of to a --. ugnos~ biofem · -a-- identification of genes important to psezllrdomonrs...

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IDENTIFICATION OF GENES IMPORTANT TO PSEUDOMONAS ---a=- A --A - - --. - AER UGNOS~ BIOFEM FOWTION Antonio Finelli A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Sciences University of Toronto BCopyright by Antonio Fineiii 200 1

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Page 1: OF TO A --. UGNOS~ BIOFEM · -A-- IDENTIFICATION OF GENES IMPORTANT TO PSEZllrDOMONRS AERUGllY(3Ls;a (P-AaIj BTO~FO~TION Degree of Master of Science Antonio FineIli lnstitute of Medical

IDENTIFICATION OF GENES IMPORTANT TO PSEUDOMONAS ---a=- A --A - - --. -

AER U G N O S ~ BIOFEM F O W T I O N

Antonio Finelli

A thesis submitted in conformity with the requirements for the degree of Master of Science

Institute of Medical Sciences University of Toronto

BCopyright by Antonio Fineiii 200 1

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The authar has granted a non- exclusive licence dowhg the National Library of Canada to reproduce, loan, distribute or seîî copies of this thesis in microform, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fkom it may be p ~ t e d or otbezwise reproduced without the author's permission.

L'auteur a accordé une licence non excIusive permettant à la BibliothQue nationale du Canada de reproduire, ptêter, distribuer ou vendre des copies de cette thèse sous la forme de microfichelfilm, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de ceIIe-ci ne doivent être imprimés ou autrement reproduits sans son

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-A--

IDENTIFICATION OF GENES IMPORTANT TO PSEZllrDOMONRS AERUGllY(3Ls;a (P-AaIj B T O ~ F O ~ T I O N

Degree of Master of Science

Antonio FineIli

lnstitute of Medical Sciences University of Toronto

ABSTRACT

A biofilm is a structural community of bacteria adherent to a s h e and enclosed

in a ~e~produced exopolysaccharide matrix. Bacteria in biofilms have a unique

muhicellular architecture and increased resistance to host defenses and antimicrobials.

The transition tiom a pianktonic existence to a biofilm involves changes in gene

expression.

An IVET (in vivo expression technology) approach was used to shdy gene

expression in Pseudomonus ueruginosa biofilms. An AmC transcriptionai regulator

homologue, PA3 782, and a GMC oxidoreductase homologue, PA37 1 O, were found to be

upregulated in biofilm bacteria and important to bionlm formation. PAS065, a u&iB

hombgue, was found to be upreguhed in biofiln bacteria and essential for d v a l

regardless of the mode of growth.

In conclusion, gene expression in P. aemginosa biofilms seems to reflect

responses to physiochemical environmental stimuli The genes identined in this study

appear to be involved in adaptation to inrreased omsmolarity and to oxygen and nutrient

limitations.

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ACKNOWLEDGEMENTS

My two years at the Center for Infection and Biomaterials Research went by so

quickly. The time was pleasurable and memorable because of the individuals I worked

with. I'd like to thank Selva, Lian, and Wendy for facilitating my integration into the

laboratory and Julie and Brian for the good times during t h i s past year.

I'd like to extend my gratitude to cornmittee members Drs. J w i , Khoury and

Brunton for their constructive feedback and direction.

1 owe a great deai to my supervisor Dr. Lon Burrows. 1 truly appreciate the time

Lori took in guiding me through rny research. She is an excellent supervisor, great role

mode1 and wonderful person.

Lastly, I'd like to thank my wife, Sky, for her ongoing support of my academic

aspirations that often cut into "our" tirne.

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TABLE OF CONTENTS

.......................................................................... ABSTRACT

ACKNOWLEDGEMENTS .......................................................

TABLE OF CONTENTS ..........................................................

LIST OF FIGURES .................................................................

.................................................................. LIST OF TABLES

LIST OF ABBREVIATIONS .....................................................

INTRODUCTION ..................................................................

1 . Antibiotic Resistance ........................................................ ............................................. 2 . Genetics of Biofilm Formation

3 . In vivo Expression Technology (WET) ...................................

............................................................................. METHODS

The IVET system based on purEK in P . aenrginosa PAK .............. Growth of PAK and PAK-AIU Biofilms Individually and in . . Competltlon .................................................................... The Biofilm Assay and Growth of the WET Library as a Biofilm ..... Selection of "Biofilm-Specific" Clones .................................... Confirmation of the Isolated Clones' Abilities to Form Isogenic

........................................................................ Biofilms Planktonic Growth of the Isolated Clones. PAK and PAK-AR2 ...... Southern Blot of the Seven Isolates and PAK withpurEK ............ Isolation of the Cloned DNA Fragments Lying Upstream ofpurEK Cloning and Secpncing of the DNA Fragment Lying Upstream of purEK .......................................................................... Sequencing and Analysis of Upstream Sequences .......................

.................................... RNA Purrification and RNA Dot-Blots Generation of a PAS065 Mutant ............................................ Codhat ion that PA5065 is Essential for Survival in P . aeruginosa .. Generation of PA37 1 0 and PA3782 Mutants ............................ Growth of PA0 1, the PA3782 and PA37 10 Mutants Planktonicalty and as Biofilms ................................................................ Competition Biofilm Assay: PA01 grown with ~ ~ 3 7 8 2 : : ~ m ~ and PA3 7 1 0: .GmR ................................................................. Scanning Electron Microscopy (SEM) of Biofilms ...................... Static Biofilm Formation Assay .............................................

. . 11

iii

iv

vi

ix

X

1

3 7 12

15

15

17 19 20

20 21 2t 24

24 27 28 30 34 37

39

40 41 41

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............................................................... . 19 SDS Resistance . . . . - - ... .. ..... .

20;- Osmotic Stress Assay .........................................................

Growth of P A . and PAK-AR2 Biofilms Individually and in . . cornpetition .................................................................... Growth of the N E T Library as a Biofilm and Selection of 56 Biofilm-Specific" Isolates .................................................. Confirmation of the Isolates' Abilities to Form Biofilms ............... Planktonic Growth of the Isolated Clones. PAK and PAKAR2 ....... Southem Blot to Detect the Number of Unique Strains Amongst the Isotated Clones ............................................................ Identification of Open Reading Fnunes Downstream of the Cloned

....................................................................... Promoters RNA Dot-Blots to C o n h Upregulation of the in biofim Induced Genes ................................................................. Generation of PAS065. PA37 10 and PA3782 Insertional Mutants ..... 8.1 PA5065 ................................................................ 8.2 PA37 10 and PA3782 Mutants ......................................

............... Phenotypic Studies of the PA371 0 and PA3782 Mutants ................................................... 9.1 Planktonic Growth

....................................................... 9.2 Biofiim Growth 9.3 Cornpetition Assay ...................................................

..................................... 9.4 Scanning Electron Microscopy ................................................. 9.5 Static Biofilm Assay

....................................................... 9.6 SDS Sensitivity ............................................... 9.7 Osmotic tolerance test

........................................................................... DISCUSSION

........................................................................ REFERENCES

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LIST OF FIGURES

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Construction of the IVET library ..................................

Biofilm growth apparatus. ..........................................

.............. Touchdown PCR of four ex-biofilm IVET strains.

......... Biofilm cornpetition assay with PAK and PAK-AR2..

Ex-biofilm adenine-requiring IVET strains grown on DMM and DMM+A agar.. .........................................

Figure 6a. Growth curves of PAK and the ex-biofiim strain 9.27A Grown planktonicdly in DMM and DMM+A.. .................

Figure 6b. Growth curves of PAK and the ex-biofilrn strain 9.278 Grown planktonically in DMM and DMM+A.. .................

Figure 7.. Growth curves of PAK and the ex-biofih strain 9.27C Grown planktonically in DMM and DMM+A.. .................

Figure 7b. Growth curves of PAK and the ex-biofilm strain 9.27D Grown planktonicdly in DMM and DMM+A.. .................

Figure 8.. Growth curves of PAK and the ex-biofilm strain 9.27E Grown planktonically in DMM and DMM+A.. .................

Figure 8b. Growth curves of PAK and the ex-biofilm strain 3.15 Grown planktonically in DMM and DMM+A.. .................

Growth curves of PAK and the ex-biofhm strain 9.29 Grown planktonicdly in DMM and DMM+A.. .................

Figure 9b. Growdi curves of PAK and PAK-AR;! grown planktonically in DMM and DMM+A.. ..........................

Figure 10. Southem blot of EcoRI-digested chromosomal DNA fiom Seven ex-biofilm adenine-requiring IVET s a s probed

........................................................... with purEK.

Figure 11. Multiplex PCR amplification of PA3782 and PA3710 using ..... DNA fiom PA0 1 and 1 5 P. aenrginosa clinical isolates. 55

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vii

Figure 12. --

Figure 13.

Fipre 14.

Figure 15.

Figun 16.

Figun 17.

Figure 18.

Figure 19.

Figun 20.

Fipre 21.

Figure 22..

Figun 22b.

Fipre 23a.

Figun 23b.

Protein alignment of PA0240 and its closest homoiogues - - with h o w n iùnctron.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protein alignment of PA3 7 1 0 and its closest homologues.. . . . Protein alignment of PASO65 and its closest homologues.. . ..

Protein alignment of PA3 782 and its closest homologues.. . . .

Dot-blots of RNA isolated fkom PAOl grown planktonically and as a biofilm probed with rpsA, rpoS, PAS065 and PA3 7 10.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SmaI digest of plasmid preparations fiom PA0 1 - P A ~ O ~ ~ : : G ~ ~ - ~ U C P ~ ~ - P A S O ~ ~ and E. coli JM 1 O9 pUCP26-PASO65.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EcoRI and HindIII digests of plasmid preparations fiom PAO 1 - P A ~ O ~ S : : G ~ ~ - ~ U C P ~ ~ - P A ~ O ~ S that had been electroporated with pUCP2O and pUCP2O-ubiBEc and grown under selection for Pipercillin resistance.. . . . . . . . . . . . . . ...

EcoRI and HindIII digests of a plasrnid prepmtion nom E. coli Ml09 that had been electroporated with a plasrnid preparation fiom PAO 1 -PASO~S : : ~ r n ~ - ~ ~ ~ ~ 2 6 - ~ ~ 5 0 6 5 . P A O I - P A ~ ~ ~ ~ : : G ~ ~ - ~ U C P ~ ~ - P A S ~ ~ ~ had previously been electroporated with pUCP20 and grown under selection for Pipercillin resistance.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR amplification of template DNA fiom PA0 1, a PA37 10 merodiploid and PAO 1 PA^ 7 1 0: : ~ r n ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR amplification of template DNA fiom PAO1, a PA3782 merodiploid, PAO 1 - ~ ~ 3 7 8 2 : : ~ m ~ . . . .., . . . . . . . . . . .. . . . . . .. Growth curves of PAOl, ~ ~ 3 7 1 0 : : ~ m ~ and ~ A 3 7 8 2 : : ~ r n ~ grown planktonically in DMM.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cornparison of PA0 1, ~ ~ 3 7 1 0 : : ~ r n ~ and pA3782::GrnR grown as a biofilm in DMM for five days.. . . . . . . . . . . . . . . . . . . . . . . Biofilm competition assay with PA0 1 and ~ ~ 3 7 1 0 : : ~ m ~ . . . ..

Biofiim competition assay with PA0 1 and ~ ~ 3 7 1 0 : : ~ m ~ (relative composition of each strain). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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viii

Figure 24s. Biofilm cornpetition assay with PAOl and ~ ~ 3 7 8 2 : : ~ r n ~ . . . . - .- - - - - - 73

F i p r e 24b. Biofilm cornpetition assay with PA0 l and ~ ~ 3 7 8 2 : : ~ m ~ (relative composition of each stmin). . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73

Figure 25. SEM of a ~ ~ 3 7 8 2 : : ~ r n ~ biofih after 24 hours of growth.. . 74

Figure 26. SEM of a PA01 biofilm after 24 hours of growth.. . . . . . . . . . . . 75

Figure 27. SEM of a ~ ~ 3 7 1 0 : : ~ r n ~ biofilm d e r 24 hours of growth. .. 76

Figure 28. Osmostic stress tolerance of PA0 1, ~ ~ 3 7 1 0 : : ~ r n ~ and ~ ~ 3 7 8 2 : : ~ m ~ over 24 hours in DMM with 2M NaCI.. . . . . . .. 78

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-..%.*-A .- ....

LIST OF TABLES

Table 1 . Primers used for PCR amplification .............................. 89

Table 2 . Probes used for Southern blot and RNA dobblots .............. 90

Table 3 . Bacterial strains and plasmids used in this study ................ 91

Table 4 . Putative biofilm-induced proteins and their homologues ...... 92

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a. a. c h DMM DMM+A SDS IVET PAK PA01 PCR OD Oh@ MCS MFS RT o h

LIST OF ABBREVIATIONS

amino acid colony forming units Davis Minimal Media with O. 1 % Glucose Davis Minimal Media with O. 1 % Glucose and 5% Adenine Sodium Dodecyl Sulfate in vivo Expression Technology Pseudomonas ueruginosa PAK Pseudomonus aeruginosa PA0 1 Pol y merase Chain Reac tion Optical Density Outer Membrane Protein Multiple Cloning Site Major Facilitator Superfarnily Room Temperature ovemight

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INTRODUCTION -- -

A biofiim is an accumulation of microorganisms embedded in a polysaccharide

matrix adherent to solid biologic or non-biologic surfaces [l]. Evidence for this microbial

mode of existence was recorded nearly 70 years ago [2]. However, a concerted effort to

study microbiai biofilms began only two decades ago [3]. Traditional micro biolog ical

research has involved studies of bacteria grown on agar plates or planktonically in broth

cultures. Although useful, these foms of growth do not accurately refiect bacterial

existence in nature. The majority of microbes persist attached to surfaces within a

s t r u c ~ d biofilm ecosystem [4]. The irnpetus to study microbid biofilms amse fiom

discoveries of their signifiant role in environmental biofouling and human infection.

Biofilms c m comprise a single microbial species or contain multiple microbial

species. Although mixed-species biofilms predominate in most environrnents, single-

specie biofilms exist in a variety of infections and on the surface of irnplanted medical

devices [l]. Pseudomonas aeruginosa has emerged as the best studied single-species

biofilm-forrning Gram wgative bacterium [5].

P. aeruginosa does not carry out fermentation, obtaining its energy fiom the

oxidation of sugars. Nevertheless, many seains can grow anaerobically using nitrate as a

terminal electron acceptor [6]. Pseudomonads have minimal nutritional requirements,

needing only acetate and ammonia as sources of carbon and nitrogen, respectively. The

complete genome sequence of P. aeruginosa strain PA01 was recently published [7]. Its

genome consists of 6.3 million base pairs and has 5570 predicted open reading fnunes

(ORFs) [7]. The genetic complexity of PA0 1 approaches that of the simple eukaryote

Succharomyces cerevisiae, whose genome encodes -6200 proteins [8]. In PA0 1, most

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- - - . . - A - -- of the predicted ORFs have the high G+C content (66.6%) characteristic of the genome - -

as a whole. PA01 has the highest proportion of predicted regdatory genes observed

among the sequenced bacterial genomes, with 468 genes contalliing motifs characteristic

of transcriptional regdators or envuonmental sensors [7]. Also, a disproportionately

large nurnber of genes, 150, are predicted to encode outer membrane proteins (OMPs)

[7]. The identification of these families of OMPs could have significant impact on the

focus of antimicrobial and vaccine research [7].

