310 330 350 370 390

times103







4 Conclusion




Acknowledgment


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M009

Positive control

Negative control

(a)

M009

Positive control

Negative control

(b)


M009

0005

gi|587023135|gb|KF8310241|







7









2143000

317100017220000

04081216

150 170 190 210 230 250 270 290 310 330 350 370 390

times103







4 Conclusion




Acknowledgment


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2143000

317100017220000

04081216

150 170 190 210 230 250 270 290 310 330 350 370 390

times103







4 Conclusion




Acknowledgment


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Pantoea sp. Isolated from Tropical Fresh ...

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