The role of exopolysaccharides in Pseudomonas aeruginosa ......2011/06/10 · 5 biofilm matrix (1,...

1

The role of exopolysaccharides in Pseudomonas aeruginosa 1

biofilm formation and architecture 2

3

Aamir Ghafoor, Iain D. Hay, Bernd H.A. Rehm* 4

Institute of Molecular Biosciences, Massey University, Private Bag 11222, 5

Palmerston North, New Zealand. 6

7

8

9

Short title: Exopolysaccharides and biofilm formation 10

11

12

*Corresponding author: [email protected] 13

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00637-11 AEM Accepts, published online ahead of print on 10 June 2011

on Septem

ber 19, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

http://aem.asm.org/

2

Abstract 1

Pseudomonas aeruginosa is an opportunistic human pathogen and has been 2

established as a model organism to study bacterial biofilm formation. At least three 3

exopolysaccharides (alginate, psl and pel) contribute to the formation of biofilms in 4

this organism. Here mutants deficient in the production of one or more of these 5

polysaccharides were generated to investigate how these polymers interactively 6

contribute to bioflm formation. Confocal laser scanning microscopy of biofilms 7

formed in flow chambers showed that mutants deficient in alginate biosynthesis 8

developed biofilms with a decreased proportion of viable cells when compared to 9

alginate producing strains, indicating a role of alginate in viability of cells in biofilms. 10

Alginate deficient mutants showed enhanced eDNA containing surface structures 11

impacting on biofilm architecture. PAO1∆pslA∆alg8 overproduced pel and eDNA 12

showing meshwork-like structures presumably based on an interaction between both 13

polymers were observed. The formation of characteristic mushroom-like structures 14

required both Psl and alginate whereas pel appeared to play a role in biofilm cell 15

density and/or compactness of the biofilm. Mutants only producing alginate, i.e. 16

deficient in both psl and pel production, lost their ability to form biofilms. Lack of psl, 17

enhanced the production of pel, and absence of pel enhanced production of alginate. 18

The function of psl in attachment was independent of alginate and pel. A 30% 19

decrease in psl promoter activity in the alginate overproducing MucA-negative mutant 20

PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this 21

study demonstrated that the various exopolysaccharides and eDNA interactively 22

contribute to the biofilm architecture of P. aeruginosa. 23

24

25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

3

Introduction 1

Pseudomonas aeruginosa has been widely studied as model organism for 2

biofilm formation. P. aeruginosa is an opportunistic human pathogen which causes 3

infection in burn wounds and in the lungs of patients suffering from the genetic 4

disease called cystic fibrosis (CF). The ability to form biofilms is critical for its 5

survival in the CF lung environment and enhances its resistance to antimicrobial 6

treatment and host defence mechanisms (9). P. aeruginosa biofilms are also common 7

on medical devices such as contact lenses and catheters (20). At least three 8

exopolysaccharides have been shown to be produced by P. aeruginosa: alginate, psl 9

and pel (27). Each of these exopolysaccharides has been found to be involved in 10

biofilm formation (27). 11

Alginates, which are overproduced by P. aeruginosa after infection of CF 12

patients, are linear polyanionic exopolysaccharides composed of uronic acids (25). 13

Alginate overproducing mutants form large finger like microcolonies when compared 14

to wildtype strains (7). Alginate has been shown to contribute to decreased 15

susceptibility of biofilms to antibiotic treatment and human antibacterial defence 16

mechanisms (19, 23). 17

The psl polysaccharide is rich in mannose and galactose and is involved in 18

initial attachment and mature biofilm formation (12). Psl is produced during 19

planktonic growth mediating attachment to surfaces and contributing to microcolony 20

formation. In mature biofilm psl is associated with the caps of mushroom like 21

microcolonies, forming a peripheral meshwork covering the cap region (12, 13). 22

Pel is a glucose-rich, cellulose-like polymer essential for the formation of a 23

pellicle at the air-liquid interface (5). Increased pel production has also been 24

associated with the wrinkled colony phenotype (6). Recently it has been shown that 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

4

pel plays a role in cell-to-cell interactions in P. aeruginosa PA14 biofilms providing a 1

structural scaffold for the community at early stages of biofilm formation (4). 2

Besides the important role of the exopolysaccharides in biofilm formation, 3

extracellular DNA (eDNA) has been shown to be an important component of the 4

biofilm matrix (1, 15). eDNA was found to mediate cell-cell interactions in biofilms 5

(1). Moreover, it was shown that removal of eDNA by DNase treatment at initial 6

stages of biofilm formation interfered with maturation of the biofilm. However, in 7

mature biofilms such DNase treatment showed little impact on biofilm architecture 8

(34). eDNA was found to be mostly present in the stalk region of microcolonies (1). A 9

recent study has suggested that psl and eDNA are spatially separated from each other 10

with psl present at the periphery of the biofilm and eDNA mostly in the psl free 11

biofilm matrix (12). 12

This study aims to shed light on how these exopolysaccharides and eDNA 13

contribute to biofilm formation and architecture particularly in view of synergistic 14

effects between the different exopolysaccharides 15

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

5

Materials and Methods 1

Bacterial strains and growth conditions. The bacterial strains, plasmids, and 2

oligonucleotides used in this study are listed in Table1. LB medium was used to grow 3

all Escherichia coli strains at 37°C. When needed, antibiotics were added to the media 4

at the following concentrations: ampicillin, 75 µg/ml; and gentamicin, 10 µg/ml. P. 5

aeruginosa PAO1 and PDO300 (14) and their isogenic mutants were grown in LB or 6

PI(A) medium (Pseudomonas isolation [agar] medium: 20 g of peptone, 10 g of 7

K2SO4, 1.4 g MgCl2. 6H2O, 25 mg of Triclosan, and 20 ml of glycerol per liter) at 8

37°C and, if required, gentamicin and carbenicillin were added at a concentration of 9

100-300 µg/ml and 300 µg/ml, respectively. 10

Isolation, analysis, and manipulation of DNA. General cloning procedures 11

were performed as described previously (28). DNA primers, deoxynucleoside 12

triphosphate, Taq, and Platinum Pfx polymerases were purchased from Life 13

Technologies (Auckland, NZ). DNA sequences of new plasmid constructs were 14

confirmed by DNA sequencing according to the chain termination method using an 15