P. aetuginosa is known for its intrinsic resistance to many Eront-line antibiotics,

due mainly to its low outer membrane permeability and to active efflux of antibiotics [9].

P. aeruginosa is a clinically significant pathogen, fiequently responsible for bacteremia

in burn victims, urinary tract infections in catheterized patients, and hospital-acquired

pneurnonia in patients on ventilators [IO]. It is also the predominant cause of morbidity

and mortality in cystic fibrosis (CF) patients, whose abnormal airway epithelia allow

long-term colonization of the lungs. These infections are impossible to eradicate because

P. aeruginosa fonns a biofih in the lungs of these patients [Il, 121. In recent yean P.

aenrginosa has become more resistant to antibiotics with 10-1 2% of clinical isolates now

resistant to ciprofloxacin [13]. The problem of resistance is enhanced when P. aeruginosa

grows as a biofilm. Nickel et al. [14] demonstrated that mature P. aeruginosa biofilms

are resistant to levels of antibiotics 1,000-fold higher than that required to kill planktonic

cells of the same strain.

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r.-A5tiiïotk R-esi'stance

The Centers for Disease Control now estimate that 65% of human bacterial

infections involve biofihs [Il. Bacteria growing in biofilms are remarkably resistant to

host defenses, including opsonic antibodies and phagocytes [1 SI. The refkactory nature of

biofilrn-related infections to antibiotic chemotherapy suggests that sessile bactena in

biofilms are highly resistant to conventional levels of antibiotics that would readily kill

planktonic cells of the same species. Recent reviews have concluded that resistance is

muitifactorial [ 16, 171.

One mechanism of biofilm resistance to antirnicrobial agents is the inability of an

agent to penetrate the full depth of a biofilm [18]. The exopolysaccharide matrix of a

biofilrn retards diffision of antibiotics [19] and studies have shown that solutes in general

difise more slowly through a biofilm thm they do in water [20]. The ability to penetratc

a biofilm varies amongst antibiotics. For example, the negatively charged

exopolysaccharide surrounding biofilm cells is very effective in protecting them fiom

positively charged aminoglycoside antibiotics [2 1,221.

in contnist, smaller, non-positively charged mtimicrobial molecules should

encounter less physical resistance to penetration. For example, fluoroquinolones, which

are relatively small moiecules, readily equilibrate across a biofilm [2 1-24]. Restricted

diffision alone should only delay penetration, but not eliminate it entirely. However,

retarded penetration can enhance the effect of other resistance mechanisms, such as P-

lactamase enzyme degradation of antibiotics [16]. This synergy between retarded

diffision and degradation provides effective resistance in P. aenrginosa biofilms

expressing a plactarnase [25].

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- . A noverassay was devetoped to stüay the rote ofpotenfial difision barriers in

Klebsiella pneumoniae biofilm resistance [23]. A filter paper disk was placed on top of

K. pneumoniae colonies that were grown on agar plates with or without antibiotic. By

performing a standard zone of inhibition assay with the recovered disk, they were able to

determine antibiotic penetration through the colony. The study showed that ampicillin

was unable to traverse the biofilm. However, ampicillin could traverse a biofilm formed

by plactamase mutants and thus, the production of Flactamase was likely responsible

for the inability of ampicillin to pass through the wild-type biofilm. Interestingly, P

lactamase mutants grown in a biofilm remained resistant to ampicillin even with

complete penetration, suggesting that other mechanisms contribute to the resistace of

these celts [23]. Similarly, fluoroquinolones have bcen found to penetrate P. aeruginosa

biofilms with efficiencies ranging from 50- 100% [22], as does imipenem [26], but

biofilm cells are more resistant to these agents than are isogenic planktonic cells. ïüere

is sufficient evidence to support the fact that the exopolysaccharide maûix is not

impenetrable and thus, additional mechanisms are responsible for the observed

antimicrobial resisbnce.

Since antimicrobials are more effective at killing rapidly dividing cells, biofilm

bacteria may be protected by their slower growth rate [27,28]. Penicillin and ampicillin

do not kill non-growing cells whereas aminoglycosides and fluoroquinolones c m kill

non-growing cells, but are more effective against rapidly dividing cells [16]. Studies

have shown that slow growing planktonic and biofilm P. aeruginosa were equally

resistant to ciprofloxacin, but as the growth rate increased, planktonic cells became more

sensitive to the antibiotic than did the biofilm cells [29]. This effect varies according to

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tIië bac€eriàraiiathe anfibiofic. Broouxï et a t [3q founCrtbatfhe srow growth rate in P:

a e r ~ ~ n o s u biofilms seemed to account for resistance to tetracycline, but the growth rate

did not seem to affect resistance to tobramycin. Thus, these results again suggest that

gmwth rate alone is not responsible for the enhanced resistance to some classes of

antimicrobial demonstrated by bacterial biofilms.

Since the physical properties of a biofilm, the diffision barrier and slower growth

rate, do not entirely explain the increased antimicrobial resistance, the concept of a

"biofilm phenotype" has emerged. Within the heterogeneous biofilm, there may be

bacteria that express active resistance mechanisms. To investigate this hypothesis,

researchers have studied the potential role of drug efflux pumps [30,3 11. Multidnig

eMux pumps cm extrude a number of chcmically welated antimicrobials. The emux

pump AcrAB, encoded by the mar operon in E. coli, is believed to be responsible for a

multiârug resistant phenotype. However, studies of mur expression with lac2 fusions

failed to demonstrate upregulation in a biofilm [3 11. Furthemore, mutations in mur did

not affect ciprofloxacin resistance [32]. P. aeruginosa has four known multi-emux

purnps and several other putative pumps. Using strains that either lacked or over-

expressed the MexAB-OprM pump, it was shown that at low concentrations of ofloxacin,

a fluoroquinolone, biofilms lacking the pump were more susceptible to this drug than

biofilms that overexpressed this pump [30]. However, for ciprofloxacin, there was no

difference. Overall, it appears that drug emux pumps are not responsible for the

enhanced resistance of biofilm bacteria.

Another proposed resistance mecbanism that c m be induced in biofilm cells is

alteration of the membrane-protein composition. This is similar to that which occurs in

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reSponse to antimicrobiars and couIdüItimatety r d t o a decreased permeability to these

compounds [ 171. Mutations in the outer membrane proteins ompF and ompC increased

the resistance of E. coli to &lactams [33]. Studies in s w i n g bacteria demonstnited that

the relative proportions of OmpF and OmpC were altered, with preferential expression of

the smaller ponn OmpC [34]. Bacteria living in biofilms are under osmotic stress [4,35]

and this environmental stimulus may be responsible for changes in the protein

composition of the outer membrane. Therefore, altered porin expression affects the

intrinsic resistance of bacteria and could possibly play a role in bacterial biofilm

resistance.

Lastly, Lewis [16] has proposed an interesthg hypothesis to explain biofilm

resistance that relates to a subpopulation of bacteria in the biofilm referred to as

''persisters". A study of dose-response killing of P. ueruginosa biofilms by the

fluoroquinolones ofloxacin and ciprofloxacin showed that the majority of cells are

effectively eliminated by low concentrations of antibiotics, similar to planktonic cells

[16]. Most of the biofilm cells were killed within a clinically achievable range of

concentrations, but afler a 3- to 44og reduction, any increase in antibiotic concentration

had no M e r effect on killiag [30]. Thus, the majority of cells in a biofilm are not

necessady more resistant to killing than planktonic cells, but a subpopulation of

persisters survive [Ml. These observations suggest a aew paradigm for explaining

biofilm resistance. However, the genetics and phenotype of persisters remahs to be

elucidated.

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- S. %-netEs- of BiofZim Formation

Recent advances in microscopy and molecular technology have made it possible

to examine bacterial biofilrns in situ in grcater detail. With the advent of confocal

scanning laser microscopy (CSLM), researchea were able to appreciate the unique

architecture of biofilms [36]. Mature biofilms consist of "mushroom-like" structures,

containhg bacteria embedded in an exopolysaccharide matrix, separated by channels that

facilitate convective flow of nutnents [4]. Such a unique and reproducible stnicture

suggests that dramatic changes in gene expression occur when bacteria switch from

single ce11 swimrning in media to a community of microbes attached to a surface. A

better understanding of the changes in bacterial physiology during biofilm formation may

help explain increased aatimicrobial resistance and aid the development of novel

preventative and therapeutic measures. Only over the past five years have studies

addressing gene expression in biofilm bactena been initiated.

Kolter and colleagues [37-39) used a simple microtitre plate mode1 to study the

effect of transposon mutagenesis on initiation of biofilm formation in E. coli and two

species of Pseudomones. The mutants that failed to form a biofilm in their 96-well

microtitre assay were referred to as swface attachment defective (sud) mutants. Using

this screen, the motility organelles, flagella and pili, were identified as important for

initiation of biofilm formation [37-391. Time-lapse microscopy demonstrated the inability

of a non-motile mutant, sud-36, to colonize an abiotic surface 1381. The sequence

flanlring the transposon insertion site in this sud-mutant was homologous toflgK, a gene

encoding the flagellar hook protein of E. coli Dg]. At this t he , it is not clear whether

flagella play a direct role in adhesion or whether they are required to produce a force-

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geaerating movemenf that is necessaryto overcome-sorface reputsive forces €401. h

should also be noted that this rnotility-associated phenornenon is surface- and substrate-

specific. A P. jluorescens flageilar mutant,jIiP-, fails to fom a biofilm on

polyvinylchloride, but foms biofilms on polystyrene and other surfaces 1381.

Testing the transposon mutant library in the microtitre plate assay also identified

genes involved in pili synthesis as being necessary for biofilm formation [37-391. Type

IV pili are important for the adherence to and colonization of eukaryotic cell surfaces by

P. aemginosa [41]. It has also been shown that type N pili are required for a surface-

associated mode of translocation known as twitching motility [42]. Twitching motility is

thought to result from extension and retraction of type iV pili, which propels the bacteria

across a surface by an undescribed mechanism 1431. Time-lapse microscopy

demonstrated that mutants lacking pili were able to colonize an abiotic surface, but failed

to fom microcolonies 1373. This is in contrast to the wild-type bactena that twitch and

corne together to form microcolonies [37]. These colonies then grow in size to form

"rnushroom-like" structures that are characteristic of mature bacterial biofilms. Thus,

through this series of elegant experiments Kolter and colleagues [37-391 demonstrated

the importance of the motility organelles, flagella and pili, in the initiation of biofilm

formation.

The distinct architecture of mature bacterial biofilms suggests that bacteria must

undergo changes in gene expression in order to form and maintain these structures. The

genetic changes that occur with the progression from microcolony formation to mature

biofilm morphology bave not yet been studied on a genomic scale. However, some

notable snidies have shed light on this area [44,45]. The exopolysaccharide rnatrix in P.

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-aemginosa biofiims consists primarÎty of atginate [46r. The gene algC is invohred in

alginate production and had been shown to be upregulated as the result of attachment to a

surface [Ml. Utilizing an algC-lac2 reporter constnict, it was shown that expression of

algC in biofilm bacteria was 19 times higher than in planktonic bacteria [44]. Also, algC

was upregulated as early as 15 min af€er attachment to the substratum [44]. However,

initial ce11 attachment to the substratum appeared to be independent of algC promoter

activity [44]. Interestingly, the sigma factor (AlgT/AlgU or d2), required for

upregulation of alginate synthesis causes dom-regulation of a key flagellar biosynthetic

gene [47]. In E. coli, a lac2 reporter system was used to show that the flagellar synthesis

gene WC) was downreplated during biofilm growth [35]. Therefore, together with the

previously described experiments of Kolter et a1.[37-391, these fmdings imply that

flagella are necessary for the initiation of biofilm development, but with formation of the

exopolysaccharide matrix, the motility organelle is no longer needed, and its synthesis is

repressed.

Switching fiom a unicellular mode of existence to becoming part of a

multicellular biofilrn community has been compared to the complex differentiation

behaviours that occur in Caufobacter crescentus, Bacillus subtilis and A@xococcus

xanthus [SI. C. crescenhcr undergoes a ce11 cycle-controlled swarmer-to-staked ce11

transition, and individual vegetative cells of B. subtilis integrate multiple extemal and

interna1 signals to synthesize a new morphological structure, a spore, that is adapted for

survival in a variety of harsh envkonments [SI. Upon starvation, M. xanthus progresses

tluough a series of stages towards formation of fniiting-bodies that are reminiscent of

biofilms [SI. In the initial stages of fhiting-body development, cells colonize a surface

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aMTcoiikscë ushg type N mediated motïiïty CgIidiBgj fo finn itl: xanthus aggregates

that resemble P. aeruginosa microcolonies 151. The aggregates then develop into

fniiting-body structures that are rerniniscent of the mushroom-like structures

characteristic of mature P. aeruginosa biofilms [SI. The formation of hit ing bodies

requires intercellular communication through the extra-cellular signaling molecules

known as A-signal and C-signal [SI.

Two intercellular signaling systems identified in P. aeruginosu are the lasR-lad

and rhlR-rhll systems [45]. LasI and RhlI are synthetase enzymes that direct synthesis of

homoserine lactones (HSLs), whereas LasR and RhlR are transcriptional activators [45].

At sufEcient population densities, these self-produced signals, HSLs, reach the

concentration required for gene activation. Thus, this type of signaling has been temed

quorum sensing [48]. A shidy of rhll and losl mutants revealed that the M mutant

fomed a flat dense biofilm that lacked the charactenstic mushroom-like structures [45].

Wben the id mutant was grown in the presence of the specific HSL that is synthesized

by LasI, it fomed biofilms with the characteristic mushroom-like structures [45]. The

lasl mutant biofilrns were aiso more sensitive to 0.2% sodium dodecyl sulfate (SDS) than

the wild-type 1451. Using CSLM in conjunction with id and rhl-unstable g@ (green

fluorescent protein) fusions, it was shown that maximal expression of both autoinducers

occurs at the biofilm-substratum interface [49]. This spatial heterogeneity may simply

reflect the increased ce11 density at the surface and diminished opportunity for the HSLs

to diffise away.

As previously mentioned, P. aeruginosa forms biofilms in the lungs of

individuals with CF, resulting in recurrent pneumonia with progressive respiratory

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failure. It was recently show that the HSL profile of P. aeruginosa isolates fiom -- - - --

patients with CF resembled that of strains grown as in vitro biofilms or cultured fiom

spuhun as compared to when grown planktonically [12]. Therefore, there is growing

evidence that intercellular signaling is involved in biofilm development.

