Coexistence of quorum-quenching and quorum-sensing in tropical marine Pseudomonas aeruginosa strain

ORIGINAL PAPER

Coexistence of quorum-quenching and quorum-sensing in tropical marine Pseudomonas aeruginosa strain MW3A

Cheng-Siang Wong ?Wai-Fong Yin ?Yeun-Mun Choo ?Choon-Kook Sam ?Chong-Lek Koh ?Kok-Gan Chan

Received:20January 2011/Accepted:30June 2011/Published online:9July 2011óSpringer Science+Business Media B.V.2011

Abstract A chemically de?ned medium called KGm medium was used to isolate from a sample of sea water a bacterial strain,MW3A,capable of using N -3-oxohexanoyl-L -homoserine lactone as the sole carbon source.MW3A was clustered closely to Pseudomonas aeruginosa by 16S ribo-somal DNA sequence analysis.It degraded both N -acylho-moserine lactones (AHLs)with a 3-oxo group substitution and,less preferably,AHLs with unsubstituted groups at C3position in the acyl side chain,as determined by Rapid Resolution Liquid Chromatography.Its quiP and pvdQ homologue gene sequences showed high similarities to those of known acylases.Spent supernatant of MW3A harvested at 8-h post inoculation was shown to contain long-chain AHLs when assayed with the biosensor Escherichia coli [pSB1075],and speci?cally N -dodecanoyl-L -homoserine lactone and N -3-oxotetradecanoyl-L -homoserine lactone by high resolution mass spectrometry.Hence,we report here a novel marine P.aeruginosa strain MW3A possessing both quorum-quenching and quorum-sensing properties.

Keywords KG medium áMarine Pseudomonas á

N -acylhomoserine lactone áAcylase áQuorum-sensing áQuorum-quenching

Introduction

Although unicellular,bacteria are highly interactive and are able to communicate with each other to regulate gene expression in a concerted way.The term ‘‘quorum-sens-ing’’(QS)refers to the ability of a population of unicellular bacteria to act as a multicellular organism in a cell-density-dependent manner,that is,a way to sense ‘‘how many are out there’’(Fuqua et al .1996,2001;Miller and Bassler 2001;Schauder and Bassler 2001).

QS involves production,release,detection,and response to small molecules termed autoinducers by bacteria.Thus far the most well studied gram negative bacterial QS molecules are N -acyl homoserine lactones (AHLs)(Chhabra et al.2005;Williams et al.2007).The concen-tration of these signalling molecules can act as a measure of population density,allowing whole bacterial communi-ties to regulate gene expression in a concerted way.QS regulates diverse bacterial physiological processes,which include,but are not limited to,bioluminescence,swarming,swimming and twitching motility,antibiotic biosynthesis,bio?lm differentiation,plasmid conjugal transfer,and the production of virulence determinants in animal,?sh,and plant pathogens (Dunny and Winans 1999;Fuqua et al.1996;Hardman et al.1998;Salmond et al.1995).

AHL molecules are highly conserved.Each AHL consists of a homoserine lactone ring unsubstituted in the b -and c -positions but N -acylated with a fatty acyl group at the a -position (Chhabra et al.2005).The acyl side-chain varies from 4to 18carbons in its lengths,mostly even-numbered,

C.-S.Wong áW.-F.Yin áK.-G.Chan (&)Division of Genetics and Molecular Biology,

Institute of Biological Sciences,Faculty of Science,University of Malaya,50603Kuala Lumpur,Malaysia e-mail:kokgan@https://www.wendangku.net/doc/7013579202.html,.my

Y.-M.Choo

Department of Chemistry,Faculty of Science,

University of Malaya,50603Kuala Lumpur,Malaysia C.-K.Sam áC.-L.Koh

Natural Sciences and Science Education AG,National Institute of Education,Nanyang Technological University,1Nanyang Walk,Singapore 637616,Singapore

World J Microbiol Biotechnol (2012)28:453–461DOI 10.1007/s11274-011-0836-x

with different saturation levels and oxidation states which

belong to the N-acyl,N-3-oxoacyl,or N-3-hydroxyacyl class.