ABI310 automatic sequencer. 16

Construction of single, double and triple deletion mutants. Two regions of 17

the pelF gene were amplified by using Pfx polymerase with primers PelF-N1, PelF-18

N2, PelF-C1, and PelF-C2. Both PCR products were hydrolyzed with BamHI, ligated 19

together, and inserted into vector pGEM-TEasy from Promega (Sydney, Australia.) 20

resulting in pGEM-TEasy: PelF-NC. A 1,100-bp fragment, containing the aacC1 gene 21

(encoding gentamicin acetyltransferase) flanked by two Flp recombinase target sites, 22

was released when vector pPS856 (8) was hydrolyzed with BamHI. The 1,100-bp 23

BamHI fragment (aacC1 gene) was inserted into the BamHI site of plasmid pGEM-24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

6

TEasy:PelF-NC, resulting in plasmid pGEM-TEasy:∆pelFGm. A 1,949-bp ∆pelFGm 1

comprising DNA fragment was amplified by Pfx polymerase using primers PelF-2

N1and PelF-C2, and the corresponding PCR product was inserted into SmaI site of 3

vector pEX100T (8, 30), resulting in plasmid pEX100T∆pelFGm. Knock out plasmid 4

was transferred into PAO1 via electroporation as previously described (3). 5

Transformants were selected on LB medium containing 100 µg of gentamicin/ml and 6

subsequently plated on Mineral Salt medium containing 300 µg of gentamicin/ml and 7

5% (wt/vol) sucrose. Those cells able to grow on this selective medium will have 8

emerged from double-crossover events. Gene replacement was confirmed via PCR 9

with primers PelF XUP and PelF XDN. E. coli S17-1 was used to transfer the Flp 10

recombinase-encoding vector pFLP2 (8) into P. aeruginosa PAO1∆pelFΩGm. 11

Transferring of plasmid, pFLP2 into recipient cells was confirmed by selecting 12

carbenicilllin resisting cells on PIA medium containing carbenicillin (300mg/ml). 13

These carbenicillin resistant bacterial cells were cultivated on PIA medium containing 14

5% (wt/vol) sucrose and cells grown on this medium were analyzed by observing their 15

sensitivity to gentamicin and carbenicillin. PCR with primers PelF XUP and PelF 16

XDN was done to confirm the loss of the gentamicin resistance cassette. 17

Consequently P. aeruginosa PAO1∆pelF and PDO300∆pelF were generated. 18

Accordingly, plasmids pEX100T∆alg8Gm (26) and pEX100T∆pslAGm (21) were 19

used for the disruption of the alg8 and/or pslA genes in P. aeruginosa PAO1. To 20

generate psl- and alginate-negative double mutants, single mutants PAO1∆pslA and 21

PDO300∆alg8 were transformed with pEX100T∆PslAGm and pEX100T∆alg8Gm, 22

respectively, and resultant markerless PAO1∆pslAalg8 and PDO300∆pslAalg8 were 23

generated as described above. To generate the respective pel deficient double and 24

triple mutants pEX100T∆pelFGm was transferred into markerless single and double 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

7

mutants PAO1∆alg8, PAO1∆pslA (21) , PDO300∆alg8 (26), PDO300∆pslA, 1

PAO1∆pslAalg8 and PDO300∆pslAalg8, respectively. The Gm cassette was removed 2

to obtain PAO1∆alg8∆pelF, PAO1∆pslA∆pelF, PDO300∆alg8∆pelF, 3

PDO300∆pslA∆pelF, PAO1∆pslAalg8∆pelF and PDO300∆pslAalg8∆pelF, 4

respectively. All mutants were stored at -80°C for future use. 5

Complementation of isogenic pelF deletion mutant. For complementation 6

of the isogenic knockout mutant P. aeruginosa PAO1∆pslA∆pelF, pBBR1-7

MCS5:pelF was constructed. The gene pelF was amplified by PCR using the primers 8

PelFEcRfor and PelFClaRev together with chromosomal DNA from P. aeruginosa 9

PAO1. The PCR product was inserted into vector pGEM-TEasy. SalI, one of the 10

multiple cloning sites provided in vector pGEM-TEasy was available upstream of the 11

gene cloned into vector pGEM-TEasy. Thus pelF gene was hydrolyzed with SalI and 12

ClaI (for which a restriction site was available in the downstream primer), and 13

inserted into the broad-host-range vector pBBR1-MCS5 hydrolyzed by SalI and ClaI. 14

In the resulting plasmid, pBBR1-MCS5:pelF, the pelF gene was arranged linear to 15

and downstream of the lac promoter. The donor strain E. coli S17-1, harboring 16

pBBR1-MCS5:pelF was allowed to conjugate with PAO1∆pslA∆pelF. Recipient 17

PAO1∆pslA∆pelF cells carrying pBBR1-MCS5:pelF were isolated by selection on 18

PIA medium containing gentamicin (300µg/ml). To confirm the successful transfer of 19

plasmid from donor to recipient strain, plasmids were isolated and digested with 20

respective restriction enzymes. 21

Construction of the broad-host-range promoter-probe vectors 22

pTZ110::Ppel. The broad-host-range promoter-probe vectors pTZ110::Ppel, was 23

constructed as follows. The primers PpelEcRfor and PpelBaRev (Table S1) and 24

genomic DNA from P. aeruginosa PAO1 were used to amplify 1,000-bp upstream 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

8

region of pelA comprising the putative promoter region of the pel gene operon. The 1

PCR product was inserted into vector pGEM-TEasy and transformed into E. coli 2

Top10 cells. Hydrolysis with EcoRI (for which a site was present in the upstream 3

primer) and BamHI (for which a site was present in the downstream primer) yielded a 4

1000-bp putative promoter region Ppel which was subsequently cloned into the 5

corresponding sites of pTZ110. In this plasmid the promoter region was upstream of 6

lacZ. This plasmid was transferred to P. aeruginosa strains through conjugation via 7

E. coli S17-1 and transconjugants were selected for on LB agar containing 300 µg/ml 8

carbenicillin. 9

β-Galactosidase assay. β-Galactosidase activity was measured as described 10

by Miller (17) and is expressed in Miller units (MU). The data presented below are 11

the results obtained from three independent experiments. The variance is indicated by 12

error bars in the figures. 13

Pellicle formation assay. Pellicle formation was assessed as previously 14

described (5). Briefly, borosilicate glass tubes (18mm x 150 mm) containing 6 ml of 15