There is evidence supporting a relationship between quorum sensing and the

alternative sigma factor rpoS [50,51], which is known to be upregulated in stationary

phase growth [52,53]. However, the reports are conflicting and it now appears that RpoS

regulates RhlR-1 instead of the vice versa [50,5 11. Nonetheless, RpoS does appear to be

involved in biofilm formation [54,55]. nie gene rpoS is upregulated in P. aeruginosa

biofilms [55] and when mutated in E. coli, biofilm structure and ce11 density was

adversely affected [54]. The concept of a transcriptional regulator being upregulated in a

biofilm is intriguing because of the potential impact it may have on numerous genes.

Given the architectural complexity of a biofilm one would hypothesize that a number of

genes would be up- andor downregulated with the transition to a biofilm mode of

existence. Research at the protein level by Brozel and colleagues [56] supports this

contention. They monitored changes in global protein expression patterns in attached

cells and found > 1 1 proteins whose levels were altered during various stages of

attachent [56].

In one of the few genome-wide screens of biofilm gene expression, a startling

38% of 885 random transposon-mediated lac2 fusions in E. coli showed more than 2-fold

differential expression (up or down) during growth of the organism as a biofilm [35].

The screen detected downregulation of the flagellar synthesis gene WiC), correspondhg

with other studies that have shown the importance of flagella for surface contact and

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initiation rather than maintenance of mature biofilms [35,40,47]. The study also found

upregulation of ompC and the pro U operon, and nikA [3 51. The porin gene ompC and the

prou operon that encodes a high affinity glycine-betaine transporter are upregulated in

high osmolarity conditions that are consistent with the biofilm environment [19,57]. It

was previously show that bactena in biofilms encounter low oxygen tension levels,

particularly at the substratum [58]. The gene nikA is highly expressed in anaerobiosis [59]

and was found to be upregulated in biofilms [35]. This ground-breaking study was the

fust to investigate genome-wide gene expression and their results suggest a complex

interplay between environmental physiochemical conditions and bacterial biofilm

development.

3, In vivo Exmession Technoloev

Broad-scale âifferential gene expression stuàies in bacteria pose unique

challenges separate nom those encountered with eukaryotic systems. Bacterial messenger

RNA (mRNA) has a short half-life compared with that of eukaryotic mRNA (3-5 min vs.

up to 30 min), which makes isolation of intact mRNA difficult [60]. As well, unlike

eukaryotic mRNA, bacterial messages only rarely have a poly-adenylation signal at the 3'

end [60]. Therefore, there is no facile way to separate prokaryotic mRNA from total

cellular RNA prior to reverse transcription. This leads to synthesis of appreciable levels

of contaminating cDNAs transcnbed fiom non-message RNA molecules. Given these

challenges, there have been limited attempts at using such techniques as differential

display PCR and subtractive hybridization [60]. With the development of microarray

technology and a sequenced PA01 genome, automated screening of thousands of genes

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at - - a t h e is now possible. This technology was mavailable for P. aeruginosa at the onset -- - - - - - - - A -

of this project and an altemate strategy, in vivo expression technology (IVET), was

chosen to study genome-wide gene expression in P. aeruginosa biofilms.

IVET is a genetic system that was initially designed to identiQ bacterial genes

specifically induced upon growth in an animal [61]. The first application was

identification of Salmonella typhimurium virulence factors that were induced in a host

[6 11. Briefly, the system is a promoter trap, whereby randornly cloned bacterial DNA

fragments containing promoters drive the expression of a gene that is required to alleviate

an auxotrophy and thus, allow sumival in the environment of interest. An advantage of

IVET is that the fusions are present in single copy in the chromosome, avoiding possible

complications arising fkom the use of multicopy plasmids or polar transposon insertions.

Wang and Jin [62] constructed an NET library of P. aeruginosa PAK based on a

purine auxotrophy. The p H operon is single copy and required for de novo synthesis

of purines. Digested chromosomal DNA is cloned into a plasmid upstream of a

promoterless purEK. The recombinant plasmids represent a pool ofpwEK transcriptional

fusions driven by promoters present in the cloned fragments. They are then re-introduced

individually, via electroporation, into a P. aeruginosa purine auxotroph (PAK-AR2),

where they become integrated in the chromosome via homologous recombination (Fig.

1). When grown in an enviroment lacking purines, ody the recombinants containing

active promoters in the cloned firagrnents become functionally prototrophic. One cm

then retrieve the cloned DNA, sequence the promoter region, and ultimately identiQ the

downstream gene(s) that is upregulated in the study environment. This particular library

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METHODS

1. IVET (in vivo Expression Technologvi

The IVET library and the purEK deletion strain PAK-AR2 were the generous gift

of Dr. Shouguaog Jin (University of Florida). Briefly, the IVET library consists of

chromosomal fragments fiom the wild-type strain, P. aeruginosa PAK (PAK), inserted

into a unique BglII cloning site upstrearn of a promoterless pwEK operon in a non-

replicative (suicide) delivery plasmid [62]. The delivery plasmid has a ColE 1 replication

ongin that is not fûnctional in P. aeruginosa, and thus is non-replicative. The purEK

operon is single copy and required for survival in purine-free environments [62]. The

recombinant plasmids represent a pool ofpurEK transcriptional fusions driven by

promoters present in the cloned fragments. They are then re-introduced individually, by

electroporation, into a P. aeruginosa purine auxotroph (PAK-AR2), w here they become

integrated in the chromosome via homologous recombination (Fig. 1). The pooled

cointegrates are then grown in the environment of interest, where only clones containing

promoters that are active under those unique conditions will be functionally protatrophic

md survive. Thus, the basis of this method is the growth of purine-requi~g P.

aeruginosa PAK in a biofilm in the absence of purines to select cells expressing purEK

fiom active promoters. Survivhg cells are M e r analyzed to distinguish biofilm-

specific promoters fiom those that are active under many growth conditions (i.e.

constitutive).

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1) ligate into suicide vector upstream of promoterless p u H

. a 2) cross into chromosome of purEK-delete strain

Pad, Pa.k cloned native

3) Chromosome with native promoter (P) driving expression of pwEK and cloned promoter driving expression of potential biofilm gene (abc)

Figure 1. Construction of the IVET library.(* and **- primers for Touchdown PCR and sequencing, respectively)

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2. Growtri ofPAK and PAK-ARS BiofXms indiwtduaITv and in Com~etition

In order to determine whether thepurEK-based IVET selection system was

applicable to our biofilm assay, the ability of a purEK deletion strain, PAK-AN, to

replicate on the silicone substratum was cornparcd to that of the wild-type P M .

PAK and PAK-AR2 were grown individually o h in 5 ml of Davis Minimal

Media with adenine (50 pg/ml) (DMM+A) at 37OC with agitation (150 rpm). The

cultures were diluted to a 0.5 McFarland standard (-10~cells/ml) and then 2.5 ml was

subcuItured into 250 ml of DMM+A. The culture flask was attached to sterile silicone

tubing (Fig. 2). The culture was flowed through the system at 50 mVh for 4 h allowing

bacterial attachment to the silicone substratum. The flow f?om the culture was shut off

and DMM @MM without adenine) was flowed through the system at 50 mVh for 5 days

(120 h). In the literature, flow rates for perfused biofilm assays range fiom 10 to 100 mVh

[65]. In our experiments a flow rate of 50 mVh was used to ensure that 1) bacteria that

rnay be growing planktonically in a slow moving media are washed away and 2) to

expedite the removal of adenine that may have remained tiom the inoculating culture.

On the f i f i day the silicone tube was retrieved and, using a 10 ml pipette, rinsed

vigorously three times with 10 ml of phosphate buffered saline, pH 7.1 (PBS) to rernove

any planktonic cells. The biofilm was scraped with a sterile spatula into 5 ml of PBS.

The suspension was vortexed for 30 sec to break up cellular clumps. Serial ten-fold

dilutions of the suspension were evenly spread onto DMM+A agar for single colonies.

To m e r assess the appropriateness of this system to select biofilm-active

promoters and to detemine the length of selection tirne required, the relative replication

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rate ofPAK &ci pK-AR2 was dëtëmiuieci wïtfi a cornpetition assay. PAK and PAK-

AR2 were grown individually 01x1 in 5 ml of D W A . The cultures were diluted to a 0.5

McFarland standard (-10~cells/rnl) and then 2.5 ml of each was subcultured into 250 ml

of DMM+A. The mixed culture was flowed through the tubing, followed by five days of

DMM. Sections of tubing, 5 cm in length, were sampled evety 24 h. The tubing was

rinsed and the biofilm harvested as described above. Serial ten-fold dilutions were plated

in parallel on Luria-Burtani (LB) agar and LB agar with streptomycin (200 pg/ml) and

spectinomycin (200 pg/ml). It was possible to differentiate the relative composition of

the biofilm by utilizing the fact that PAK-AR2 was streptomycinR/spect inomycinR

whereas PAK was sensitive to these antibiotics. The cfu/cm2 determined from colony

counts on the LB Strep/Spec 200 plates was subtracted fkom that determined From counts

on the non-selective LB plates to determine the number of colonies representing PAK.

3. The Biofilm Assav and Growth of the IVET Librarv as a Biofilm

The IVET library freezer stock consisted of a minimum of 2 x 104 pooled colonies

[62]. With a stenle toothpick an aliquot of the IVET library freezer stock was used to

inoculate 5 ml of DMM+A and grown oln at 37OC with agitation (1 50 rpm). The media

was supplemented with adenine to alleviate the purine auxotrophy and ensure survival of

al1 cointegrates. The 5 ml culhue was then sub-cultured into 250 ml of DMM+A

containhg spectinomycin (200 pg/ml), streptomycin (200 pg/rnl), carbenicillin (1 50

@nl) and tetracycline (100 )ig/ml) and grown for 4 b at 37°C. The culture flask was

attached to stenle silicone tubing as described above and the culture was flowed through

the system at 50 mi/h for 4 h allowing bactenal attachment to the silicone substratum.

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Tlie flow fiom the cuihve was shut offand DMM was fiowed through the system at 50

mlh for 5 days (120 h).

Qf the cointegrates in the IVET library, only those with cloned sequences

containing active promoters were able to overcome the purine auxotrophy. On the fifth

day the silicone tube was retrieved and rinsed vigorously as described above. The biofilm

was harvested and serial ten-fold dilutions were evenly spread ont0 DMM+A agar for

single colonies. Plating on DMM+A ensured growth of al1 the clones in the er biofim

environment.

4. Selection of "Biofilrn-S~ecific" Clones

Single colonies from the DMM+A agar plates were picked and streaked in

parallel on DMM and DMM+A agar plates for o h growth at 37°C. Colonies that failed

to grow, or grew poorly on the DMM plates, but grew well on the DMM+A plates were

re-plated in parallel to confirm the differential growth. Those that continued to grow

poorly were assumed to have biofilm-specific or -enhanced promoter activity and thus

were selected for furthet analysis. Seveo clones with these properties were identified

afler streaking 10,000 colonies in parallel. They were labeled 9.27A-E, 9.29 and 3.15.

5. Confirmation of the Isolated Clones' Abilities to Form Isoaenic Biofilms

Afler detennining which clones appeared to contain biofilm-specific promoters

upstnam of purEK, we tested their ability to fom isogenic biofilms as described above.

This experiment was used to rule out any suMval secondary to spurious acquisition of

purines h m neighbouring clones.

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6. Planktonic Growth of the Isolated Clones. PAK and PAK-AR2

Planktonic growth rates of the isolates (927A-E, 929 and 3 15) in DMM was

assessed to detennine whether growth in a non-biofilm environment was impaired

relative to that of the wild-type (PAK), suggesting reduced promoter activity in this mode

of growth. PAK, PAK-AIU and the isolates were grown individually o/n in 5 ml of

DMM+A. The cells were harvested by centrüùgation (Beckman TJ-6), 5 min at 3000xg,

and washed twice in 1 ml of DMM to remove any residual adenine. The cells were

diluted to a 0.5 McFarland standard (-l~'cellslml). Serial ten-fold dilutions of each

stmin were plated for oln growth to confvm that cell counts in the initial cultures were

equivalent. Each strain was grown in triplicate in either DMM+A or DMM in the

following manner. Forty pl aliquots of each strain were added to 360 pl of broth and the

400 pl cultures were transferred to the wells of a Bioscreen-C (Leb System) automated

growth rate analyzer. The machine was set for 24 h of growth at 37OC with continuous

shaking. The O.D. of 600 nm was determined every 20 min and plotted to generate a

growth c w e . (BioLink Software, Lab System).

7. Southem Blot of the Seven Isolates and PAK with DWEK

To generate a pwEK probe for Southem blot analysis, primes were designed

based on the published sequence ofpurEK (GenBank Accession #U58364) using the

sofhvare program GENE RUNNER (Hastings Software). PurEKup and PurEKdown

primers (Table 1) were used to ampli@ a 785 bp sequence within purEK.

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- Tbe Gene Amp PCR (Potynierase C6ab Reactîon~Sysfem 2400 thennocycler

(Perkin Elmer) was used for al1 PCR amplifications. HotStar Taq (Qiagen) and supplied

buffers were used as directed by the manufacturer for al1 reactions ( H o t ~ t a r ~ a ~ ~ PCR

Handbook, Qiagen). Deoxynucleotide-triphosphates (dNTPs) were diluted fiom 100 mM

stocks (Ameaham) and pooled to generate a final 10 mM mixture. The following PCR

program was used: a 1 5 min 9S°C "ho-start", followed by 30 cycles with denaturation at

9S°C, amealing at 60°C, and extension at 72°C for 45 sec. There was also a final

extension cycle for 7 min at 72OC. The amplicon was confirmed to be correct by size and

by restriction enzyme (RE) digest with h I I . The PCR reaction mixture was separated

on a 1% agarose gel and the amplicon was gel purified according to the manufacturer's

instructions (QIAEXII, Qiagen).

The amplicon, purEK', was labeled by priming with random hexamers and

extending in the presence of digoxigenin @IG) labeled dUTP, using Klenow large

fiagrnent as directed by the manufacturer @IG DNA Labeling Kit, Boerhinger-

Mannheim) and stored at -20°C. Twenty pl of DNA was denatured by boiling for 10

min and then placed on ice. The reaction mixture contained 1 pl of Klenow enzyme (2

unitdu1 DNA Pol 1, Klenow large fkagment), 2 pl of dNTP labeling mixture (1 mM

dATP, 1 m M dCTP, 1 mM dGTP, 0.65 m M dTTP and 0.35 mM DIG-dUTP), and 2 pl of

a random hexanucleotide mix. The reaction was carried out o h in a water-bath at 37°C.