Stereochemistry at the a-centre of the homoserine lactone

(HSL)ring has been unequivocally established to be the L-isomer of N-3-oxohexanoyl-L-homoserine lactone(3-oxo-C6-HSL)produced by Erwinia carotovora(Bainton et al.

1992)and by analogy it is deduced that all other natural AHLs

have the same con?guration(Bainton et al.1992;Williams

et al.2007).It has been reported that synthetic D-isomers lack

autoinduction activity(Chhabra et al.1993,2005).

The AHL signalling system has been regarded as a

promising target for developing novel approaches to con-

trolling bacterial infections by regulating the virulence of

the population as a whole without affecting the viability of

the individual cells.This means that there is less selection

pressure for the evolution of resistance than in the case in,

for example,antibiotic therapy(Hentzer and Givskov

2003).The disruption of bacterial QS is known as‘quo-

rum-quenching’(QQ).

The bacterial QS system can be grouped into three key

functional components:signal generation,signal percep-

tion,and signal transportation,which links the?rst two

components.Therefore QQ can be achieved by targeting

one or more of these components,and the straightforward

way is to degrade the AHL molecules,hence reducing the

‘quorum’and preventing QS-mediated gene expression.

Two QQ genes,pvdQ and quiP that encode AHL acylases,

have been identi?ed from Pseudomonas aeruginosa by Huang

et al.(2003,2006)and Sio et al.(2006).Huang et al.(2006)

suggested that these two AHL acylases play a critical role in

the degradation and utilization of AHLs,including the

P.aeruginosa native N-3-oxododecanoyl-L-homoserine lac-

tone(3-oxo-C12-HSL),to control different ratios of long-and

short-chain AHL signalling molecules during different phe-

notypic stages,for example,bio?lm and planktonic states.

Although the marine ecosystem is rich in microbial

diversity,roseobacters seem to be the dominant QS bac-

teria in the marine environment and they produce various

types of AHLs(Cicirelli et al.2008;MacLeod1965;

Wagner-Do¨bler et al.2005).Recently,Romero et al.

(2011)reported15cultivable bacterial strains with QQ

activity from three different marine samples.None of the

15strains belonged to the genus Pseudomonas.Here we

describe the characterisation of a marine P.aeruginosa

with QS and QQ activities and its QQ genes.

Materials and methods

Modi?ed Luria–Bertani(LBm)medium

LBm medium was modi?ed from LB broth(Sambrook

et al.1989).It consisted of1.0%w/v tryptone,0.5%w/v yeast extract,and2.5%w/v NaCl.For extraction of AHLs, cells were grown in LB buffered with50mM3-(N-mor-pholino)propanesulfonic acid(MOPS)to pH6.5(LB MOPS) to prevent alkaline lactonolysis(Yates et al.2002).

Bacterial strains,media and culture conditions

Marine bacteria were grown in LBm medium at28°C. Escherichia coli DH5a and lux-based biosensor E.coli [pSB1075]were grown at37°C in LB and LB supple-mented with tetracycline(10mg l-1),respectively.E.coli [pSB1075]responds to exogenous long-chain AHLs by emitting light(Winson et al.1998).Broth culture was incubated with shaking(220rev min-1).

Modi?ed KG(KGm)medium

KGm medium was modi?ed from KG medium(Chan et al. 2009).It comprised a basal medium containing NaCl (1.25g l-1),KCl(0.75g l-1),Na2SO4(0.25g l-1), KH2PO4(7.5g l-1),MgCl2(0.5g l-1),CaCl2 (0.25g l-1),NH4Cl(0.3g l-1)(supplied unless otherwise stated),and2-(N-morpholino)-ethanesulfonic acid(MES) (1.0g l-1).After the basal medium was autoclaved and cooled,?lter-sterilized(0.22-l m pore size)FeCl3,MnCl2, and ZnCl2solutions were added to it to?nal concentrations of5mg l-1, 2.5mg l-1,and0.6g l-1,respectively. Finally,3-oxo-C6-HSL was added to50mg l-1as the sole carbon source.