PI Medium (broth) were inoculated with each mutant. Tubes were incubated without 16

shaking at 37ºC. Pellicles formation at air–liquid interface after 4 days was assayed by 17

visual inspection 18

Congo red binding assay. The assay to assess the ability of mutants to 19

produce Pel polysaccharide was adapted from Spiers et al. (31). Each mutant was 20

incubated in 2 ml of PI medium for 48 h at 37°C without shaking. The bacterial 21

content along with polysaccharides produced by bacterial cells was collected by 22

centrifugation, and supernatant was discarded. The pellet was washed with PI medium 23

and transferred into 2ml microfuge tubes. Pellet was re-suspended in 1 ml of 20 mg 24

ml-1

Congo red in PI medium and incubated for 90 minutes while shaking. Bacterial 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

9

content and bound Congo red were sedimented by centrifuging at 15870 x g for 5 1

minutes. The supernatant was collected and the OD at 490 nm was noted. The total 2

Congo red percentage left in supernatant was measured. 3

Solid surface attachment assay. Attachment to a solid surface was assessed 4

as previously described (16) and with some modifications. In brief, pertinent strains 5

were grown overnight in PI broth medium and OD at 600nm wavelength was 6

measured. An appropriate amount of overnight culture was added into PI broth to 7

obtain 1:100 dilutions. Eight wells of a of 96 well plate was inoculated with 100 µL 8

of diluted culture of a particular strain and incubated at 37°C for 2 -96 hours. In order 9

to remove planktonic/non-adherent bacteria at the end of each incubation time plates 10

were washed either using vigorous or gentle washing procedure as previously 11

described (7) 12

DNase treatment of biofilms. P. aeruginosa PAO1∆pslA∆alg8 and P. 13

aeruginosa PAO1∆pelF∆alg8 were grown in flow cells for 96 hours. DNaseI from 14

Sigma (St. Louis, MO) was dissolved in medium at a concentration of 500 µg/ml and 15

injected in to the flow cell. This was incubated for 30 minutes without flow. Flow was 16

restored to 0.3 ml / min for 15 minutes after which DNase treated biofilm were 17

stained as described below. 18

Continuous-culture flow cell biofilms. For biofilm analysis, each mutant was 19

grown in continuous-culture flow cells for 4 days at 37°C as previously described (2). 20

The flow cells used in this study had dimensions of 4 mm by 40 mm by 1.5 mm. Each 21

channel was filled with PI medium and inoculated with a total of 0.5 ml overnight 22

culture of the respective mutant, containing approximately 2 × 109 cells per ml and 23

incubated without flow for 4 h at 37°C. PI medium was then allowed to flow at a 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

10

mean flow of 0.3 ml min−1

, corresponding to a laminar flow with a Reynolds number 1

of 5. The flow cells were then incubated at 37°C for 96 h. Biofilms were stained using 2

the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Inc., Eugene, 3

OR) and visualized using confocal laser scanning microscopy (Leica SP5 DM6000B). 4

Images of P. aeruginosa and all of its mutants were captured using 63x objective 5

lens. Images were analyzed using IMARIS (Bitplane, Inc) software. 6

Quantitative analysis of biofilms using IMARIS image analysis software 7

(Bitplane, Inc). Biofilm appearance, biofilm volume, dead to live ratio, thickness, and 8

compactness of biofilms were the parameter used to compare architectural differences 9

of biofilms formed by the various mutants. To obtain volume per unit area (µm³/µm²) 10

a ratio between total volume and total area covered by biofilm was calculated. The 11

compactness of the biofilm was assessed as total fluorescence per volume of biofilm. 12

To obtain the ratio between the number of dead cells to living cells per biofilm 13

volume the ratio between red and green fluorescence was calculated. To obtain the 14

roughness of the biofilm, the surface area of all biofilms was calculated. It is known 15

that increased projections and pits on a surface increase the area of a surface. A ratio 16

between surface area and flat area on which biofilm was grown was calculated and 17

used as indicator of the roughness of the biofilm. In each biofilm, thickness of the 18

base and microcolonies was measured separately and standard deviations were 19

calculated. 20

21

22

RESULTS 23

Generation of isogenic pelF deletion mutants deficient in pel 24

polysaccharide production. Deletion of the pelF gene in PAO1 resulted in a pel 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

11

deficient phenotype. Pellicle formation at the air-liquid interphase was absent in the 1

∆pelF mutants PAO1∆pelF, PAO1∆pelF∆alg8, PAO1∆pslA∆pelF and 2

PAO1∆pslA∆pelF∆alg8 (Fig.1A). The Congo red binding assay also showed that pel 3

deficient mutants bind less Congo red leaving most of it in the cell-free supernatant, 4

whereas pel polysaccharide producing mutants PAO1, PAO1∆alg8 PAO1∆pslA, 5

PAO1∆pslA∆alg8 showed increased binding of Congo red leaving less Congo red in 6

the cell-free supernatant (Fig. 1B). 7

Similar Congo red staining results were shown for pel deficient mutant 8

PDO300∆pelF, PDO300∆alg8∆pelF, PDO300∆pslA∆pelF, 9

PDO300∆pslA∆alg8∆pelF (data not shown). 10

Complementation of the ∆pelF deletion mutant. To confirm that disruption 11

of the pelF gene had no polar effect on genes downstream of pelF within the pel 12

operon, a plasmid pBBR1MCS-5:pelF was constructed and transformed into P. 13

aeruginosa PAO1∆pslA∆pelF. Since the lack of pel production of the pelF mutant 14

was particular obvious in the psl negative background the double mutant 15

PAO1∆pslA∆pelF was used for complementation experiments. The mutant 16

PAO1∆pslA∆pelF harboring plasmid pBBR1MCS-5:PelF was restored in its ability 17

to produce pel which was shown by the formation of a pellicle at air-liquid interphase 18

and increased Congo red binding. (Fig. 1B) 19

Generation of various isogenic deletion mutants deficient in the 20

production of various exopolysaccharides. To study the synergistic or antagonistic 21

effects of the three polysaccharides on biofilm formation, single, double and triple 22

mutants of strains PAO1 and PDO300 were generated. Previously, a PAO1∆pslA 23

mutant was generated and characterized and was shown to be deficient in attachment 24

to solid surface at early stage of biofilm formation (21). PAO1∆pslA was used to 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