Chromosomal DNA was prepared fiom each of the seven isolated clones and

PAK using DNAZOLO (GibcoBRL) as per the manufacturer's instructions. The DNA

was quantified with spectrophotometry (260/280 nm) and 3 pg digested with EcoRI for 1

h at 37OC. The entire reaction volume, 20 pl, was loaded ont0 a 1% agarose gel and

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~e~atafecîby gei erectrophoresis af 3UV for 8;iZ h. TI% gel was incubated in 0.25M HCI

at RT for 15 min to depurinate the DNA. The gel was then washed in denaturation buffer

(OSM W H , 1 S M NaCI) for 15 min at RT. Lady the gel was placed in a neutraîization

buffer (OSM Tris-HCI, pH 7.5, 1.5M NaCl) to arrest denaturation. The gel was washed

in this buffer twice for 15 min at UT. The DNA was transferred to a nylon membrane

(Zeta-Probe, BioRad) o h by capillary action 1661. To fix the DNA, the membrane was

baked at 120°C for 30 min.

Pre-hybridization, hybridization and detection were carried out according to the

manufacturer's directions (The DIG System Users' Guide for Filter Hybridization,

Boerhinger-Mannheim). The membrane was placed in a heat sealed hybridization bag

(Rose Scientific) with pre-hybridization solution for 2 h at 42OC in a shaking water bath.

The prehybridization solution consisted of 5xSSC (NaCI:NaCitrate, IO: l), 50%

formamide, 0.1 % Na-laurylsarcosine, 0.02% SDS and 2% Blocking Reagant

(Boehringer-Mannheim). Hybridization with the DIG-dUTP labeled 785 bp purEK '

probe was performed at 42OC for 18-24h. The membrane was then washed according to

the manufacturer's instructions (Boerhinger-Mannheim). The membrane was initially

washed twice at RT with 2x SSC and 0.1% SDS and then twice at 68°C with O. lx SSC

and 0.1% SDS. The membrane was then washed twice at RT in maleic acid buffer. This

was followed by incubation in a 1% blocking solution (10% Blocking Reagant diluted in

maleic acid buffer) for 60 min at RT. The hybridized DNA probe was then detected

ushg an anti-DIG polyclonal antibody conjugated to alkaline phosphatase and its

cherniluminescent substrate AMPPD (0.23 5 mM 3 -(2'-spiroadamantane)-4-methoxy-

4(3"-p hosphory loxy )-pheny 1- 1,2-dioxetane) (Boebrioger-Mannheim) followed by

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-?-a- - - -

éxps"uie to Sray filin (KodkkJ as déscnié6by manufctlTm. Xkay fTms were

developed using an automated developer (X-OMAT 480 RA Processor, Kodak).

8. isolation of the Cloned DNA Framents Lyine U~stream of mrEK

To identiQ the cloned promoter regions driving purEK expression, a single

primer was designed based on the intervening sequence between the BglII cloning site for

the IVET library and the 5' end ofpurEK. The primer, PurEKreverse (Table I), was

designed to ampli@ the sequence upstream of the 5' end ofpurEK and thus, permit

determination of the cloned sequence containing the promoter of interest. Chrornosomal

DNA was prepared from the biofilm isolates as described above, and used as template

DNA. "Touchdown PCR" (single primer PCR) [67] was used to ampli f y this reg ion using

the following PCR program: a 15 min. 95OC "hot-start" was followed by 20 cycles with

an annealing temperature starting at 60°C, but decreasing by 1°C per cycle to a final

annealing temperature of 40°C. An additional 20 cycles at an annealing temperature of

40°C were carried out. Denatunition and extension for al1 cycles was at 95OC for 30 sec

and at 72°C for 2 min, respectively. Forty-five pl of the PCR reaction was separated on a

0.7% agarose pl, the largest and/or most prominent band was excised and the DNA

extracted with a gel purification kit (QIAEXII, Qiagen)(Fig. 3).

9. Clonina and Seauencina - of the DNA Framnent Lvina Upstream o f ~ u r E K

The purified DNA was cloned hto TOPO-pCR2.1 (hvitrogen) as per the

manufacturer's instructions. Briefly, the system is based on a plasmid, pCR2.1, which

contains the P-lactamase encoding gene, bIu and a multiple cloning site (MCS) in the

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Figure 3. Touc hdown PCR of four ex-bio fi lm adenine-requiring IVET strains separated on a ethidium bromide stained agarose gel. 1 Kb DNA ladder (Gibco, BU). Schematic of primer location based on the known 5' end of purEK.

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TücZa iÏagment of taie fFgafacfosidase gene Tor a-comptementation. TOP1 O ce 11s are

transfonned with the cloning reaction and thus, some cells will take up pCR2.1 and

others the plasrnid containing the cloned fragment. The colonies are differentiated by

blue-white selection using the chromogenic substrate X-gal(5-bromo-4-chloro-3-indolyl-

PD-galactoside). The plasmid pCR2. I is supplied Iinearized with single 3'-thymidine

overhangs and covalently bound to Topoisornerase 1 (Invitrogen). Taq polymerase has a

non-template-dependent terminal transferase activity that adds a single deoxyadenosine

to the 3'-ends of a subset of the PCR products and thus, 3'-thymidine overhangs facilitate

ligation of PCR products (Invitrogen).

Seven pl of the PCR reaction, 1 pl of pCR2.1, 1 pl of buffer and 1 pl H20 were

mixed and placed at RT for 10 min. Three pl of the reaction mixture was added to 50 pl

of competent cells (TOP10 E. coli) and incubated on ice for 30 min. The reaction was

then placed at 42°C for 2 min to heat shock the cells and induce uptake of the plasmid.

Rich media, LB, was added to the cells and the mixture was then allowed to grow at 37°C

with agitation (150 rpm) for one hour. Forty FI of X-gal(20 mg/ml) was spread plate

ont0 LB plates containing 50 pg/rnl of ampicillin (Arnp 50) and placed at 37OC. Fiw pl

of the culture was spread ont0 the LB Amp 50 plates and allowed to grow o h at 37OC.

After o h growth, up to 20 white colonies and two blue colonies (controls) were re-plated

onto LB Amp 50 plates. The plates were incubated at 37OC for 4-6 h or until adequate

growth was present.

A rapid "E-lyse" protocol was then used to examine plasmid DNA fiom white

colonies to assess whether the PCR amplicon had been successfully incorporated into

pCR2.1[68]. Briefly, bactena are treated with lysozyme to weaken the bacterial ce11 wall

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and'tfien tyseà complefely within the welk o f a SDÇcon~inïng agarose gel. A small

amount of each strain from the LB Amp 50 plates was suspended in 15 pl of TE (10 mM

Tns-HCI and 1 m M EDTA, pH 8.0). Fifteen pl of SRL (25% Sucrose in Tris-Borate - (TB) buffer, 2 U of wAse per ml, and 1 mg/ml of bsozyme) was then added to the

mixture. Twenty pl of each reaction was loaded into a 1% SDS agarose gel. Cunent was

not applied for the fiai 15 min. Thereafter the voltage was set at 25 V for 15 min, then

80-100 V for 2 h. The gel was stained in ethidium bromide (EB), (2 pV10hl from a

stock solution of 10 mg/ml), and viewed under UV light. When compared with vector

controls, the larger plasmids containing the cloned PCR product migrate more slowly.

Several positive clones were re-plated and grown o h ai 37OC.

A commercial plasmid purification kit was used as per the manufacturer's

instructions (QIAprepB Spin Miniprep Kit, Qiagen). Briefly, the process consists of

preparation of the bacterial lysate with SDS, RNAse and sodium-acetate (NaAc)

containing solutions that precipitate chromosomal DNA, RNA and protein. The mixture

is then centrifuged at 13,000 xg in a microcentrifuge for 15 min clearing the supernatant

of this precipitate. The supernatant is then transferred to a colunm where plasmid DNA

is adsorbed to the QIAprep membrane after centrifugation. The coiumn is then washed

with ethanol (EtOH) to remove any contarninating salts. Lastly the plasmid was eluted in

stenle H20 or the supplied buffer (10 mM Tris-Cl, pH 8.5). The plasmid DNA was then

sent to the Molecular Core Facility of York University (North York, ON) for sequencing.

10. Seauencin~ and Analvsis of U~stream Semences

A nested primer, IVETsequence (Table 1), was used to sequence the cloned

insert, generating the sequence of the DNA immediately upstream ofpurEK. DNA

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sëipence ànaIysysis was performedushg BigDye Trmkator Cycre Sequencing on an AB1

377-DNA Sequencer (Applied Biosystems). Sequences were compared to the P.

aeruginosa PA0 1 genome (www.pseudomonas.com) [7]. A region approximately 0.5- 1

kb downstream of the putative promoter-containing sequence was then examined for

potential open reading -es (OWs) in al1 six reading fiames using the software GENE

RUNNER (Hastings Software). BlastP [69] analysis using conceptual products fiom

translation of ORFs that were in the same orientation as the purEK operon was then used

to search al1 protein sequences in GenBank.

The genome for PAK is unavailable, but cornparison of the sequenced fragments

to the PA01 sequences consistently yielded 2 95% shared identities at the nucleotide

level. To examine the distribution of these ORFs amongst various strains of P.

aemginosa, PCR amplification of two of the identified sequences, PA3710 and PA3782,

was carried out with template DNA Rom 1 5 clinical strains of P. aemginosa. Utilizing

the pnmers, AraCup and dom, and BetAup and dom (Table l), PA3782 and PA37 10

were PCR amplified in a multiplex reaction, respectively. Reagants used in the reaction

were as described above. The following PCR program was used: a 15 min 9S°C "hot-

staq" followed by 30 cycles with denaturation at 9S°C for 30 sec, annealing at SOC, and

extension at 72OC for 60 sec. There was also a final extension cycle for 7 min at 72OC.

1 1. RNA Purification and RNA Dot-Blots

PAK was grown o h in a 5 ml culture with DMM+A. Two aliquots of 2.5 ml

were subcultwed to a total volume of 2Sml of DMM grown to log phase over 4 h at 37°C

with agitation. The cells were harvested by centrifugation at 5000 xg for 10 min in a

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JA203Sor (Becban Mode1 JZ-21) andthe supemafàatdîscarCred. The celt pellet was

resuspended in lm1 of Trizol (Gibco) in a Fasthep tube (BIO101). As previously

descrîbed, PAK was also grown as a biofilm over 5 days with continuous, fresh DMM

media. The biofilm was retrieved as described, however after rinsing with PBS it was

scraped into lm1 of Trizol in FastPrep tubes. The tubes were agitated at a setting of 6 for

18 sec. in the BI0 1 O l FastPrep machine disrupting cells and releasing proteins and

nucleic acids. Chlorofonn (200 pl) was added to each tube, mixed by inversion and

Uicubated at RT for 2 min. The tubes were then centrifuged at 1 LOO0 xg for 15 min at

4OC. The aqueous phase containing the RNA was transferred to clean RNAse-free

Eppendorf tubes. In order to precipitate the RNA, 250 pl of isopropanol was added to the

tubes, mixed by inversion and incubated at RT for 15 min. Also, to reduce

polysaccharide contamination, 250 pl of High Salt Precipitation Solution (MRC Inc.) was

simultaneously added. The mixture was then centrifuged at 13000 xg at 4OC for I O min.

The supernatant was removed and the pellet washed with 1 ml of 80% EtOH by

centrifugation at 13000 x g for 5 min at 4OC. The pellet was air dried and then

resuspended in 100 pl of diethyl pyrocarbonate @EPC)-treated H20. To precipitate the

RNA, 2x volume of 100% EtOH and 1/10 volume of 3M NaAc were added and the

mixture was placed at -70°C for 2-4 h. The sample was then centrifuged at 13000 xg for

5 min at 4OC. The pellet was then washed with 70% EtûH and centrifùged at 10000 xg

for 5 min. The pellet was air dned and resuspended in 50- 100 pl of DEPC-treated &O.

The RNA was quantified with spectrophotomeüy (260/280 nm) and assessed

quaiitatively with agarose gel electrophoresis to ensure equal loadhg of intact total RNA

onto the blots.

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A vacuum manifold bot-b tot-apparatus (Bio-Rad) was used to spot RNA ont0 a

nylon membrane (Zeta-probe, Bio-Rad). Five pg of RNA was diluted to 100 pl with

DIG-RNA dilution buffer (Boehringer-Mannheim). Serial two- fold dilutions fiom 5

pghl to 3.9x10-~ pg/ml of RNA were carried out. Fi@ pl of each dilution was spotted

onto the membrane and vacuum suction applied until fluid in each well was no longer

visible, The membrane was then baked at 120°C for 30 min to fix the RNA.

Hybridization with the DIG-dUTP labeled DNA probe of interest was c h e d out using

DIG EasyHyb solution (Boehringer-Mannheim) at 42OC as described by the

manufacturer. The probes consisted of the identified ORFs downstream of the cloned

promoten. DNA-RNA hybridization was detected as described above for the Southem

blots. Differences in expression were quantified using densitometry and Fiuorchem

software (Alpha Innotech Corp.).

12. Generation of a PA5065 Mutant

To construct chromosomal mutants a DNA fragment containing the ORF of

interest, PAS065, and flanking regions was amplified with PCR. Primers, AarFKOup

and AarFKOdown, were designed to ampli@ a 2.2 kb region that included PA5065

(Table 1). The following PCR program was used: a 15 min 95OC bbhot-start", followed by

30 cycles with denaturation at 9S°C, annealing at 62*C, and extension at 72OC for 2 min.

There was also a final extension cycle for 7 min at 72OC. The amplicon was cloned into

pCR2.1 and transformed into TOP 10 E. coli as previously described. E-lyse was used to

screen for successfÙl cloning and transformation, Purified plasmid DNA, pCR2.1-

PA5065, was digested with XbaI and Hindm to release a -2.2 kb hgment that contained

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PAS065 The mutti-cwy pUCT9-dêniëii pUCP26, a pT~i&containing a MCS Ùi TacZa

and a tetracycline resistance cassette, was purified and digested with Xbal and HinaII.

The insert of pCR2.1 and the linearized pUCP26 vector were gel purified as described

above. A 1 0 ~ 1 reaction containing the purified PA5065 product and pUCP26,Sx buffer,

1 pl of T4 DNA ligase (lU/pl, Gibco) and H20 were mixed. The ligation reaction was

carried out at RT for 5 min.

Competent E. c d Ml 09 cells were prepared in the following mannet. Briefly, a

5 ml culture of E. coli MI09 in LB was grown o/n at 37OC with agitation (150 rpm). A

1/40 subculture into 20 ml of LB was grown for 2-4 h at 37OC with agitation. The cells

were hawested by centrifugation. The 30 ml culture tube was placed in a JA20 rotor and

centrifuged for 5 min at 5000 xg (Beckrnan Mode1 J2-2 1). The supematant was

discarded and the cells were resuspended in 50 mM of cold sterile CaC12 and placed on

ice for 30 min. The suspension was centrifuged as above and the supematant discarded.