Enrichment and isolation procedures

Seawater was collected at subsurface level(5cm beneath water level)in November2007in Malacca(2°1101200N, 102°1501200E),Malaysia.The sample was collected in a sterile plastic container,transported to the laboratory and processed immediately.The marine water sample(1ml) was added to3ml of KGm medium supplemented with ammonium chloride(300mg l-1)and3-oxo-C6-HSL (50mg l-1)as sole sources of nitrogen and carbon, respectively.This mixture was incubated at28°C with shaking(220rev min-1).After48h,150l l of the sus-pension was inoculated into3ml of fresh enrichment medium.The same procedure was repeated four times.At the?fth enrichment cycle,a diluted suspension was plated onto LBm agar and a plate of3-oxo-C6-HSL-containing KGm agar to isolate individual colonies.

PCR ampli?cation of pvdQ and quiP homologue genes and16S rDNA genes

For PCR of both quiP and pvdQ genes,the thermal cycling programs for PCR consisted of an initial denaturation at

94°C for10min,followed by35cycles of94°C(30s), 57°C(30s),72°C(1min),and a?nal primer extension at 72°C for5min.For quiP gene,the forward and reverse primers used were quiP F(50-ATTAGAAGCTTATGGCCT CGCCAGCCTTC-30)and quiP R(50-ATTACTCTAGATC AGCGAGCGGGAGTG-30),respectively(Huang et al. 2006).For pvdQ gene,the forward and reverse primers used were pvdQ F(50-AGGCCAAGCTTATGGGGGATGC GTACCGTACTG-30)and pvdQ R(50-GTTATATAGCG GCCGCTAGGATTGCTTATCATTCG-30),respectively (Huang et al.2003).

PCR of16S rDNA genes was carried out as described previously(Chan et al.2007),using27F(50-AGAGTTTG ATCMTGGCTCAG-30)and1525R(50-AAGGAGGTGW TCCARCC-30)as the forward and reverse primers,respec-tively.For all PCR,negative controls were included by replacing the DNA template with sterile deionized water. PCR products were gel excised,column-cleaned,and ligated into the pGEM-T Easy vector(Promega,USA)as per the manufacturer’s instructions,followed by transformation into E.coli DH5a(Sambrook et al.1989). Phylogenetic analysis

Sequences were analysed using Chromas Lite version2.01 (Technelysium Pte Ltd,Australia)to exclude primer-binding sites.Phylogenetic and molecular evolutionary analyses were conducted with MEGA version4.0(Tamura et al.2007)as described previously(Chan et al.2007, 2009).Bootstrap analyses for1,000re-samplings were always performed to provide con?dence estimates for tree topologies.

Whole-cell AHL inactivation assays

Washed overnight-grown cells suspended in PBS buffer (pH6.5)were used as direct source of whole-cell inacti-vation assay as described previously(Chan et al.2009, 2011a).Aliquots of AHL in absolute ethanol were dis-pensed into sterile tubes and the solvent was evaporated to dryness under sterile conditions.A resting cell suspension was used to rehydrate the AHL to a?nal concentration of 0.5l g l l-1.The mixtures were incubated at room tem-perature for24h with shaking(220rev min-1).The reactions were stopped at appropriate intervals by the addition of ethyl acetate,which also served to extract any remaining AHL.Ethyl acetate containing the residual AHL was removed and evaporated to dryness.The residue was reconstituted in an appropriate volume of acetonitrile. Extracted AHL was detected by Rapid Resolution Liquid Chromatography(RRLC).Reverse-phase RRLC analysis of AHL degradation

Reverse-phase RRLC analysis of AHL was carried out as reported(Chan et al.2010)using an Agilent Technologies 1200Series Rapid Resolution LC system(Agilent Technol-ogies,Germany)equipped with a vacuum degasser,a binary pump,and a diode-array detector.Ten microlitres of extracted AHL from AHL inactivation assay was applied onto an ana-lytical C18reverse-phase column(Agilent ZORBAX Eclipse XDB-C18,4.6mm950mm,1.8l m particle size).RRLC was run on isocratic pro?le of acetonitrile–water(35:65,v/v, for short-chain AHL;60:40,v/v,for long-chain AHL)for 3min at a constant?ow rate of0.7ml min-1and the spectrum was monitored at210nm.Agilent Chemstation(version B.04.01)was used for data collection and analysis.The resultant retention time and spectral properties were compared to those of a series of synthetic AHL standards obtained from Sigma–Aldrich.AHLs incubated with washed E.coli DH5a cells and PBS were used as negative controls.The percentage of AHL inactivated and the speci?c activity were determined by estimating the amount of AHL(by comparison of the reduction in peak areas for a given retention time)with respect to AHL solutions of known concentrations(Chan et al.2010).