12

generate psl/alginate, psl/pel and psl/alginate/pel deficient mutants. The resultant 1

mutants PAO1∆pslA∆alg8, PAO1∆pslA∆pelF and PAO1∆pslA∆alg8∆pelF showed 2

impaired attachment in the SSA (Fig. 2A). PDO300∆pslA, PDO300∆pslA∆alg8, 3

PDO300∆pslA∆pelF and PDO300∆pslA∆alg8∆pelF generated in this study also 4

showed impaired solid surface attachment (Fig. S1). 5

PDO300∆alg8 has already been previously characterized as deficient in 6

alginate production (26). All alg8 knock out mutants of PDO300, 7

PDO300∆pslA∆alg8, PDO300∆alg8∆pelF and PDO300∆pslA∆alg8∆pelF were non 8

mucoid when grown on PIA plates and showed no alginate production when assessed 9

for alginate quantification (Table S2). Isogenic mutants PAO1∆alg8, 10

PAO1∆pslA∆alg8, PAO1∆alg8∆pelF and PAO1∆pslA∆alg8∆pelF were generated as 11

outlined in Supplemental Material (Table S1). An overview of all strains used and 12

generated in this study, their exopolysaccharide biosynthesis relevant genotype and 13

phenotype is presented in Table 1. 14

Effect of anti-sigma factor MucA on transcription of the pel and psl 15

operon. All the mutants in this study were derivatives of PAO1 and its isogenic 16

mucA negative mutant PDO300. MucA is a membrane anchored anti-sigma factor 17

which sequesters AlgU, an alternative sigma factor required for transcription of the 18

various genes including those in the alginate operon (29). To assess the impact of this 19

mucA mutation in the alginate overproducing PDO300 on the expression of the psl 20

and pel biosynthesis genes, the levels of transcription from the psl and pel promoters 21

were analysed in the PAO1 strain and its mucA negative derivative PDO300. To 22

analyse the activity of the psl and pel promoter, respectively, plasmids pTZ110::Ppel 23

and pTZ110::Ppsl (21) were introduced into PAO1 and PDO300, respectively. The 24

measured β-galactosidase activity was used to deduce the respective promoter 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

13

activity. The pel promoter in PAO1 and PDO300 mediated a β-galactosidase activity 1

of 683 ±158 and 564 ±68 Miller units, respectively. The psl promoter in PAO1 and 2

PDO300 mediated a β-galactosidase activity of 2704 ±477 and 1923 ±183 Miller 3

units, respectively. 4

Analysis of the role of the various exopolysaccharides with respect to 5

attachment to solid surfaces. SSA was conducted to observe the attachment of cells 6

at initial and mature stages of biofilm development. After 2, 4 and 6 hours of 7

incubation the strains producing psl (PAO1, PAO1∆pelF, PAO1∆alg8, 8

PAO1∆pelF∆alg8) showed increased attachment when compared to psl negative 9

mutants. After 4 days of growth, psl negative, pel producing,mutants PAO1∆pslA 10

and PAO1∆pslA∆alg8 showed increased attachment when compared to pel deficient 11

mutants. Results are summarized in Figure 2. Similar results were found for PDO300 12

and derived isogenic mutants (Supplemental material, Fig. S1). 13

Analysis of biofilms formed by mutants deficient in the production of one 14

or more exopolysaccharides. Representative biofilm images of respective 15

exopolysaccharide negative mutants of P. aeruginosa were obtained using CLSM 16

(Figs. 3; S2 and S3 (supplemental material]). The CLSM stack images were 17

reconstructed into 3D images using IMARIS software. The results showed that 18

PAO1∆alg8 was not able to form mushroom-like structures. Mutants including 19

PAO1∆pelF and PAO1∆pelF∆alg8 capable of producing psl and alginate or only psl, 20

respectively, formed mushroom-like structures. Interestingly, PAO1∆pelF, which 21

produces psl and alginate formed mushroom-like structures with higher density of 22

living cells when compared to PAO1∆pelF∆alg8 biofilms which additionally lacked 23

alginate and formed mushroom-like structures with the caps almost devoid of living 24

cells (Fig. 3 C & E). PAO1∆pslA and PAO1∆pslA∆alg8 were still able to form 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

14

biofilms but these biofilms were flat and much more compact than biofilms formed by 1

all other studied mutants as well as both live and dead cells were present in these 2

biofilms (Fig. 3 D & F.). Interestingly, it was observed that PAO1∆pslA∆pelF and 3

PAO1∆pslA∆pelF∆alg8 were not able to form any biofilm after 96 hours and only a 4

layer of single cells was observed (Fig. S2). Similar results as obtained for the various 5

PAO1 mutants were obtained when analyzing the biofilms of the respective PDO300 6

mutants (Figure S3 A-G) except for PDO300∆pelF which could not be assessed due 7

to the excessive formation of exopolymeric matrix material and the subsequent 8

blocking of the flow cell. The levels of alginate produced by this strains were found to 9

be about 10-fold increased when compared with parent strain PDO300 (Supplemental 10

material, Table S2). Although mutant PDO300∆pelF∆pslA showed a similar 11

overproduction of alginate, biofilms could still be grown in flow cells without 12

blockage. 13

DNase treatment of mutants PAO1∆pslA∆alg8 and PAO1∆pelF∆alg8. The 14

biofilms of the PAO1∆pslA∆alg8 and PAO1∆pelF∆alg8 strains showed increased 15

levels of red fluorescence indicating dead cells and eDNA. To assess the role of 16

eDNA in biofilms, DNase treatment was conducted. This resulted in almost complete 17

removal of red fluorescent structures (Fig. 4 A-D) as was indicated by a decrease of 18

the red fluorescence to green fluorescence ratio from 1.08 to 0.45 in case of 19

PAO1∆pslA∆alg8 and 0.8 to 0.2 in case of PAO1∆pelF∆alg8. 20

Quantitative analysis of biofilms. The biofilm volume, dead to live ratio, 21

thickness, and compactness of biofilms were assessed using IMARIS software. To 22

obtain the volume per area ratio the total volume (x×y×z=µm3) and total area 23

(x×y=µm2) covered by each biofilm was measured. The highest volume to area ratio 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