The cells were resuspended in 50 m M CaCI2 and 10% glycerol. Aliquots of50 pl were

placed in sterile Eppendorf tubes and stored at -70°C. A 50 pl aliquot of chemically

competent E. coli SM 109 was mixed with 3-5 pl of the ligation reaction and placed on ice

for 30 min. The mixture was then placed at 42OC for 2 min to heat-shock the cells and

facilitate transformation. LB was then added to the mixture and the cells were incubated

at 37OC with agitation ( 1 50 rpm) for 2 h. E. coli JM 109 (genotype: A(lac-proAB),

F'(traD36, proAB, lacIZAM 1 S), requires a-complementation and a gratuitous inducer to

activate the lac operon present on the F' episome (Table 3). After 2 h of growth, PTG

(isopropylthio-p-D-galactoside), an artïficial inducer of lac, was added to the mixture.

The culture was then plated on LB agar containing tetracycline (15 pg/ml) and 2% X-gal

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for o h growth at 37OC. in the presence of IPTG andkgal, bacteria containing

pUCP26-PA5065 would produce a white colony as compared to blue colonies produced

by bacteria that took up a recircularized pUCP26. Bacteria that were not transformed

were counter-selected by teûacycline. Several white colonies and two blue colonies,

controls, were selected and re-streaked on LB Tet 15 agar and grown at 37OC for 4-6 h.

E-lyse was used to screen for successful ligation and transformation. Purified plasmid

DNA, pUCP26-PA5065, was digested with BglII that linearized the plasrnid since a

unique RE site was present within PAS065.

The plasmid, pUCGm, contains a ~ r n ~ cassette (aacCl) flaaked by multiple pairs

of RE sites [70]. The basis of constnicting P. aeruginosa mutants is to insert this cassette

into the ORF of interest. The plasmid pUCGm was digested with BamHI, releasing an

875 bp sequence containing the ~m~ cassette. The (2rnR cassette was lipted to pUCP26-

PA5065 linearized with BgnI, which has compatible sticky ends, and transformed into E.

coli SM l O (genotype: thi-l thr leu tonA lac Y supE recA RP4-2-Tc: :Mu, k n R ) as

described above. Successful transforrnants were selected by oln growth on LB agar

containing gentamich (1 5 pg/ml). Puritied plasmid DNA was prepared fiom colonies

that grew on the selective media and digested with f i a l and HindIII. The products were

separated by gel electrophoresis to ascertain correct synthesis of the construct.

The plasmid pEXl8Ap is a novel gene replacement vector conîaining bla, the

counterselectable SUCB marker and a MCS in lacZa for blue-white screening [7 11.

Purified pEX 1 SAP was digested with XbaI and HindIII. The -3 kb product,

~ ~ 5 0 6 5 : : ~ r n ~ released fiom pUCP26 by Xb4I and Hindm, was gel purified and ligated

to the linearized pEX18AP as described above. Competent E. coli SM10 cells were

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msf i rmea as descriiaabove. LITwas thën ad'ed7o the rnixhue and the ce11s were

incubated at 37OC with agitation (150 rpm) for 4-6 h. The culture was then plated ont0

LB agar containing gentamicin (1 5 pg/ml) and grown oln at 37°C. Cells that had been

successfully transformed were then mated with PA0 1 to conjugally transfer pEX 1 8Ap-

~ ~ 5 0 6 5 : : ~ r n ~ . E. coli SM10 expresses chromosomally encoded tra fûnctions that

facilitate conjugation. Briefly, broth cultures of PA01 and E. coli SM 10 (the donor

strain) are grown oln. Egual volumes of the cultures are mixed and -1 ml aliquots are

placed in Eppendorf tubes. The cells are harvested by low speed centrifugation in a

microcenüifbge (4000 xg for 5 min). The supernatant is discarded and the cells are

gently resuspended in 30 pl of LB. The 30 pl aliquots are spotted ont0 a LB sgar plate

and incubated oln at 37OC.

After mating, cells were scraped from the plate with a sterile toothpick,

resuspended in 200 pl of LB and plated on Pseudomonus isolation agar (PIA) containing

200 pg/ml gentamicin to counterselect the donor while selecting for gentarnicin resistant

P. aeruginosa. Resulting PA01 colonies were plated on LB containing gentamicin 200

pg/ml and 5% sucrose to identie isolates that had lost the vector-associated sacB gene

and thus, had become resistant to sucrose.

To c o n h loss of the vector-associated DNA, surviving isolates were plated in

parallel on LB Gm 200 and LB agar containhg Piperacillin (1 5 pglml). Piperacillin

sensitivity c o n f h s successful double crossover with loss of vector-associated markers

and the disrupted gene replacing the native functional copy. However, after several

atternpts, none of the CimR sucroseR colonies were Piperacillin sensitive. This indicated

that the native fhctional copy of PAS065 had been retained in tandem with the cloned

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~~5065::~rn~ and sucrose resïstance IEkefy ar6se thoufi spontaneous mutation in sacB.

Lack of a double crossover under selective pressures suggested that disruption of PASO65

would prove lethal. In order to prove that PA5065 is an essential gene, the following

experiments were carried out.

13. Confirmation that PA5065 is Essential for Survival in P. aenrainosa

To explore the lethality of a PAS065 mutation, we attempted to mutate the

chromosomal copy of PA5065 in the presence of a plasmid containing a bctional

PA5065. Electrocompetent PA0 1 cells were prepared in the following marner. Briefly,

a 250 ml culture of PA01 in DMM was grown o/n at 37OC with agitation (1 50 spin). A

1/50 subculture into 200 ml was grown at for 4 h 37°C with agitation. The cells were

harvested by centrifugation. The 250 ml culture bottle was placed in a JA14 rotor and

centrifuged for 5 min at 5000 xg (Beckman ModelJ2-2 1). The supematant was removed

and the cells were washed in 200 ml sterile cold 10% glycerol. The suspension was

centrifùged as above and the supematant discarded. The cells were resuspended in -1 mI

of 10% glycerol that remained and 50p1 aliquots were placed in Eppendorf tubes and

stored at -70°C.

Purified pUCP26-PA5065 was transfomed into electrocompetent PA01 in the

following manner. A 50 pl aliquot of electrocompetent cells was transferred to a 2 rnm-

gap width electroporation cuvette (BIO/CAN) and placed on ice. Purified pUCP26-

PA5065 (3 pl of -250 pg/pl plasmid DNA) was added to the cells. The cuvette was then

placed in the electroporation device (E. coli Pulser, Bio-Rad) and pulsed at a setthg of

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2.5 kV (25pF capacitance) generating a h e constant of 4.5-5 msec. Two hundred pl of

LB was added to the cells after transfomation and the bacteria grown for 2 h at 37°C

with agitation (150 rpm). The cells were then plated on LB Tet 50 agar. et^ colonies

arising represented successfil transformation of PA01 with pUCP26-PA5065. The

identity of the plasmid in PA01 was confirmed by plasmid purification and RE digest. A

commercial plasmid purification kit (QIAprepB Spin Miniprep Kit, Qiagen) was used as

described above with the following modification. The bacterial lysate was prepared as

per the manufacturer's instruction, but to enhance plasmid yield, four preparations were

sequentially centrifbged through the sarne adsorption column.

The next step was to mutate the chromosomal copy of PA5065 in this

recombinant stmin of PA0 1. Previously prepared E. coli SM I O-pEX 1 8Ap-

~ ~ 5 0 6 5 : : ~ m ~ was mated with PA01-pUCP26-PA5065 The ceIl mixture was plated on

PM Tet 50 Gm 200 to counterselect the E. coli donor strain. Resulting colonies were

plated in parallel on LB Gm 200 and LB Pip 15. Al1 colonies grew on LB Gm 200, and

al1 were Piperacillin sensitive. As previously described, plasmid purification and RE

digest were used to confm that the undismpted copy of PA5065 was present on

pUCP26. PCR with purified template DNA from P A O ~ - P A ~ O ~ ~ : : G ~ ~ - ~ U C P ~ ~ -

PA5065 and the AarFKO prirners (Table 1) produced only the 2.2 kb amplicon

representing undisrupted PAS065, Iikely due to the high copy number of the plasmid-

borne gene template. Therefore to confim that the chromosome contained

P A S O ~ S : : G ~ ~ , pnmers were designed to ampli@ the ~m~ cassette, GmUp and GmDown

(Table 1). The expected amplicon of 700 bp was produced, confinning that the cassette

was present in the chromosome.

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1 -

The expression ofPA5065 in trans~aTToweiisurviv;irof tIïe PA01 mutant. The

ubi& gene (formerly yigR) of E. cofi is a homologue of PASO65. To test whether it was

a functional homologue, the following expenments were conducted. RE-linked prirners,

YigRKOup and YigRKOdown (Table 1) were designed to PCR ampli@ a 1.8 kb region

of the E. coii chromosome containhg ubiBEc . The following PCR program was used: a

15 min 9S°C "hot-start", followed by 30 cycles with denahuaiion at 9S0C, annealing at

60°C, and extension at 72°C for 2 min. There was also s final extension cycle for 7 min

at 72OC. The PCR reaction was separated by gel electrophoresis, and the amplicon was

excised and gel purified as described above. The B-lactamase encoding cloning vector

pUCP20 has the same origin of replication as pUCP26 and thus, the two are

incompatible. The ubiBEc-containing amplicon was ligated into the MCS of pUCP20 and

the resulting construct transfonned into E.coli JM 109 as described above. The cells were

grown at 37OC for 1 h with agitation (150 rpm) and plated on LB Amp 50 agar. E-lyse

and a RE-digest of purified plasmid DNA confhed successful ligation and

transformation. PAO 1 - P A ~ O ~ ~ : : G ~ ~ - ~ U C P ~ ~ - P A ~ O ~ ~ was made electrocompetent in

the manner above. Purified pUCP20 and pUCP2O- ubiBE, were transfomed in psrdllel

into these competent cells. LB was added to the mixture and the cells were grown ai

37°C with agitation for 4 h. Fifty pl of each culture was plated ont0 LB Pip 15 agar

plates to select for pUCP20 and against pUCP26 and grown o h at 37OC. Since both

plasmids have the same origin of replication they are incompatible and thus, the bacteria

should retain oaly one type of plasmid, especially under selective pressure. Swiving

colonies were re-plated and grown for 46h at 37OC, plasmid DNA was purified and RE-

digested with EcoRI and HindlII, as previously descriied. The reaction was separated by

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gel efec@ophorësis. Expression ofib;iBEc in han^ waS able to comptement the

inactivated chromosomal~~5065::~m~ and the pUCP26 construct was lost during

Piperacillin selection. However, cells transfomed with pUCP2O retained both pUCP2O

and pUCP26-PA5065 even in the presence of Piperacillin selection, suggesting that

PAS065 is essential for survival. To m e r veriQ that both plasmids had been retained,

purifid plasmid DNA was used to transfomi E. coli M109. The transformed cells were

plated separately ont0 LB Tet 15 and LB Amp 50 agar plates. Plasrnid DNA was then

purified fkom the E. coli transformants, RE-digested and separated by gel electrophoresis,

confuming both pUCP2O and pUCP26-PA5065 were present in the plasmid DNA

isolated fiom the PA0 1 transformants.

14. Generation of PA3710 and PA3782 Mutants

A modified and more expeditious approach to that descnbed above was used to

constmct PA3782 and PA3710 mutants. A DNA fiagrnent containing the ORF of interest

(PA3710 or 3782) a d flanking regions to -2.8 kb was amplified with PCR. Primers

were designed to include an EcoRi site (forward) and a HindIII site (reverse) (Table 1).

The following PCR program was used to ampli@ PA3782 and adjacent DNA with

AraCKOup and AraCKOdown pnmen (Table 1): a 15 min 95OC "hot-start" followed by

30 cycles with denaturation at 9S°C for 30 sec, annealing temperature of 60°C, and

extension at 72°C for 2.5 min. There was also a fmal extension cycle for 7 min at 72OC.

PA37 10 was amplified in a similar fashon using BetAKOup and BetAKOdown primers

(Table 1). The PCR program was the same as that used for PA3782 except the amealing

temperature was 6S°C. The amplicon and pEX l8Ap were digested with EcoRI and

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-llidiE Boa prducfs were separateir 6y ge te~cti'opiiorësis andgel purified as

previously described. The amplicon, pEX 18Ap, 5x buffer, T4 DNA ligase (Gibco) and

H20 were mixed and the ligation reaction was allowed to proceed at RT for 30 min to

improve efficiency. Competent E. coli M l 0 9 cells were mixed with 3-5 pl of the

ligation reaction and placed on ice for 30 min. The mixture was then placed at 42OC for 2

min to heat-shock the cells and facilitate transformation. LB was then added to the

mixture and the cells were incubated at 37OC with agitation (150 rpm) for 1 h.

Thereafler, IPTG was added to the mixture and the culture was plated on LB Amp 50

plates containing X-gal and grown oln at 37OC. As described above, E-lyse was used to

screen for successfil ligation and transformation. To M e r confm correct ligation and

transformation, plasmid DNA was purified and digested with EcoRi and HindIII, and

separated b y gel electrophoresis.

The 2.85 kb amplified region containing PA3782 has a unique Pst1 site 788 bases

fiom the start of the ORE The plasmids, pUCGM and pEX 18Ap-PA3782, were digested

with P d releasing an 875 bp (3rnR cassette from the former and linearizing the latter.

The DNA was separated by gel electrophoresis and the fragments of interest were gel

purified. The gel-purified products were ligated using T4 DNA ligase as described

above. Competent E. coli SM 10 cells were transformed as described above. LB was then

added to the mixture and the cells were incubated at 37°C with agitation (150 rpm) for 4-

6 h. The culture was then plated ont0 LB agar containhg gentamich 15 pglml and

grown o h at 37OC. Correct orientation of the ~ r n ~ cassette and its promoter within the

gene of interest, to prevent polar effects on downstream genes, was c o n h e d by

restriction analysis.

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CëKs 16at liad k e n successfinjl tiansfo~ehweië then mated with PA0 1 to

conjugally iransfer p ~ ~ l 8 ~ p ~ ~ 3 7 8 2 : : G m ~ . After mating, cells were plated on PIA

containing gentamicin 200 pg/ml to counterselect the E. coli donor. Resulting P.

aenrginosa colonies were plated on LB containing 200 pg/ml of gentamicin and 5%

sucrose to identiQ isolates that had lost the vector-associated sacB gene and thus, had

become resistant to sucrose. To m e r confirm loss of the vector associated DNA and

the native chromosomal copy of the gene, surviving isolates were plated in parallel on LB

Gm 200 and LB Pip 15. Gentamicin cesistance and Piperacillin sensitivity (loss of the

vector-associated marker) confirmed successful gene replacement. PCR utilizing the

initial primers was perfomed to verim that correct gene replacement had occurred.

Correct gene replacement would produce an amplicon equal in size to the initially

amplified region plus the ~m~ cassette. Similar techniques were used to inactivate

PA3710, however the ~ r n ~ cassette was cloned into a unique BamHI site within PA3710.