Extraction of AHL

For extraction of AHL,spent supernatant of P.aeruginosa strain MW3A(200ml)was collected by centrifugation (7,0009g,10min)at8-h post inoculation,extracted twice with an equal volume of acidi?ed ethyl acetate(0.01%v/v glacial acetic acid in ethyl acetate).The organic layer was collected,dried by adding an excess of anhydrous MgSO4,?ltered through a Whatmann3MM paper,and rotary evaporated.The extract residue was resuspended in200l l of acetonitrile and analysed by luminometer–spectropho-tometer used in conjunction with lux-based biosensor E.coli[pSB1075]and liquid chromatography mass spec-trometry(LC–MS).

Measurement of bioluminescence

Bioluminescence was measured as a function of cell density using a combined automated Tecan luminometer–spectro-photometer(In?nite,Tecan).An overnight culture of E.coli [pSB1075]was diluted with LB to OD6000.01and0.2ml of this dilution was added to each well of a96-well optical bottom microtitre plate.E.coli[pSB1075]cells were grown at37°C for24h in luminometer–spectrophotometer.When required,AHL solvent(ethyl acetate),AHL extracted from P.aeruginosa strain MW3A,and synthetic N-3-dodecanoyl-L-homoserine lactone(C12-HSL)(100mM,Sigma Aldrich) were added.Growth measurements and bioluminescence

were the means of triplicate experiments.Data were plotted as RLU(Relative Light Units)/OD against time.

Mass spectrometry analysis of AHLs

The Agilent RRLC1200system was used as the LC delivery system.The column used in the LC analysis was Agilent ZORBAX Rapid Resolution HT column (100mm92.1mm,1.8l m particle size).Analysis was carried out at60°C with?ow rate of0.3ml min-1.The volume injected was20l l.Mobile phases A and B were 0.1%v/v formic acid in water and0.1%v/v formic acid in acetonitrile,respectively.The gradient pro?les were as follows(time:mobile phase A:mobile phase B):0min: 60:40,5min:20:80,7and10min:5:95,and11and 13min:60:40.

The high-resolution electrospray ionization mass spec-trometry(ESI–MS)and electrospray ionization tandem mass spectrometry(ESI–MS/MS)analyses were performed with the Agilent6500Q-TOF LC/MS system.The MS experiment was performed in the ESI-positive mode.The probe capillary voltage was set at3kV,desolvation tem-perature at350°C,sheath gas at11ml h-1,and nebulizer pressure at50psi.Nitrogen gas was used as the collision gas in the collisionally induced dissociation mode for the MS/MS analysis,with collision energy set at20eV.The MS data were analysed using the Agilent MassHunter software.

Nucleotide sequence accession numbers

The16S rDNA,pvdQ,and quiP nucleotide sequences of MW3A were deposited at GenBank under the GenBank accession numbers GQ180117,GQ423677,and GQ423680, respectively.All other sequences were from the GenBank database.

Results

Enrichment and isolation of bacteria from marine water Within48h after inoculation with a marine water sample, 3-oxo-C6-HSL-containing KGm medium became turbid. The pH of the marine water sample was7.88.No obvious turbidity was visible in a control tube without3-oxo-C6-HSL.Subsequently,a bacterial isolate,designated as MW3A and grown on3-oxo-C6-HSL-containing KGm agar,was re-streaked on LBm plate.It yielded white, transparent,convex colonies with colony diameter of 5mm.Each colony was essentially round with entire edges and appeared smooth and shiny.MW3A appeared as long rod-shaped cells with cell length of approximately2.0l m and its cells were stained Gram-negative(data not shown). The ability of MW3A to degrade3-oxo-C6-HSL was ver-i?ed by RRLC analysis,which suggested that approxi-mately40%of3-oxo-C6-HSL was degraded by MW3A after24h of incubation(Fig.1).MW3A was selected for further analysis.