15

was found for PAO1 and PAO1∆pslA∆alg8 biofilms. The PAO1∆alg8 has showed 1

lowest volume to area ratio (Table 2). 2

The biofilms formed by PAO1∆pslA, PAO1∆pslA∆alg8 and PAO1∆alg8, 3

respectively, were found to be more compact when compared to the other biofilms. 4

Intensity of fluorescence per unit biofilm volume was used to compare the 5

compactness of biofilms. All biofilms formed by pel producing PAO1, PAO1∆alg8, 6

PAO1∆pslA, and PAO1∆pslA∆alg8 showed 1.83x103, 1.43x10

3, 3.75 x10

3 and 7

1.79x103 relative light intensity per µm

3, respectively, which is greater than compared 8

to biofilms formed by pel deficient mutants PAO1∆pelF and PAO1∆pelF∆alg8 9

showing 1.07x103

and 8.82x102 relative light intensity per µm

3, respectively . The 10

ratio of dead cells to live cells in the various biofilms showed that in the absence of 11

alginate biofilms contain more dead cells and extracellular DNA. Dead to live cell 12

ratios in biofilms produced by alginate negative mutants PAO1∆alg8, 13

PAO1∆pslA∆alg8 and PAO1∆pelF∆alg8 were higher, than alginate producing PAO1, 14

PAO1∆psl and, PAO1∆pelF biofilms (Table 2). 15

Biofilms formed by all strains contained a base layer of various heights. Psl 16

producing PAO1, PAO1∆pelF and PAO1∆pelF∆alg8 formed structured biofilms 17

having base layer one-fourth to one-fifth the total height of the microcolony and the 18

microcolony could be clearly differentiated into stalk and cap structure. On the other 19

hand psl deficient mutant PAO1∆pslA and PAO1∆pslA∆alg8 formed unstructured flat 20

biofilms where the only discernible features were short dome like structures. 21

Interestingly, PAO1∆alg8 which produces psl and pel showed an unstructured flat 22

biofilm similar to pel overproducing, psl deficient mutants. (Fig. 5). 23

24

25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

16

Discussion 1

In this study the role of single exopolysaccharides and various combinations of 2

exopolysaccharides in biofilm formation by P. aeruginosa was investigated. Three 3

exopolysaccharides, psl, pel and alginate have been described to be produced by P. 4

aeruginosa as well as have been found to play a role in biofilm formation and biofilm 5

architecture (24). To elucidate the role of each exopolysaccharide, key biosynthesis 6

genes required for the production of the respective exopolysaccharide were disrupted 7

independently and in various combinations. Hence, isogenic deletion mutants of 8

strains PAO1 and its mucA negative derivative PDO300 deficient in the production of 9

one or more exopolysaccharides were generated for comparative analysis. 10

Previous work had established that knocking out alg8 from P. aeruginosa 11

results in the complete loss of alginate production as shown by the lack of alginate 12

and uronic acids in culture supernatants (26). Similarly, deletion of pslA had been 13

shown to abolish psl production which impaired surface attachment (21). Hence 14

mutants deficient in alginate and/or psl were generated in this study by knocking out 15

alg8 and pslA, respectively. Previously, it was shown that a third exopolysaccharide, 16

pel, is produced by P. aeruginosa and was found to be important for the formation of 17

static biofilms by various strains (5). Seven genes contained in a single operon (pelA-18

G) are required for production of the pel exopolysaccharide (5, 33). The pelF 19

sequence analysis suggested that it encodes for a glycosyltransferase, presumably 20

involved in the polymerization of pel exopolysaccharide(5). Deletion of pelF from the 21

genome P. aeruginosa PAK significantly reduced biofilm formation when compared 22

to the parent strain (33). Inability of mutants to produce pel had been characterized by 23

lack of pellicle formation at air-liquid interphase and reduced Congo red binding (5, 24

11). Here, pelF was deleted from PAO1 and PDO300 in order to generate isogenic 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

17

mutants deficient in pel exopolysaccharide biosynthesis. All pelF mutants showed 1

reduced Congo red binding and the absence of pellicle formation. Although the 2

specificity of this Congo red binding assay has not been established, it has been 3

widely used as a indicator of pel production (5, 32, 33). Our results showed that all 4

mutants deficient in pellicle formation at air liquid-interphase also showed reduced 5

Congo red binding (Fig. 1). 6

Previous studies showed that psl is involved in initial attachment of cells (2, 6, 7

10, 21) whereas pel plays a role in the later stages of biofilm maturity (6). Our results 8

were consistent with these findings. Mutants, deficient of psl, showed reduced crystal 9

violet staining of static biofilms at early stage of biofilm development. Furthermore, 10

neither alginate nor pel appeared to compensate this deficiency (Fig. 2 A-B). However 11

in mature biofilms, increased crystal violet staining of the pel overproducing mutant 12

PAO1∆pslA∆alg8 suggested that pel at later stages of biofilm development mediates 13

entrapment of more cells (Fig. 2). Since pel has been shown to contribute to cell-to-14

cell interactions in P. aeruginosa PA14 (4), increased pel production could have 15

further increased cell-cell interactions. PA14 is a naturally psl deficient strain and the 16

here generated psl deficient mutants are similar to PA14 strain in this respect. 17

Therefore it is conceivable that increased crystal violet staining resulted from an 18

increased number of bacterial cells in the biofilm held together by excessive pel 19

produced by these mutants. 20

Here it was shown that mutants lacking alginate production showed an 21

increased ratio of dead to live cells (Table 2; Fig. 3). However, propidum iodide also 22

stains eDNA (18). PAO1∆pslA∆alg8 and PAO1∆pelF∆alg8 biofilms showed 23

extensive surface structures predominantly composed of eDNA as was suggested by 24

DNase treatment (Fig. 4). Previous studies suggested that eDNA plays an important 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