15. Growth of PAOI. the PA3782 and PA3710 Mutants Planktonicall~ and as Biofitms

Single colonies of PA0 1, ~ ~ 3 7 10::GmR and ~ ~ 3 7 8 2 : : ~ m ~ were inoculated into

5 ml of DMM and grown o h at 37OC with agitation. The cultures were standardized to a

0.5 MacFarland standard (-10~cells/ml). Each strain was grown in triplicate in DMM in

the following manwr: 40 pl aliquots of each strain were added to 360 pl of broth and the

400 pl cultures were transferred to the wells of a Bioscreen-C (Lab System) automated

growth rate analyzer. The machine was set for 24 h of growth at 37OC with continuous

shaking. The O.D. of 600 nm was determined every 20 min and plotted to generate

growth curves. (BioLiok Software, Lab System).

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For biotilm growtb, 2.5 ml of each culture was subcuitured into separate flasks of

250 ml of DMM and flowed through silicone tubing in parallel, as previously described.

Colony counts of the initial standardized cultures were performed to confirm that the

starting inocula were sirnilar. Afier 4 h fresh DMM was flowed through the system at 50

mVhr for five days. On the fifi day, 5 cm of the silicone substratum was rinsed three

times with 10 ml of PBS and the biofilm scraped with a sterile spatula into 5 ml of PBS.

Serial ten-fold dilutions were plated onto LB agar and grown o h at 37OC. Colony counts

were detemined and expressed as cfu/cm2.

16. Cornpetition Biofilm Assav: PA0 1 mown with P ~ 3 7 8 2 : : ~ r n ~ and ~ ~ 3 7 10 : :~ rn~

Single colonies of PAOl, ~ ~ 3 7 10::emR and ~ ~ 3 7 8 2 : : ~ m ~ were itioculated into

5 ml of DMM and grown oln at 37OC with agitation. The cultures were diluted to a 0.5

McFarland standard (-10~cellslml). Equal volumes, 2.5 ml each of PA0 1 and

~ ~ 3 7 10:GrnR were subcultured into 250 ml of DMM and flowed through the biofilm

apparatus at 50 m l h for 4 h. In parailel, and in a similar fashion, a 250 ml 111 00

subculture of PA0 1 and ~ ~ 3 7 8 2 : : ~ m ~ was used to inoculate the silicone substratum.

After 4 h, fiesh DMM was flowed through the system at 50 ml/h for five days. At 24 h

intervals, 5 cm of tubing was reûieved, and the biofilm harvested as previously described.

Serial ten-fold dilutions were plated in parallel on LB agar and LB agar with gentarnicin

200 pg/ml. It was possible to diflerentiate the relative composition of the biofilm by

screening for ~m~ encoded by the cassette that had been used to create the mutants. The

cfulcm2 detemined from colony counts on the LB Gm 200 plates was subtracted fiom

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-- - tbat determineci from counts on the non-setective taptates to detemine the number of

colonies representing PA0 1.

17. Scannine Electron Microscoov (SEM of Biofilrns

PAOI, ~ ~ 3 7 10::~rn~ and ~ ~ 3 7 8 2 : : ~ r n ~ were grown as individual biofilms.

Sections of tubing were excised at 4,8,24, and 48 h. The tubing was rinsed three times

with 10 ml of PBS and then placed in 5 ml of 2% gluteraldehyde. The samples were then

processed in the SEM department at the Hospital for Sick Children. Briefly, each sarnple

was washed three times in PBS. The sarnples were then fixed in 0 s04 for 1 h at RT and

washed three times in PBS, followed by a nnse in ddH20. The samples were then

dehydrated in a graded ethanol senes. The samples were cntical point dried and mounted

on SEM stubs. The specimens were viewed with a EOL 820 SEM and Iridium software

was used to capture the images.

18. Static Biofilm Formation Assav

A recently described biofilm assay utilizing growth in 96-well microtitre plates

followed by crystal violet (CV) staining [38] was used to compare biofilm formation

amongst the mutants and the parent strain, PA0 1. Bacteria grown in this assay have been

shown to fom biofilms on the walls of the wells. CV stains the peptidoglycans in

bacterial ce11 walls. Thus, the measured absorbance of solubilized CV directly correlates

with the extent of biofilm formation.

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- - FAOI, ~ ~ 3 7 1 0 : : ~ r n ~ and~A3782::~m"werë Brown sepiüatety o h in 200 pl of

LB at RT. T'en pl of each culture was subculhired in triplicate into 190 pl of LB in a 96-

well microtitre plate. The culture was incubated o h at RT. A LOO pl volume of the

planktonic culture was transferred to sterile 96-well microtitre plate to be used for 0 . D

600 measurements to standardize results for ceIl density. The remaining volume was

decanted. The plate was then rinsed under a gentle strearn of tap water three times to

remove any nsidual planktonic culture. The wells were thm stained by the addition of

200 pl of 1% CV. After 15 min of incubation at RT the CV was discarded and the plates

were rinsed three times with tap water as above. The retained CV was solubilized with

200 pi of 95% EtOH.

The O.D. at 600 nm of each plate (cells and CV stained) was then measured using

an automated 96-well plate reader (Versaw, Microplate Reader) and the data compiled

with Sofùnax PRO software (Molecular Dynamics).

19. SDS Resistance

The SDS resistance of PA01 and the mutants was tested with the static biofilm

assay. The 96-well microtitre plates were prepared as above. A second plate was

cultured in parallel to test for SDS resistance. AAer the plated was nnsed, 200 pl of 0.2%

SDS was dispensed into each well and incubated at RT for 5 min. The SDS was

discarded and then wells were gently rinsed with three times with tap water. The plates

were then stained and read as described above.

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2 O T Osmotic Stress Assav

Single colonies of PAOl, ~ ~ 3 7 1 0 : : ~ r n ~ and ~ ~ 3 7 8 2 : : ~ m ~ were inoculated into

25 ml of DMM and grown o/n at 37OC with agitation. The cells were then harvested by

centrifugation for 5 min at 10,000 xg in a JA20 rotor. The supernatant was discarded and

the cultures were diluted to a 0.5 McFarland standard (-l~'cells/ml). One hundred pl of

PA0 1, PA37 1 0 : : ~ r n ~ and ~ ~ 3 7 8 2 : : ~ r n ~ were individually subcultured into 1 O ml of

DMM containing 2M NaCl and grown at 37OC with agitation (1 50 rpm). Serial ten-fold

dilutions were plated on LB agar to confirm equal bacteriai inocula for each culture.

Sarnples from each culture were taken at 2,6 and 24 h, serially diluted and plated for ceIl

counts to detennine survival in this hyperosmotic environment.

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RESULTS

1. Growth of PAK and PAK-AR2 Biofilms Individuallv and in Cornpetition

In order to determine whether the purEK-based IVET selection system was

applicable to Our biofilm assay, the ability of a purEK deletion strain, PAK-AR2, to

replicate on the silicone substratum was compared to that of the wild-type PAK.

Although PAK-AR2 could survive and form a biofilrn, the ce11 counts (cfu/cm2) obtained

after five days were four logs lower than those obtained from the PAK biofilm. To further

assess the appropriateness of this system to select biofilm-active promoters, a PAK vs.

PAK-AR2 competition assay was carried out. By 96 hours, the biofilm that was

inoculated with equal amounts of PAK and PAK-AR2 consisted solely of PAK (Fig. 4).

Therefore, growth of the IVET library for five days in the absence of purines sliould

select for clones that contain biofilm-active promoten upstream ofpurEK. Also, this

provides additional proof that non-purine producing strains were unlikely to survive

through acquisition of purines fiom neighbouring bacteria.

2. Growth of the iVET Librarv as a Biofilm and Selection of "Biofiim-Specific" Isolates

The IVET library was grown as a biofilm on a silicone substratum in a flowing

system (see methods). After 5 days, the biofilm was harvested as described in the

methods and senal ten-fold dilutions were plated on DMM+A. The resulting ce11 count

of 1 018 cfu/cm2 was intermediate to the counts obtained ûom PAK and PAK-AR2

biofilms. Given this information, appropriate dilutions of the IVET library biofilm were

plated on DMM+A agar to obtain single colonies. Colonies arising afler biofilm growth

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Biofilm Competition h a y - PAKIARP vs. PAK

1 ~ 1 0 ' ~

Figure 4. PAK and PAK-AR2 grown in a mixed biofilm with equal inocula at Day O. Afier 24 h PAK-AM represented less than 1% of biofilm bacteria and was eliminated by Day 4.

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rë@resented isotates with bïofi-spëcific or constitutFvë promoters cloned upstrearn o f

purEK. Over 10,000 clones were streaked in parallel on DMM and DMM+A agar plates.

Those that grew poorly on DMM were re-tested in parallel on DMM and DMM+A agar

plates (Fig. 5). Seven isolates, numbered 9.27A-E, 9.29 and 3.15, consistently grew

poorly on DMM as compared to growth on adenine-supplemented plates.

3. Confknatioa of the Isolates' Abilities to Form Biofilms

Afier identifjhg clones that appeared to contain biofilm-specific promoters

upstream ofpurEK, their ability to form isogenic biofilms was tested as described in

methds. This experiment was used to rule out survival of attenuated clones by spurious

acquisition of purines fiom neighbouring strains. Each of the isolates formed a biofilm,

with ceIl counts ranging fiom 5-7x 10" cfu/cm2.

4. Planktonic Growth of the Isolated Clones. PAK and PAK-AR2

Planktonic growth rates of the ex biofilm isolates (927A-E, 929 and 3 15) in DMM

was assessed to determine whether growth in a non-biofilm environment was irnpaired

relative to that of the wild-type ( P M ) and the deletion strain (PAK-ARS). We

hypothesize that these clones may have reduced promoter activity, and thus reduced

growth rates due to adenine deficiency, in this non-biofilm mode of growth. An

automated shaker/incubator system (Bioscreen-C, Lab System) recorded O.D. readings at

600nm every 20 min and generated growth curves (Figs. 6-9). Planktonic growth rates of

PAK, PAK-ARS and the isolates in D W A , were similar. Intetestingly, PAK-AR2

was able to grow in the absence of adenine via an unknown mechanism, but a significant

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Figure 5. Ex-biofilm, adenine-requiring P. aeruginosa lVET strains. Clones plated on DMM (A) and DMM+Adenine (B).

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P M and 9.27A Grown in OMM +/- Adenine

PAK and 9.278 Gmwn in OMM +/-Adenine

1 ;

; + 9.278 in DMM -c- PAK in DMM+

, --- * PAK in OMM

Figure 6. Growth curves for P. aeruginosa PAK and the ex-biofilm ~trains A) 9.27A and B) 9.278 grown planktonically in DMM with and without adenine.

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PAK and 9.27C Grown in DMM +\- Menine

PAK and 9.270 Gmwn in DMM +/-Admine

1 7

r-9.27~ in DMM; 1 -9.27D in DMM j +PAK in DMM+ i -PAK in DMM i _ _ - - - - -

Figure 7. Gmwth of P. aemginosu PAK and the ex-biofilm strains A) 9.27C and B) 9.27D grown planktonically in DMM with and without adenine.

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PAK and 9.27E Grown in DMM +I- Adenine

P M and 3.1 5 Grown in DMM +I- Adenine

1 :

1-3.15 in M M ) + PAK in DMM+ : + t P A K inMM ----

Figure 8. Growth of P. aemginosa PAK and the ex-biofilm sûains A) 9.27 E and B) 3.15 grown planktonically in DMM with and without adenine.

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PAK and 9.29 Grown in DMM +I-Adenine

-9.29 in DMM i .- PAK in M M +

PAK and PM-AR2 Grown in DMM +l- Adenine

-PAK-AR2 in DMM+ + PAK-ARZ in DMM -PAK in DMM+ +PAK in DMM

Figure 9. Growth of P. aeruginosu PAK and the ex-biofilm strains A) 9.29 and B) PAK-AR2 grown planktonically in DMM with and without adenine.

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-- - - - - ragplïase, -20 ii, was observed (Fig. 9B). Tn compa6son, the SE of the seven ex biofilm

isolates demonstrated lag phases, v a m g fiom 4 to 12 hours, followed by growth rates in

log phase similar to the wild-type strain. Attenuated or delûyed planktonic growth in the

absence of adenine is consistent with reduced promoter activity and lower expression of

purEK. It should be noted that strain 9.29 had a lag phase of 19 hours and had a growth

curve that was nearly superimposable on that of PAK-AR2

5. Southern Blot to Detect the Number of Uniaue Strains Amongst the Isolated Clones

Total chromosomal DNA was prepared fiom the isolates and digested with

EcoRi. Southem hybndization with a labeled purEK probe (Table 2) was used to screen

for unique hybridization pattems. Six unique patterns were identified (Fig. 10). 9.278

and C appeared sirnilar whereas 9.27A,-D,-E, 9.29 and 3.15 had unique pattems. This

suggested that at least six unique strains were present arnongst the seven isolates.

6. Identification of O ~ e n Reading Frames Downstream of Cloned Promoters

DNA upstream ofpurEK containing the putative promoter of interest was

amplified by "Touchdown PCR" (see methods) and its sequence determined. The

sequences thus acquired were used as in silico probes to locate the promoter regions in

the PA01 genome and to search for downstream ORFs. BlastP 1691 with the conceptual

products from translation of ORB in the same orientation as purEK and downstream of

the cloned promoter was then used to search al1 prokaryotic protein sequences in

GenBank. PA desipations from the P. aeruginosa genome project

(http://www.pseudomonas.com) were used to identiQ the ORFs of interest. Although the

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Figure 10. Southern blot of EcoRI-digested chromosomal DNA ûom 7 ex- biofilm, adenine-cequiring IVET strains, probed with purEK.

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P. ahginosa PAK genome has not k e n sequëncedtfie cronedDNA shared 295%

nucleotide identity with PAOl. Also, PCR amplification of two of the identified ORFs

using chromosomal DNA from 15 P. aeruginosa clinical isolates as templates yielded

amplicons equal in size to those of PAK in each case (Fig. 1 1). This suggested that the

genes were present and the sequences of the primer binding sites were highly

homologous and conserved in al1 strains of P. aemginosa.

9.27A (PA0241, probable MFS (Major Facilitator Superfamily) transporter

and PA0240, porin channel homologue). The cloned promoter fiom 9.27A lay

upstream of PA0241 and PA0240, two ORFs separated by 26 nucleotide base pain.

PA0241 is a probable MFS transporter that shares homology with a number of putative

MFS transporters (Table 4.). Of MFS transporters with known fiuidion, PA024 1 shares

28% identity with ExuT of E. coli over 380 a.a. ExuT is a hexuronate transporter and

part of the complex Exu regulon in E. coli [72].

PA0240 is a probable porin that shares 40% identity over 39 1 a.a. with PhaK of P.

putida (Table 4). PhaK is a membrane protein that imports phenylacetic acid (PhAc)[73].

P. aemginosa has a number of other more highly homologous putative porins; PA04898,

PA22 13 and PA0755 for example. However, of other proteins with known functions,

PA0240 is homologous to OprD, OprE 1 and OprE3 of P. aeruginosa (Fig. 12). OprD

foms a channel that contains a binding site for imipenem, basic amino acids and peptides

[74], and OprE 1 is an anaerobically induced porin [75].