Web-based similarity searches against GenBank using the16S rDNA nucleotide sequence(1,479nucleotides)of MW3A indicated its similarity to the genus Pseudomonas, sharing at least99.8%sequence identities with the16S rDNA of P.aeruginosa PAO1.Further phylogenetic analysis supported the conclusion that MW3A was a spe-cies of Pseudomonas clustered closely to P.aeruginosa PAO1(Fig.2).

Degradation of various AHLs

Further studies were carried out to determine degradation of AHLs other than3-oxo-C6-HSL(Fig.1)by P.aeru-ginosa strain MW3A.RRLC analyses showed that*55 and*40%of N-hexanoyl-L-homoserine lactone(C6-HSL) (Fig.3a)and N-octanoyl-DL-homoserine lactone(C8-DL-HSL)(Fig.3b),respectively,were degraded after24h of incubation with MW3A.It seems that MW3A preferen-tially degraded unsubstituted AHLs with shorter N-acyl side chain.In all AHL-degradation analyses,no obvious degradation of AHLs was observed when the AHL-inac-tivation assays were repeated with E.coli DH5a and PBS buffer(100mM,pH6.5)(data not shown).

Molecular cloning of the pvdQ and quiP genes

PCR of the pvdQ and quiP homologue genes of MW3A yielded amplicons of*2.5kb(Fig.4).Web-based BLAST similarity searches against the GenBank using the partial nucleotide sequence of the pvdQ gene(671nucle-otides)of MW3A indicated that the nucleotide sequence was highly similar to that of a gene encoding penicillin acylase in P.aeruginosa UCBPP-PA14(Fig.5).Similarly, phylogenetic analysis of the quiP homologue gene(1,459 nucleotides)of MW3A indicated that it was highly similar to quiP of P.aeruginosa UCBPP-PA14(Fig.6).

Detection of AHLs produced by P.aeruginosa MW3A

When E.coli[pSB1075]was incubated with ethyl acetate, the AHL solvent,no bioluminescence was detected (Fig.7).However,when it was incubated with the AHL extract of P.aeruginosa MW3A,bioluminescence was detected(Fig.7),suggesting the presence of long chain AHLs that activated the expression of luxCDABE

transcriptional fusion in E.coli [pSB1075](Winson et al.1998).

Mass spectrometry analysis of AHL

Results of mass spectrometry con?rmed the presence of N -3-oxotetradecanoyl-L -homoserine lactone (3-oxo-C 14-HSL)(m/z 326.2339)(Fig.8a)and C 12-HSL (m/z 284.2221)(Fig.8b)in the spent supernatant of MW3A processed at 8-h post inoculation.The ESI–MS/MS spectrum of C 12-HSL (m/z 284.2221,Fig.8b)shows fragments (m/z 95.0880,102.0946,and 109.1025,Fig.8c)typical of an AHL-moiety (Ortori et al.2007).

Discussion

KG medium has been successfully used to select and enrich QQ bacteria from different environments (Chan et al.2009,2010).Here,we successfully used a modi?ed KG medium to enrich QQ bacteria from a sample of seawater.In KGm medium,the concentrations of all salts were increased to produce higher salinity to adjust for the osmotic pressure for halophilic bacteria in the marine environment.KGm medium was buffered to pH 6.5with MES to prevent lactonolysis of AHLs under alkaline conditions.The iso-lation of the marine P.aeruginosa strain MW3A,pos-sessing both QQ and QS properties,con?rmed that

KG

Fig.1RRLC analysis of

3-oxo-C 6-HSL after 0(blue )and 24h (red )incubation with

a MW3A,

b E.coli DH5a ,and

c PBS buffer (100mM,pH 6.5);arrow shows 3-oxo-C 6-HSL degradation by MW3A after 24h of

incubation

Fig.216S rDNA gene-based phylogenetic tree,generated using Neighbour-Joining algorithm,showing the phylogenetic position of strain MW3A;the horizontal bar at the bottom represents evolution-ary distance as 0.02change per nucleotide position,determined by measuring the lengths of the horizontal lines connecting the species;the numbers (bootstrap values as percentages of 1,000replications)provide support for the robustness of the adjacent nodes;

Burkholderia sp.strain MSMB43was used as outgroup;GenBank accession numbers (in parentheses):P.aeruginosa PAO1(AE004091[rrs 722096–723631]),P.aeruginosa PA7(CP000744[rrs 807093–808589]),P.?uorescens strain IHB B 142(GU186124),P.putida strain IHB B 1369(GU186116),and Burkholderia sp.strain MSMB43(EF114404)

medium,with or without modi?cation,was effective in selecting QQ bacteria from various environments including the marine ecosystem.