18

role in biofilms as a cation chelating and antibiotic resistance inducing matrix 1

component (18, 34). Since large amounts of eDNA were only observed in mutants 2

lacking alginate production, increased cell death might have led to more eDNA being 3

released functionally and structurally replacing the polyanionic alginate. 4

The role of alginate for entrapping live cells in the cap of the mushroom-like 5

structure was underpinned by the observation that the caps in biofilms formed by 6

PAO1∆pelF∆alg8 (produces only psl) were almost devoid of live cells whereas those 7

formed by psl and alginate producing strains PAO1, PAO1∆pelF (Fig. 3) and 8

PDO300 (Fig. S3A) showed a high-density of live cells in cap region..This suggested 9

that the caps of the mushroom-like structures are not made up of only psl but that 10

alginate plays an important role in retaining live cells and/or supporting the viability 11

of cells in these caps. 12

Interestingly, analysis of biofilms formed by PAO1∆pslA∆alg8 suggested that 13

pel and eDNA together with live bacterial cells constituted a connected meshwork 14

showing increased cell-to-cell interactions and hence increased compactness of the 15

biofilms (Figs. 3 and 4). This is consistent with a study that suggested that increased 16

pel production enhanced cell-to-cell interactions as well as increased biomass of 17

biofilms (4). This compactness might be the reason why even when psl is produced 18

(PAO1∆alg8) elevated mushroom like structure were not formed (Fig. 5). Increase in 19

volume and height (Fig. 5) and decrease in compactness (Fig. 3C & E) of biofilms 20

produced by pel deficient mutants, PAO1∆pelF and PAO1∆pelF∆alg8, as compared 21

to the parent strain provided further evidence that pel contributes to the compactness 22

of the biofilm Unlike pel, psl along with eDNA in biofilms of PAO1∆pelF∆alg8 did 23

not show such cell-to-cell interactions. This is in accordance with previous studies 24

which suggested that psl and eDNA in a biofilm are not located in close vicinity (12). 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

19

Interestingly, the psl negative mutant showed more pel production in static 1

biofilms (Fig. 1) and the pel deficient mutant showed increased alginate production 2

when grown on solid media (Table S2) when compared with the respective wild type 3

strains. This could be due to competition of the biosynthesis pathways of the pel, psl 4

and alginate with respect to metabolic precursors. Polymer biosynthesis pathways in 5

P. aeruginosa had been suggested to be competitive with respect to common 6

precursors (22, 24). However, it can not be excluded that the absence of one or two 7

exopolysaccharides impacts at regulation level on the biosynthesis of the still 8

produced polysaccharide or polysaccharides.. 9

To assess how the anti-sigma factor MucA impacts on the ability to produce 10

pel and psl at the transcriptional level, the activation of the pel and psl promoters 11

were assessed in the PAO1 and PDO300 strains, respectively. This showed the lack of 12

MucA did not significantly impact on the pel promoter activity but decreased the psl 13

promoter by about 30%. It is known that deficiency of MucA results in increased 14

alginate production and transcriptional activation of the alginate operon (29). Here it 15

seemed that this increase in alginate production and/or the unleashed AlgU had a 16

negative effect on the transcriptional activity of psl operon. This suggested some 17

regulatory cross talk between the regulation of the biosynthesis of various 18

exopolysaccharides. Further investigations are required to shed further light on 19

regulation of polysaccharide biosynthesis. 20

Overall, this study showed that thick structured biofilm are still formed by 21

mutants producing one or two polysaccharides except when only alginate is produced. 22

Only when both psl and pel (PAO1∆pslA∆pelF) were not produced, cells were not 23

able to form biofilms (Fig. 4G-H).. Experimental evidence was provided that the 24

deficiency in one or two polysaccharides enhanced production of the remaining 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

20

polysaccharides or polysaccharide, respectively (Fig. 1; Table S2). These data shed 1

new light on how the three polysaccharides and eDNA interactively contribute to the 2

P. aeruginosa biofilm formation and architecture. 3

4

Acknowledgment 5

This study was supported by research grants to B.H.A.R. from Massey University. 6

A.G. was supported by Higher Education Commission of Pakistan. The authors are 7

grateful to Adrian Turner (School of Biological Sciences, University of Auckland) for 8

his permission to use the IMARIS software. 9

10

References 11

1. Allesen-Holm, M., K. B. Barken, L. Yang, M. Klausen, J. S. Webb, S. 12

Kjelleberg, S. Molin, M. Givskov, and T. Tolker-Nielsen. 2006. A 13

characterization of DNA release in Pseudomonas aeruginosa cultures and 14

biofilms. Mol Microbiol 59:1114-28. 15

2. Campisano, A., C. Schroeder, M. Schemionek, J. Overhage, and B. H. 16

Rehm. 2006. PslD is a secreted protein required for biofilm formation by 17

Pseudomonas aeruginosa. Appl Environ Microbiol 72:3066-8. 18

3. Choi, K. H., A. Kumar, and H. P. Schweizer. 2006. A 10-min method for 19

preparation of highly electrocompetent Pseudomonas aeruginosa cells: 20

application for DNA fragment transfer between chromosomes and plasmid 21

transformation. J Microbiol Methods 64:391-7. 22

4. Colvin, K. M., V. D. Gordon, K. Murakami, B. R. Borlee, D. J. Wozniak, 23

G. C. Wong, and M. R. Parsek. 2011. The Pel Polysaccharide Can Serve a 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

21

Structural and Protective Role in the Biofilm Matrix of Pseudomonas 1

aeruginosa. PLoS Pathog 7:e1001264. 2

5. Friedman, L., and R. Kolter. 2004. Genes involved in matrix formation in 3

Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol 51:675-90. 4

6. Friedman, L., and R. Kolter. 2004. Two genetic loci produce distinct 5

carbohydrate-rich structural components of the Pseudomonas aeruginosa 6

biofilm matrix. J Bacteriol 186:4457-65. 7

7. Hay, I. D., K. Gatland, A. Campisano, J. Z. Jordens, and B. H. Rehm. 8

2009. The impact of alginate overproduction on attachment and biofilm 9

architecture of a supermucoid Pseudomonas aeruginosa. Appl Environ 10

Microbiol. 11

8. Hoang, T. T., R. R. Karkhoff-Schweizer, A. J. Kutchma, and H. P. 12

Schweizer. 1998. A broad-host-range Flp-FRT recombination system for site-13

specific excision of chromosomally-located DNA sequences: application for 14

isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77-86. 15

9. Hoiby, N., O. Ciofu, and T. Bjarnsholt. 2010. Pseudomonas aeruginosa 16

biofilms in cystic fibrosis. Future Microbiol 5:1663-74. 17

10. Jackson, K. D., M. Starkey, S. Kremer, M. R. Parsek, and D. J. Wozniak. 18

2004. Identification of psl, a locus encoding a potential exopolysaccharide that 19

is essential for Pseudomonas aeruginosa PAO1 biofilm formation. J Bacteriol 20

186:4466-75. 21

11. Lee, V. T., J. M. Matewish, J. L. Kessler, M. Hyodo, Y. Hayakawa, and S. 22

Lory. 2007. A cyclic-di-GMP receptor required for bacterial 23

exopolysaccharide production. Mol Microbiol 65:1474-84. 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