9.278 and 9.27C (PA3710, AkJ homologue) BetA of E. coli and A M of P.

putida have 40% and 55% shared a.a identity with PA3710, respectively (Table 4). The

AlkJ flavoprotein putatively functions as an alcohol dehydrogenase, but is highly

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, 5 Clinical Isolates Y $ ' 1s

Figure 11. Multiplex PCR amplification of PA3782 and PA37 10 using DNA from P A 0 1 and 15 P. aeniginnosa clinical isolates separated on a ethidiurn bromide stained agarose gel. 1 Kb DNA ladder (Gibco, BRL).

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P .putida PhaK ~.aexugi~osa-ûpr~l P.aeruginoaa-PA0240 P. aeruginoea-ûprD

P.putida-PhaK P. aeruginosa OprEl P. aeruginoaa~~A024 O P.aeruginoaa-OprD

P. putida PhaK P. aerugiïiosa-0pr~l P. aeruginoea PA0240 P. aeruginosaIOpr~

P. putida PhaK P. aeruginosa OprEl P. aeniginosa-PA0240 ~.aeruginosd~~pr~

P. put i da PhaK P. aenigi~oea ûprE1 P. aeruginosa-PA0240 P. aeruginosaIûpr~

P. putida-PhaK P. aeruginoad OprEl P. aeruginoaa-~~0240 P. aeruginoaalopr~

P. putida PhaK ~.aerugiÏïoea OprEl p.deruginosa-~~0240 P. aeruginoea~~pr~

P. putida PhaK ~.aenigiÏïoea-ûpr~l P.aeruginosa PA0240 P. aeruginoaaIOpr~

P. puti da PhaK P. aeniginosa-OprEl P.aeruginosa-PA0240 P. aeruginoaa-OprD

PQAKAGEWGQGFTWSGPTEGPVGPGMAMGQLGIKLDS------ SRDRRNTGLLPFG SPSKQE~OQoFILNyQSGFTQo~GKiVDAfXiUOVRLDGGGRAGKSG~RQPGTVFPL S ~ ~ K A E E W T ~ ~ F I ~ G P ~ Q G S V G ~ L D V L G L Y S L K L M ; - - - - - - - GKGTAGTQLLPI DR- - -VDWTQOFLTmESGFTQûTVGFGVDAF'GYLûLKLDG- - - - - - - TSDKTGTGNLPV

* * * .***** ***est *. * * 4

PNSiEPVDDYSELGLTGKIRVSKSTLRLGTLQPILPVVVYNDTRLLASTFQGGLLTSQDV ESNGEP~FAçLGLTAKAKVSNTE:FRYG~QFKLPVVTYNDGRLLPVTFE~VTSTDL HDDORPADDFGRLAVAGIILRVSNSEIiKIGEWMPVLPILRÇDM3RÇLPQTFRGGQLSANEI MNDGKPRDDYSRAGGAVKVRISKTMLKWGnMQPTAPWAAGGSRLFPQTATGFQLQSSEF

*. . ' ..*.. .. * * . . . . DGLTPNAGRLTKANWIDSSGRDDIGYGAAS-------- SDHLDFGGGSYAITPQTSVS -Y KDFTLVAGQLEHSKGRNSTDNRSLSIAGANGSSASSRDSNKFYYAOGDYKVNKDLTLQ-Y

-SNG~RDTDITWIQSGPFKDVSLRWRNVTFRSGNGLTN------ AVüENRLIIGYT -DgS-EWsRoISLAWIPDoTFKGLOfiwKNASFRSGLPAAGSSNNQRDQDENRLIVS~ -DbGSEWGRESELGYTLQSGAFI(RLNVRWRNSSQRRDWGSNTR------ FDENRLIVSYP GEDûKHHETNLEAKYWQSGPAKDLSFRIRQAWHRANADQGEG- - - - - DQNEFRLIVDYP

* . ' * . . . 4 .* te*. . LALW LPLL LSLL LSIL * .

Fig. 12. Protein alignment of PA0240 and its closest homologues with known fhnction. Identical residues (*) and conserved substitutions (.) are noted.

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-& - A

homologous tu the choline dehydrogenase encded 6y beLI [76J BetA is involved in the

glycine-betaine osmoregulatory pathway [77]. A l imen t of PA37 10 with BetA and

Alkl demonstrates a high degree of homology, especially in the conserved glycine box

and ADP binding region characteristic of this group of proteins [76] (Fig. 13).

9.27D and E (PAS065, UbiB homologue) The ubiB gene product (previously

aurF [78 1) in E. cofi and PA5065 have 5 5% ama. identity (Table 4). UbiB is required for

the fmt monoxygenase step in ubiquinone biosynthesis [79]. The presence of

homologues in E. coli, Providencia shiarfii, Neisseria meningitidis and Vibrio cholerae

may suggest conservation of function for this gene product among Gram negative

organisms. Protein alignment of PASO65 with its three closest homologues demonstrates

their significant homology (Fig. 14).

9.29 (PA2247, BkdAl homologue) The b M l gene in P. putida encodes the

alpha-E 1 subunit of a branched chain ketoacid dehydrogenase [80]. The protein

sequences of PAZ247 and BkdAl share 88% identity (Table 4). Expression of this gene is

affected by nitrogen and carbon sources [8 1,821. The planktonic growth curve of 9.29 in

DMM was nearly identical to that of PAK-AR2 (Fig. 9). The lag time in reaching

exponential growth approached 20 h, similar to that demonstrated by PU-AR2. This

suggests that the cloned hgment lying upstream ofpurEK may not contain an active

promoter and that the unknown mechanism relievhg the awtotrophy in PAK-AR2 at 20 h

is also responsible for the growth of 9.29 at that point in time.

3.15 (PA 3782, AdpA homologue) PA3782 shares 49% a.a. identity with AdpA

(Table 4). AdpA is a member of the AraC family of transcriptional regdators (831.

PA3782 and its Streptomyces homologues contain 15 of the 17 amino acids that are

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P. aerugniosa- PA3 71 O P-putida-AlkJ E.coli-BetA H. elongata-BetA

P.aerugnioea-PA3710 P.pucida-AlkJ E. col i -Be- H. elongata-BetA

P.aerugniosa-PA3710 P.pucida-AlkJ E . coli -Be- H.elongata-BetA

PAGTCRMGQGPQAVMAQLRVHGI PGLRIADAS IMPSLTSGNTCS PVLVIAEKAAQMX W PVGTCRMGKDPASWOPCMVRGLRNIRVVDAS IMPNLVAGNTNAPTIMI AENAAEI IVR

Fig. 1 3. Protein aligament of PA37 10 and its closest homo10 es. The Glycine Box and pap-structure of the putative FAD-binding region d s conservecl.

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P. aeruginosa- PA5065 E.coli-übiB P. stuartii-AarF N.meningitidie-AarF

ERVSSEARWtPffsWSDYEKirIVDEL13LLREAANAçQtRRNFEGS PLLYVPQVYWDWCR PRLLPMjRRLRPTEVMlEYE:~LIDELmLRESANAIQLRRNPEDSPMLY I PEVYPDYCS AKALPEARRtKPVEVVRlEYEKTUOELDLRREAANAIQLRRNPENSEELWPEVLTDFCN ERLFADOKRLKPREWAEFDKY~ELDLMREAANASQW3RNFQNSDMLIVP~YCT . . . .+.. +++ . , * + . + * + . a ++,*++ + + .++* 0 + -1 . . + . *

Fig. 14. Protein alignment of PAS065 with its closest homologues.

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--- --- J- conseWeb in the Cterminus of AraCfamiy pt0tehs (Fig. tS) [ml. This family of

transcriptional regulators has diverse bct ions ranging fiom production of virulence

factors to regdation of stress responses [83].

7. RNA Dot-Blots to Confirm Uprermlation of the in-biofilm Induced Genes

As described in the methods, RNA dot blots were prepared with total RNA

isolated fiom P. aenrginosu PAK grown planktonically to log-phase and fiom PAK

grown as biofilm. Intemal fkagrnents of PASO65, PA3710 and PA3782 were used to

probe the blots (Table 2). The alternative sigma factor gene rpoS and the ribosomal

protein S 1 gene psA served as reference standards. The genes rpoS and rpsA have been

shown to be upregulated in biofilms and log-phase growth, respectively [55]. PA5065

and PA37 10 showed increased expression in the biofilm environment (Fig. 16).

Densitometry measurements suggested that there were 6x and 2x greater transcript levels

in the biofilm versus planktonic cells for PA5065 and PA3710, respectively. Blots

probed with PA3782 were consistently negative for both populations regardless of altered

experimental conditions, iocluding loading greater amounts of RNA and decreasing the

hybndization temperature.

8. Generation of PA5065 PA37 10 and PA3782 Insertional Mutants

8.1 PAS065

Insertional mutations were created in PA01 as opposed to PAK because of the

available genome sequence for PA0 1. A PA5065 insertional mutation was attempted,

however, after several unsuccessfbl attempts it became clear that the gene was likely

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S.grieeus-AàpA -MsQDSAAATEAARKLTGRRRREWAVLLFSGGPI FESSIPLSVFGIDRQDAGVPRYRLL S.cotlecolor-AraC-like MçWSTAAPEAAARKtSGRRRKEIVAVLLFSGGPIPESSIPLSVFGIDRQDAGVPRYRU

S .griseus AdpA VCGGEEGPLRTKiGLELTAPYGLEAISRAGTVWPAWRS ITS PPPAEALDALRRAWEEGA ~.coelecoror-Arac-like VCAGEDGPLR~LELTAPQGL~ISRAGTVVVPAWRÇITSPPPEEALDALRRAHEEGA P. aerugiinosa PA3702 VCTAEFGRLRTTVGFTIEAE~LEALAEAQTVI VPSWRDPHERPEQALLDALIAARARGA E. coli-Arac- like ICAEKPGNMSAPGFSVTATWYTAVIQADIVIIPYWGTITQKPPQKLLEAL~ARDNGA

S.griseus AdpA RIVGL~LLDGRPATTHWMYAPTLAKRYPSVHVDPRELFVDDGDVLTSAGT S. coelecoror -Arac- 1 i ke R 1 V G L ~ L L M ; R P A T T H W m A ~ Y P S V H V D P R E L ~ i l G D V L T S A G T

S .grieeus MpA AAGIDLCLHIVRTDHGTEAAGALARRLWPPRRS~GQERYLDRSLPEEIGSDP~AW S. coelecoTor-Arac- like AAGIDLCLHIVRTDH~GALARRLWPPRRsGGQERYmRsLPEEIWP~AW P.aerugiinosa PA3782 V A G I D C C L H L V R Q ~ W A P H R Q G G Q A Q F I E Q P L P D S A Q D G R L O E L L W

S.griseus MpA ALEHLHEQ FDVET WLI S. coeleco~or ara^- like AtEHLHEQFDVET W WLI P.aerugiinosa PA3782 LRQNLDQAHSLDS PT lm E . coli ara^- 1Ike LRQNIAQQHDLDS LT WLI

. . * . +..** . * * * * e s * * * t*,. .** , , C s , + . . . .

S.griseus AdpA SGRRGSTLSSAAVAVAASVGSGELSLPGPDAWPGRPALPGQRÇAP S. coelecofor-Arac- like SDPPASLAPENAVPFQTRR- - - - - - - - TATPMPAGAAsVPûQRSAP

Fig. 15. Protein alignment of PA3782 and its closest homologues. Highlighted residues are conserved amongst members of the AraC family of transcriptional regdators. PA3782 contains 15 of the 17 conserved residues.

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Figure 16. RNA dot-blots. rpsA and rpoS interna1 controls, upregulated in planktonic (P) and biofilm (B) cells, respectively. PAS065 (ubiB homologue) and PA37 10 (betA homologue) upregulated in biofiim. RNA equall y loaded according to spec trophotometry and gel electrophoresis (not shown).

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- eSsentiaTfir survivat ofTAOf. Therefoe, toconfÎiim fie Tetbality of such a knockout,

supporthg experiments were conducted. First, we constructed an insertional mutant with

a functional copy of the gene being expressed in trans. PCR and restriction enzyme (RE)

digests of pwified plasmid preparations confimed that PA0 1 could survive a

chromosomal mutation by expressing PA5065 in trans (Fig. 17). An EcoRI and HindIII

digest of plasmid DNA from PA01 - P A S O ~ ~ : : G ~ ~ pUCP26-PA5065 produced the

expected 2.2 kb product representing the undisrupted PA5065 Also, PCR with the

AarFKO primers (Table 1 ) and purified template DNA from PA0 1 -PASO~S : : ~ r n ~

pUCPZdPA5065 yielded on1 y an amplicon representing undisrupted PAS065 (2.2 kb),

likely due to the high copy number of the plasmid-borne gene. Therefore, to confinn that

the chromosome contained ~ ~ 5 0 6 5 : : ~ m ~ , primers were designed to ampli@ the ~ r n ~

cassette, GmUp and GmDown (Table 1). The expected arnplicon of 700 bp was

produced, confirming that the cassette was present in the chromosome. We then tested

the ability of a homologue, ubiBEc (yigR) of E. d i , to confer survival if expressed in

tram. Utilizing a plasmid replacement-strategy based on plasmid incompatibility and

antibiotic selection, displacement of pUCP26-PA5065 separately by either pUCP2O or

pUCP20-ubiBEc was attempted.

Although pUCP26-PA5065 was readily displaced by pUCP2O- ubif?, pUCP2O

could not displace pUCP26-PA5065. The ~~5065: :Cirn~ bacteria retained both plasmids

despite antibiotic selection and plasmid incompatibility (Figs. 18). Thus, ubiB expressed

in tram is sufficient for survival.

To m e r confinn that PAO 1 - ~ ~ 5 0 6 5 : : ~ m ~ retained both pUCP20 and

pUCP26-PA5065 under a Piperacillin selective pressure, purified plasmid DNA fiom this

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Figure 17. Restriction enzyme digest (SmaI) of plasmid preparations fiom PA0 1 -PA5065::GmR-pUCP26-ubiB ( 1 4) and E.coli JM 109 pUCP26-PA5065 (5-control) separated on a ethidium bromide stained agarose gel. 1 Kb DNA ladder (Gibco, BE).

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Figure 18. Restriction enzyme digest (EcoRI and HindIII) of plasmid preparations From P A O ~ - P A S O ~ ~ : : G ~ ~ - ~ U C P ~ ~ - P A ~ O ~ ~ that had been electropotated with pUCP20 (1) and pUCP2O-ubiB,, (2) and grown on LB Pip 15 agar. The products fiom the RE digest were separated in a ethidium bromide stained agarose gel. 1 Kb DNA ladder (Gibco, BRL). Note retention of pUCP26-PAS065 in lane 1 regardless of antibiotic selection and incompatibility of the two plasmids.