P.aeruginosa is a common bacterium found in diverse environmental conditions owing to its metabolic versatility.Consequently,it is ubiquitous in the soil and water

ecosystems including the marine ecosystem.In this study,the marine P.aeruginosa strain MW3A was shown to degrade 3-oxo-C 6-HSL,C 6-HSL,and C 8-HSL with different ef?ciencies.The ability to degrade AHLs with a C 6acyl side chain,with or without a 3-oxo substituent,is additional to the reported capability of P.aeruginosa PAO1to degrade AHLs with acyl chains of eight or more carbons.Spent supernatants of P.aeruginosa strain MW3A were assayed for AHLs at 24-h post inoculation,but neither 3-oxo-C 14-HSL nor C 12-HSL was detected by ESI–MS (data not shown).This led us to speculate that AHLs produced by P.aeruginosa strain MW3A were self-degraded.Hence,the extraction of AHLs from the supernatants was performed at 8-h post inoculation.Homologues of both pvdQ and quiP genes,showing high nucleotide sequence similarities to those of known acylase genes,were detected in P.aeruginosa strain MW3A.

Currently,relatively little information is available on the AHL-based QS systems of marine bacteria compared

to

Fig.3RRLC analysis of a C 6-HSL and b C 8-DL-HSL after 0(blue )and 24h (red )of

incubation with MW3A;a drop of peak shows degradation of signalling

molecules

Fig.4PCR-ampli?ed a pvdQ and b quiP homologue genes of MW3A (lane 2)and P.aeruginosa PAO1(lane 3);arrow shows the 2.5kb amplicons;M =1-kb DNA ladder;lane 1=negative

control

Fig.5Phylogenetic tree,generated using Neighbour-Joining algo-rithm,showing the phylogenetic position of pvdQ homologue gene of P.aeruginosa strain MW3A;the horizontal bar at the bottom represents evolutionary distance as 0.2change per nucleotide position,determined by measuring the lengths of the horizontal lines connecting the species;GenBank accession number:P .aeruginosa

LESB58pvdQ (218768969:3206318–3208606),P.aeruginosa PA7pvdQ (150958624:2955521–2957803),P.aeruginosa UCBPP-PA14pvdQ (115583796:3004988–3007276),P.aeruginosa PAO1pvdA gene (Z25465:1129155),P.aeruginosa PAO1pvdQ (110227054:2636517–2638805)

those of bacteria from the terrestrial environment,although the marine Vibrio ?scheri is the ?rst bacterium that pro-vides the evidence of an AHL-based QS mechanism (Eb-erhard 1972).From marine snow,different AHL-producing Roseobacter strains have been isolated (Gram et al.2002).Synthesis of a novel AHL molecule with a C 14N -acyl side chain and a double bond at C-7,namely 7-cis -N -(tetrade-cenoyl)-HSL,has been reported in the free-living marine bacterium Rhodobacter sphaeroides (Puskas et al.1997).A marine Mesorhizobium sp.that produces structurally novel AHLs,i.e.,5-cis -3-oxo-C 12-homoserine lactone (5-cis -3-oxo-C 12-HSL)and 5-cis -C 12-HSL,has been reported (Krick et al.2007).In addition,a diverse range of AHLs (with N -acyl side chains from C 4to C 14)detected in Pro-teobacteria associated with marine sponge are mostly

produced by a -proteobacterial isolates mainly from the Silicibacter –Ruegeria subgroup of the Roseobacter clade (Mohamed et al.2008).

Recently,Romero et al.(2011)reported that QQ is a common feature among cultivable bacteria isolated from dense marine bacterial communities.Fifteen isolates,capable of inactivating AHLs,were characterized and they belong to nine different genera.However,none of them belong to the genus Pseudomonas .