22

12. Ma, L., M. Conover, H. Lu, M. R. Parsek, K. Bayles, and D. J. Wozniak. 1

2009. Assembly and development of the Pseudomonas aeruginosa biofilm 2

matrix. PLoS Pathog 5:e1000354. 3

13. Ma, L., K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak. 4

2006. Analysis of Pseudomonas aeruginosa conditional psl variants reveals 5

roles for the psl polysaccharide in adhesion and maintaining biofilm structure 6

postattachment. J Bacteriol 188:8213-21. 7

14. Mathee, K., O. Ciofu, C. Sternberg, P. W. Lindum, J. I. Campbell, P. 8

Jensen, A. H. Johnsen, M. Givskov, D. E. Ohman, S. Molin, N. Hoiby, and 9

A. Kharazmi. 1999. Mucoid conversion of Pseudomonas aeruginosa by 10

hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis 11

lung. Microbiology 145 ( Pt 6):1349-57. 12

15. Matsukawa, M., and E. P. Greenberg. 2004. Putative exopolysaccharide 13

synthesis genes influence Pseudomonas aeruginosa biofilm development. J 14

Bacteriol 186:4449-56. 15

16. Merritt, J. H., D. E. Kadouri., and G. A. O'Toole. 2005. Growing and 16

analyzing static biofilms, p. 1B.1.1-1B.1.7. In R. Coico, T. Kowalik, J. 17

Quarles, B. Stevenson, and R. Taylor (ed.), Current protocols in microbiology, 18

vol. 1. J. Wiley & Sons, Hoboken, NJ. 19

17. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor 20

Laboratory Press, Cold Spring Harbor, NY. 21

18. Mulcahy, H., L. Charron-Mazenod, and S. Lewenza. 2008. Extracellular 22

DNA chelates cations and induces antibiotic resistance in Pseudomonas 23

aeruginosa biofilms. PLoS Pathog 4:e1000213. 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

23

19. Nivens, D. E., D. E. Ohman, J. Williams, and M. J. Franklin. 2001. Role of 1

alginate and its O acetylation in formation of Pseudomonas aeruginosa 2

microcolonies and biofilms. J Bacteriol 183:1047-57. 3

20. O'Toole, G., H. B. Kaplan, and R. Kolter. 2000. Biofilm formation as 4

microbial development. Annu Rev Microbiol 54:49-79. 5

21. Overhage, J., M. Schemionek, J. S. Webb, and B. H. Rehm. 2005. 6

Expression of the psl operon in Pseudomonas aeruginosa PAO1 biofilms: PslA 7

performs an essential function in biofilm formation. Appl Environ Microbiol 8

71:4407-13. 9

22. Pham, T. H., J. S. Webb, and B. H. Rehm. 2004. The role of 10

polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in 11

rhamnolipid and alginate production as well as stress tolerance and biofilm 12

formation. Microbiology 150:3405-13. 13

23. Pier, G. B., F. Coleman, M. Grout, M. Franklin, and D. E. Ohman. 2001. 14

Role of alginate O acetylation in resistance of mucoid Pseudomonas 15

aeruginosa to opsonic phagocytosis. Infect Immun 69:1895-901. 16

24. Rehm, B. H. 2010. Bacterial polymers: biosynthesis, modifications and 17

applications. Nat Rev Microbiol 8:578-92. 18

25. Remminghorst, U., I. D. Hay, and B. H. Rehm. 2009. Molecular 19

characterization of Alg8, a putative glycosyltransferase, involved in alginate 20

polymerisation. J Biotechnol 140:176-83. 21

26. Remminghorst, U., and B. H. Rehm. 2006. In vitro alginate polymerization 22

and the functional role of Alg8 in alginate production by Pseudomonas 23

aeruginosa. Appl Environ Microbiol 72:298-305. 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

24

27. Ryder, C., M. Byrd, and D. J. Wozniak. 2007. Role of polysaccharides in 1

Pseudomonas aeruginosa biofilm development. Curr Opin Microbiol 10:644-2

8. 3

28. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning :a 4

laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press Cold Spring 5

Harbor, NY. 6

29. Schurr, M. J., H. Yu, J. M. Martinez-Salazar, J. C. Boucher, and V. 7

Deretic. 1996. Control of AlgU, a member of the sigma E-like family of stress 8

sigma factors, by the negative regulators MucA and MucB and Pseudomonas 9

aeruginosa conversion to mucoidy in cystic fibrosis. J Bacteriol 178:4997-10

5004. 11

30. Schweizer, H. P., and T. T. Hoang. 1995. An improved system for gene 12

replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 13

158:15-22. 14

31. Spiers, A. J., J. Bohannon, S. M. Gehrig, and P. B. Rainey. 2003. Biofilm 15

formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 16

wrinkly spreader requires an acetylated form of cellulose. Mol Microbiol 17

50:15-27. 18

32. Vasseur, P., C. Soscia, R. Voulhoux, and A. Filloux. 2007. PelC is a 19

Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of 20

proteins involved in exopolysaccharide transport. Biochimie 89:903-15. 21

33. Vasseur, P., I. Vallet-Gely, C. Soscia, S. Genin, and A. Filloux. 2005. The 22

pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and 23

late stages of biofilm formation. Microbiology 151:985-97. 24

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

25

34. Whitchurch, C. B., T. Tolker-Nielsen, P. C. Ragas, and J. S. Mattick. 1

2002. Extracellular DNA required for bacterial biofilm formation. Science 2

295:1487. 3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

26

Figure legends 1

Figure 1 Assessment of pel formation in various exopolysaccharide deficient mutants 2