The presence of P.aeruginosa strain MW3A in the marine environment may be important in inactivating AHLs secreted in marine waters,thereby controlling AHL-regulated phenotypes in marine QS bacteria such as Vibrio sp.,Roseobacter sp.,and Mesorhizobium sp.(Eberhard 1972;Gram et al.2002;Krick et al.2007;Mohamed et al.2008).This QQ activity may have an impact on aquatic ?sh and human opportunistic pathogens such as Aeromonas spp.(Chan et al.2011b ;Swift et al.1996).Since the supernatant incubated for 24h showed no detectable AHLs as compared to that incubated for 8h,this led us to spec-ulate that the QQ activity of P.aeruginosa strain MW3A may be important in the self-perception and regulation of the AHLs it produces.Once the QS-mediated phenotypes have been expressed,the AHLs that it produces are degraded.

Two major AHLs produced by P.aeruginosa strain MW3A were characterized by mass spectrometry.The data obtained are interesting because P.aeruginosa has been reported to produce two major AHLs,namely 3-oxo-C 12-HSL and N -butanoylhomoserine lactone (C 4-HSL)(Lati?et al.1996;Schuster and Greenberg 2006),and two minor AHLs,C 6-HSL and 3-oxo-C 6-HSL (Rumbaugha et al.2000;Winson et al.1995).In contrast,P.aeruginosa strain MW3A was shown to produce 3-oxo-C 14-HSL

and

Fig.6Phylogenetic tree generated using Neighbour-Joining algo-rithm,showing the phylogenetic position of quiP homologue gene of P.aeruginosa strain MW3A;the horizontal bar at the bottom represents evolutionary distance as 0.1change per nucleotide position,determined by measuring the lengths of the horizontal lines connecting the species;GenBank accession number:P.aeruginosa UCBPP-PA14quiP (115583796:4527711–4530254),P.aeruginosa

LESB58quiP (218768969:4725132–4727675),P.aeruginosa PA7(150958624:4478165–4480708),Pseudomonas syringae pv.syringae B728a (63253978:4609176–4611650),P.putida KT2440(24987239:1265544–1267985);P.?uorescens Pf-5(68342549:1438348–1440777),P.aeruginosa PAO1quiP (110227054:1119674–1122217),Pseudomo-nas mendocina ymp

(145573243:1577468–1579981)

Fig.7Bioluminescence and OD were measured throughout growth for 24h at 37°C in the presence of C 12-HSL (diamond )(positive control),ethyl acetate (square )(negative control),and AHL extracted from P.aeruginosa strain MW3A (circle );in all experiments,RLU and OD were determined as described in Sect.‘‘Materials and methods ’’;the graphs represent assays of two independent experi-ments,each with triplicate sets of similar results;RLU,relative light units

C 12-HSL,which have not been reported previously.No C 4-HSL was detected in this study and this may be because AHLs were extracted at 8-h post inoculation when hardly any or very little C 4-HSL was produced.In addition to the AHL-based QS system,P.aeruginosa is known to produce 2-alkyl-4-quinolone signal molecules called Pseudomonas quinolone signal (PQS;2-heptyl-3-hydroxy-4-quinolone)that functions as a link between the las and rhl QS systems (Pesci et al.1999).Indeed,we have detected PQS from P .aeruginosa strain MW3A (data not shown).

Further investigation is required to clarify the role of P .aeruginosa strain MW3A,possessing both QS and QQ properties,in the marine environment.

Acknowledgments This work was supported by a High Impact Research Grant (J-00000-73552)and the Malaysia Ministry of Sci-ence,Technology and Innovation under the Science Fund (12-02-03-2085)to KG Chan and a Postgraduate Research Grant (PS256/2008A)to CS Wong from the University of Malaya.