PAO1; ∆F, PAO1∆pelF; ∆8, PAO1 ∆alg8; ; ∆A, PAO1∆pslA; ∆A∆F, 3

PAO1∆pslA∆pelF; ∆A∆8, PAO1∆pslA∆alg8; ∆8∆F, PAO1 ∆alg∆pelF8; ∆A∆F∆8, 4

PAO1∆pslA∆pelF∆alg8 A) Pellicle formation at air-liquid interphase when each 5

mutant was grown in 10ml of PI Medium for 4days as static biofilm. (B) Congo red 6

binding assay: All strains grown as static biofilm for 4 days were mixed with 20 7

mg/ml Congo red, Biomass was sedimented and unbound Congo red was detected at 8

490 nm. Percentage of Congo red left in supernatant is shown here. 9

10

Figure 2 Attachment of various P. aeruginosa PAO1 strains to solid surface. The SSA 11

was used to assess the impact of various exopolysaccharide deficiencies on 12

attachment. PAO1; ∆F, PAO1∆pelF; ∆8, PAO1 ∆alg8; ; ∆A, PAO1∆pslA; ; ∆A∆F, 13

PAO1∆pslA ∆pelF; ∆A∆8, PAO1∆pslA∆alg8; ∆8∆F, PAO1 ∆alg∆pelF8; ∆A∆F∆8, 14

PAO1∆pslA∆pelF∆alg8; Media, uninoculated Pseudomonas Isolation medium 15

control; OD600, optical density at 600 nm.(A) Differences during early attachment 16

phase at 2, 4 and 6 hours time points; (B) differences between loosely and tightly 17

attached 4 days old biofilms (adherent biofilms after soft and vigorous washing, 18

respectively). Values and error bars represent the averages and standard deviations, 19

respectively, for twenty four independent replicates. 20

21

Figure 3 Confocal laser scanning microscopic images of P. aeruginosa biofilms 22

grown in a continuous-culture flow cell for 4 days. XY (central), XZ (bottom) and YZ 23

(left) plans of each image are shown. A; PAO1, B; PAO1∆alg8, C; PAO1∆pelF, D; 24

PAO1∆pslA, E; PAO1∆alg8∆pelF , F; PAO1∆pslA∆alg8, 25

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

27

1

Figure 4 DNase treatment of high amount of extracellular DNA producing mutants 2

biofilms.A; Images of PAO1∆pslA∆alg8 without DNase treatment, B; Images of 3

PAO1∆pslA∆alg8 aftert DNase treatment, C; Images of PAO1∆pelF∆alg8 without 4

DNase treatment, D; Images of PAO1∆pelF∆alg8 aftert DNase treatment 5

6

Figure 5. Height of microcolonies and base layer of each biofilm forming mutant 7

measured from 3D pictures of 96 hour grown biofilms. White part of the bars 8

represents height of base of the biofilm. PAO1,; ∆F, PAO1∆pelF; ∆8, PAO1∆alg8; 9

∆A, PAO1∆pslA; ∆A∆8, PAO1∆pslA∆alg8; ∆8∆F, PAO1 ∆alg∆pelF8 10

11

12

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

28

1

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Table 1 Exopolysaccharide biosynthesis relevant genotypes and phenotypes of strains

used and generated in this study.

*Psl quantification was not done due to unavailability of a specific and differentiating

detection method. However, its production was indicated by determining the total

exopolymers produced by alginate/pel deficient mutant.

-, Polysaccharide production not detectable

+, Polysaccharide production detectable with increased number of + indicating

increased relative production

Exopolysaccharides Produced by strains Strains

Alginate Pel Psl*

PAO1 + + +

PAO1∆pelF + - +

PAO1∆alg8 - ++ +

PAO1∆pslA + +++ -

PAO1∆pslA∆pelF + - -

PAO1∆pslA∆alg8 - +++ -

PAO1∆pelF∆alg8 - - +

PAO1∆pslA∆pelF∆alg8 - - -

PDO300 +++ + +

PDO300∆pelF ++++ - +

PDO300∆alg8 - ++ +

PDO300∆pslA ++ +++ -

PDO300∆pslA∆pelF ++++ - -

PDO300∆pslA∆alg8 - +++ -

PDO300∆pelF∆alg8 - - +

PDO300∆pslA∆pelF∆alg8 - - - on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

Table 2 Characteristics of biofilms formed by mutants

Biofilm forming

strains

Volume

(µm³/µm²)

Dead:Live

Ratio*

PAO1 47.21 0.33

PAO1∆pelF 35.63 0.53

PAO1 ∆alg8 28.91 0.41

PAO1∆pslA 25.09 0.65

PAO1∆pslA∆alg8 49.37 1.08

PAO1∆pelF∆alg8 42.57 0.81

*Ratio between red and green fluorescence shown by each biofilm forming mutant.

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

0

20

40

60

80

100

120

PAO1 ∆F ∆8 ∆A ∆A∆F ∆A∆8 ∆F∆8 ∆A∆F∆8 ∆A∆F

(MCS-

5:pelF)

∆A∆F

(MCS-5)

Media

% C

on

go

re

d in

su

pe

rna

tan

t

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

PAO1 ∆F ∆8 ∆A ∆A∆F ∆A∆8 ∆F∆8 ∆A∆F∆8 Media

OD

60

0

2 hrs

4 hrs

6 hrs

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

0

2

4

6

8

10

12

PAO1 ∆F ∆8 ∆A ∆A∆F ∆A∆8 ∆F∆8 ∆A∆F∆8 Media

OD

60

0

Vigorous

Soft

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

0

10

20

30

40

50

60

70

80

PAO1 ∆F ∆8 ∆A ∆A∆8 ∆F∆8

Biofilm forming strains

Hei

gh

t (µ

m)

on Septem


.asm.org/

Dow

nloaded from

http://aem.asm.org/

The role of exopolysaccharides in Pseudomonas aeruginosa ......2011/06/10 · 5 biofilm matrix (1,...

Documents

Transcript of The role of exopolysaccharides in Pseudomonas aeruginosa ......2011/06/10 · 5 biofilm matrix (1,...