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′mara M,Koh CL,Williams P (2011a)Characterization of N -acylhomo-serine lactone-degrading bacteria associated with the Zingiber of?cinale (ginger)rhizosphere:co-existence of quorum-quench-ing and quorum-sensing in Acinetobacter and Burkholderia .BMC Microbiol

11:51

Fig.8Mass spectrometric analysis of AHLs:a ESI–MS spectrum of 3-oxo-C 14-HSL (326.2339,8.33min),b ESI–MS spectrum of C 12-HSL (284.2221,6.90min),and c ESI–MS/MS spectrum of C 12-HSL (284.2221,6.90min)

Chan KG,Puthucheary SD,Chan XY,Yin WF,Wong CS,See Too WS,Chua KH(2011b)Quorum-sensing in Aeromonas species isolated from patients in Malaysia.Curr Microbiol62:167–172 Chhabra SR,Stead P,Bainton NJ,Salmond GPC,Stewart GSAB, Williams P,Bycroft BW(1993)Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homoserine lactone.J Antibiot46:441–454 Chhabra SR,Philipp B,Eberl L,Givskov M,Williams P,Camara M (2005)Extracellular communication in bacteria.Top Curr Chem 240:279–315

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关于时间管理的英语作文 manage time

How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.

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脐带间充质干细胞的研究进展 间充质干细胞（mesenchymal stem cells,MSC S ）是来源于发育早期中胚层 的一类多能干细胞[1-5]，MSC S 由于它的自我更新和多项分化潜能，而具有巨大的 治疗价值 ,日益受到关注。MSC S 有以下特点：(1)多向分化潜能，在适当的诱导条件下可分化为肌细胞[2]、成骨细胞[3、4]、脂肪细胞、神经细胞[9]、肝细胞[6]、心肌细胞[10]和表皮细胞[11, 12]；(2)通过分泌可溶性因子和转分化促进创面愈合；(3) 免疫调控功能，骨髓源（bone marrow ）MSC S 表达MHC-I类分子，不表达MHC-II 类分子，不表达CD80、CD86、CD40等协同刺激分子，体外抑制混合淋巴细胞反应，体内诱导免疫耐受[11, 15]，在预防和治疗移植物抗宿主病、诱导器官移植免疫耐受等领域有较好的应用前景；(4)连续传代培养和冷冻保存后仍具有多向分化潜能，可作为理想的种子细胞用于组织工程和细胞替代治疗。1974年Friedenstein [16] 首先证明了骨髓中存在MSC S ，以后的研究证明MSC S 不仅存在于骨髓中，也存在 于其他一些组织与器官的间质中：如外周血[17]，脐血[5]，松质骨[1, 18]，脂肪组织[1]，滑膜[18]和脐带。在所有这些来源中，脐血(umbilical cord blood)和脐带(umbilical cord)是MSC S 最理想的来源，因为它们可以通过非侵入性手段容易获 得，并且病毒污染的风险低，还可冷冻保存后行自体移植。然而，脐血MSC的培养成功率不高[19, 23-24]，Shetty 的研究认为只有6%，而脐带MSC的培养成功率可 达100%[25]。另外从脐血中分离MSC S ，就浪费了其中的造血干/祖细胞(hematopoietic stem cells/hematopoietic progenitor cells,HSCs/HPCs) [26, 27]，因此，脐带MSC S (umbilical cord mesenchymal stem cells, UC-MSC S )就成 为重要来源。 一．概述 人脐带约40 g, 它的长度约60–65 cm, 足月脐带的平均直径约1.5 cm[28, 29]。脐带被覆着鳞状上皮，叫脐带上皮，是单层或复层结构，这层上皮由羊膜延续过来[30, 31]。脐带的内部是两根动脉和一根静脉，血管之间是粘液样的结缔组织，叫做沃顿胶质，充当血管外膜的功能。脐带中无毛细血管和淋巴系统。沃顿胶质的网状系统是糖蛋白微纤维和胶原纤维。沃顿胶质中最多的葡萄糖胺聚糖是透明质酸，它是包绕在成纤维样细胞和胶原纤维周围的并维持脐带形状的水合凝胶，使脐带免受挤压。沃顿胶质的基质细胞是成纤维样细胞[32]，这种中间丝蛋白表达于间充质来源的细胞如成纤维细胞的，而不表达于平滑肌细胞。共表达波形蛋白和索蛋白提示这些细胞本质上肌纤维母细胞。 脐带基质细胞也是一种具有多能干细胞特点的细胞，具有多项分化潜能，其 形态和生物学特点与骨髓源性MSC S 相似[5, 20, 21, 38, 46]，但脐带MSC S 更原始，是介 于成体干细胞和胚胎干细胞之间的一种干细胞，表达Oct-4, Sox-2和Nanog等多

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英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn

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