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- traltrsformant was use& to transform E. cotiJMtW(see methods); Tbe transformeci

M l 0 9 cells were selected on either LB Tet 15 or LB Amp 50. Purified plasmid DNA

fiom these transformants was digested with EcoRI and HindIII, and separated by gel

electrophoresis, M e r c o n h i n g that both pUCP20 and pUCP26-PA5065 had been

present in the recombinant PAO 1 - P A S O ~ S : : G ~ ~ strain grown on LB agar with

Piperacillin (Fig. 19).

These experiments showed that ubiBEC was a functional homologue of PASO65,

and that expression of PA5065 or a functional homologue was necessary for survival if

the chromosomal copy of PA5065 had been inactivated.

8.2 PA3710 and PA3782 Mutants

The novel gene replacement vector, pEXlgAp, and ~ r n ~ cassette containing

pUCGm, were utilized to construct chromosomal mutants (see methods). To confinn

successful mutation of these genes, the primers AraCup and AraCKO dom, BetAKOup

and BetAdown (Table 1) were used to PCR-ampli@ template DNA from ~ ~ 3 7 8 2 : : ~ m ~

and P A ~ ~ I O : : G ~ ~ , respectively. PCR amplification of the mutants' DNA resulted in an

amplicon that was equal to the sum of the amplicon produced with PCR amplification of

wild-type DNA and the cloned (3rnR cassette. During selection for the mutants, bacteria

that were ~m~ and pipR represented cells that had retained pEX18Ap plasmid DNA and

thus, werc merodiploids of the gene of interest. PCR amplification of the DNA from

these putative merodiploids resulted in two amplicons, since they contained both the

native functional copy and a mutated copy of the gene (Figs. 20-2 1).

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Figure 19. E. coli JM109 was transformed with a plasmid preparation fiom PA0 1 -pUCP26-PAS065 that had ken electroporated with pUCP2O and grown on LB Pip 1 S agar. Evidence that both pUCP26-PA5065 (1 and 2) and pUCP2O (3 and 4) wen retained. Restriction enzyme digest of plasmid preparation with EcoRl and HindIU separated on a ethidium bromide stained agarose gel. 1 Kb DNA ladder (Gibco, BRL).

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Figure 20. PCR amplification of PA37 10 with template DNA fiom PA01 - PA37 10::GmR (1 4), a PA37 10 merodiploid (5) and PA0 l (6) separated on a ethidium bromide stained agarose gel. 1 Kb DNA ladder (Gibco, BRL).

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Figure 21. PCR amplification of PA3782 with template DNA fiom PAOI- PA3782::GmR(1-3), a PA3782 merodiploid (4) and PA01 (5) separated on a ethidium bromide stained agame gel. 1 Kb DNA ladder (Gibco, BRL).

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X PEenoWbi'c StudKes o f the PA3710 iihdPA37üTMUfanfs

9.1 Planktonic Growth

The planktonic growth rates of the wild-type PA0 1 strain and its PA^ 7 10::~rn~

and ~ ~ 3 7 8 2 : : ~ m ~ mutants were compared in order to assess whether these putative

"biofilm genes had an effect on non-biofilrn growth. As described in the methods, equal

inocula were used and the O.D. at 600 nm was measured every 20 min. using an

automated incubator/shaker. The growth curves generated were essentially

superimposable, suggesting that the inactivated genes were not required for planktonic

growth (Fig. 22A).

9.2 Biofilm Growth

In order to assess the mutants' abilities to fonn a biofilm, each strain and the wild-

type were individually grown as biofilms for five days. At five days, the biofilms were

harvested and the c wcm2 determined. The wild-type, PA0 1, formed the densest biofilm

compared to the mutants whose ce11 counts were >2 logs less than those obtained fiom

the PA0 1 biofilm (Fig. 22B).

9.3 Com~etition Assav

To further investigate biofilm fitness, cornpetition assays were designed, pitting

PA0 1 against each mutant. Equal quantities of each sedin were CO-cultured and flowed

through the biofilm apparatus. PA01 + ~ ~ 3 7 1 0 : : ~ r n ~ and PA0 1 + ~ ~ 3 7 8 2 : : ~ m ~ mixed

biofilms were grown, in parallel. The biofilms were sampled at 24 h intervals and

relative quantities of each strain were detennhed by comparing total plate counts (both

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Figure 22. Growth of the PA37 10 and PA3782 mutants compared to P A 0 1 A) planktonically and B) as a biofilm for five days.

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~ ~ 3 7 1 0 : : ~ r n ~ vs. PA01

1 ~ 1 0 ~ -- -

O t 2 3 4 5 6

A) Days

Figure 23. Biofihn competition assay with PA37 1 O::GmR and PA0 1. A) colony forrning units for each strain and B) relative composition.

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~ ~ 3 7 8 2 : : ~ r n ~ vs. PA01

Figure 24. Biofilm competition assay with P~3782::Gm~ and PAOI. A) colony forming nits for each s t r a h and B) relative composition.

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Figure 25. SEM of P ~ 3 7 8 2 : : G m ~ biofilm after 24 h of growth. A) 250X and B) 1 OOOX magnification.

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Figure 26. SEM of PA01 biofilm af?er 24 h of growth. A) 250X and B)lOOOX magni fication.

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Figure 27. SEM of PA3710::GmR biofilm afier 24 h of growth. A) 250X and B) 1 000X magnification.

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w& grown in fnplicafe at RTfor 24h. After the cultures were discarded, the ptate was

stained with crystal violet. The absorbance of solubilized crystal violet at 600 nrn was

recorded for each well. There was no difference in biofilm formation between the parent

strain and the mutants based on this assay (data not shown).

9.6 SDS Sensitivity

Bacteria in biofilms are known to be more resistant to biocides, such as SDS, than

their planktonic counterparts [84]. With the static biofilm assay, SDS sensitivity was

tested afier 24 h of growth. Afler the planktonic cells were discarded and the wells rinsed

with H20, the biofilms were exposed to 0.2% SDS for 5 min. Absotbance readings of

solubilized CV were compared to those fkom untreated plates. There was no difference

in SDS sensitivity between PA0 1 and the mutant strains.

9.7 Osmotic tolerance test

PA3710 is homologous to BetA, a protein involved in osrnoprotection. In order

to assess the possible role of PA37 10 in osmoprotection, PA0 1, ~ ~ 3 7 8 2 : : ~ r n ~ and

~ ~ 3 7 1 0 : : ~ r n ~ were subjected to growth under osmotic stress in 2M NaCl DMM, and ceIl

sunival was assessed at periodic intervals [85]. ~ ~ 3 7 1 0 : : ~ r n ~ was most sensitive to

osmotic stress resulting in a decline fkom 10' to 1 o2 cWml in 24 h. PA0 1 was the most

resistant and ~ ~ 3 7 8 2 : : ~ m ~ was intermediate in its ability to survive in this hyperosmotic

environment (Fig. 28).

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1 SURVNAL IN DMM + PM NaCl

Figure 28. Osmotic stress tolerance of PA0 1, P ~ 3 7 1 O::GmR and PA3782::GmR over 24 hours in DMM with 2M NaCl.

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DTSCUSSfON

Traditionally, bacteria have been viewed as individual organisms and have been

grown and studied as a planktonic population. However, bacteria in natural envùonments

are usually found as a community of sessile organisrns organized in a biofilm [86].

Notably, bacteria in biofilms are a significant cause of morbidity because of enhanced

resistance to antibiotics. To date, studies of the initiation of biofilm formation in P.

aemginosa have revealed the importance of flagella and type N pili 1371. Also, there is

evidence of increased alginate production [44] and intercellular signaling during bio film

maturation [45]. Until recently there had not been an attempt to study gene expression in

mature P. aemginosa biofilms on a global level[87]. We utilized an IVET system to

study gene expression in five-day old P. aeruginosa biofilms grown on a silicone

substratum.

The IVET selection system is based on the fact that the medium used to grow the

biofilm lacked purines and thus, isolates surviving the selection process must synthesize

their own via expression ofpurEK. In order to determine the feasibility of using this

IVET system to study biofilm gene expression, a PAK vs. P M - A R 2 cornpetition study

was conducted. By 96 h, biofilm consisted solely of PAK, suggesting that growth of the

library for five days in the absence of adenine should select for strains that contain

biofilm-active promoters.

The biofilm was harvested and plated ont0 DMM+A agar. The surviving clones

had promoters upstream ofpurEK that were presumed to be active in the biofilm

environment. The promoters were either biofilrn-induced or constitutive. Approximately

10,000 single colonies were tested for their ability to grow in the absence of adenine by

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-pkthgon DMM anbDMiW+A in paraiEer. S m h s thafgrew poorly in the absence of

adenine were more likely to house biofilm-induced promoters and thus, were selected for

further analysis. It should be noted that promoters that are active in both the biofilm and

agar plate environments, such as those that are surface-induced, would be missed by this

selection.

The ability of each of the isolates to form a biofilm individually confirmed theù

ability to survive independently in a purine-fiee environment. This eliminated the

possibility of survival secondary to spurious acquisition of adenine from neighbouring

clones. Planktonic growth rate analyses in DMM+A and DMM M e r supported the

notion that the isolates contained promotea with reduced activity during planktonic

growth. Rates were nearly equivalent for PAK in both DMM and DMM+A, and the

isolates in DMM+A. However, the isolates showed attenuated growth in DMM

demonsûated by an increased lag phase, suggesting that this planktonic environment was

not stimulating expression from the promoter upstream ofpurEK. This observation

M e r supported the notion that the isolated promoters were relevant to a biofilm mode

of existence. Interestingly, PAK-AR2 should be unable to grow in the absence of

adenine, however aller -20 h in lag phase, it overcame the awotrophy and entered log

phase growth. The mechanism by which this occurred is unclear at this time.

Regardless, the biofilm cornpetition assay of PAK vs. PAK-AR2 showed that the wild-

type dominated, resulting in the elimination of detectable PAK-AR2 by the fourth day.

This result suggests that clones with active promoters would be the dominant species in

the biofilm.

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Affer anaïyzihg six of the sevë5 isoktes, we itentifiëcïhve different ''in bioflm-

inducible" genes. PA0 1 genome analy sis with the cloned promoter fiom 9.27A

suggested that both PA0241 and PA0240 were under its control. PA0241 is a probable

MFS transporter and PA0240 is a putative porin channel that is homologous to a number

of other putative porin channels in the PA0 1 genome. Likewise, PA0241 shares

homology with a number of other putative MFS transporters. Of proteins with known

function, PA0241 is most homologous to ExuT of E. coli. ExuT is a member of the

anion:cation symporter (ACS) family in the subclassification of the MFS [88]. Proteins

in the ACS family are widely distributed in nature and present in both gram-negative and

gram-positive bacteria 1881. ExuT is a hexuronate (D-Glucoronate and D-Galachironate)

transporter [72]. Once inside the cell, hexuronate, an altemate carbon source, is degraded

to pyruvate and glycenildehyde-3-phosphate through a series of enzymatic reactions [89].

PA0241 may be upregulated in biofilms because the nutritional limitations experienced in

this environment may result in greater dependence on altemate carbon source utilization

by bacteria.

Of proteins with known function, PA0240 shares -40% a.a. identity with the

porins Ph&, OprD and OprE 1. Prolonged imipenem treatment of patients with P.

aeruginosu infections results in selection for imipenem-resistant mutants that either lack

OprD or have significantly reduced OprD levels [74]. Interestingly, it was found that

eluate fkom siliconized latex catheters induced imipenem resistance in P. aeruginosa

[go]. P. aeruginosa grown in siliconized latex catheter eluate did not express oprD and

expressed a new outer membrane protein of about 50 kDa [go]. The fuaction of this new

outer membrane protein is unhiown, however this work M e r supports the notion that

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bacieria alter outer membrane protein expression in response to their environment and as

a means of resisting antimicrobials. Perhaps PA0240 represents an OMP that is

upregulated in biofilms and confers antibiotic resistance or is simply upregulated to

provide a basic transport function instead of a porin such as OprD that is known to

facilitate imipenem entry.

Further studies are required to detennine the significance of PA024 1 and PA0240

in biofilm development. Initially, upreplation at the RNA levei should be confirmed.

Thereafter, it would be interesting to determine any effect mutations may have on biofilm

formation and/or antimicrobial resistance in the case of PA0240.

PA37 10 is homologous to AkJ of P. purida and BetA of E. coli. PA37 10 was

shown to be upreplated two-fold in a biofilm as compared to planktonic growth. Also,

the PA37 10::(3rnR mutant was found to be defective in biofilm formation. It was less fit

in biofilm competition experiments with P A 0 1 and SEM demonstrated delayed biofilm

development. Also, the ~ ~ 3 7 1 0 : : ~ m ~ mutant was significantly more sensitive to

osmotic stress than the wild-type as tested by growth in DMM supplemented with 2M

NaCI. To exclude the possibility of an ionic effect, this experiment should be repeated

with a non-ionic compound such as sucrose.

A dehydrogenase with more homology than PA3710 to BetA is present on the

PA01 genome, suggesting PA37 10 is not BetA. The properties of AkJ and BetA are

similar to those of the dye-Iinked alcohol dehydrogenase of P. aeruginosa that was later

shown to be a periplasmic pyrroloquinoline quinone-containing enzyme, linked to the

electron-transfer chah through a dedicated newly-characterized c ytochrome [76]. A M

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- an&BetA may use coenzyme-Qas the5 e n w point for the erectron-transfer chah, sirnitu

to the succinate, lactate and NADH dehydrogenases [76].

Therefore, the exact function of PA3710 is uaknown at this time.

Protein alignment demonstrates conservation of the N-terminal pap-structure of the

putative FAD-binding ngion amongst PA37 10, Alkl of P. purida and BetA of E. d i .

The N-terminal amino acid sequence contains the so-called glycine box (Gly-X-Gly-X-

X-Gly) and other conserved amino acids that are typical for the binding site of the ADP

moiety of FAD and NAD [76,77].

The alkl gene encodes a 59 kDa protein that shows significant homology with the

flavin protein choline dehydrogenase [76]. AIW is membrane bound and cowerts

aliphatic medium-chain-length alcohols into aldehydes [76]. The properties of AlkJ

suggest that it is linked to the electron transfer chain [76]. The betA gene is part of a

complex regulon in E. coli that is responsible for the osmoregulatory choline-glycine

betaine pathway [77]. BetA is 6 1.9 kDa choline dehydrogenase that catalyzes the

oxidation of choline to betaine aldehyde as the first step of the glycine betaine synthetic

pathway [77]. Accumulation of glycine betaine is an osmoprotective response that

enables bacteria to resist dehydration when exposed to a hyperosmotic environment [9 11.

In E. coli, the prou operon encodes a high afinity transport system for glycine betaine

[19]. Utilizing transposon-mediated lac2 fusions, Prigent-Cornbaret showed upregulation

ofprou in E. coli biofilms [35]. Upregulation of osmoprotective mechanisms implies that

bacteria in biofilms must adapt to a heterogeneous environment in which they encounter

higher-osrnolarity conditions, as well as lower O2 levels and higher ce11 density [4,35].

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- - -- Deositomeûy readiags of RNA dot-Mots demotistrate&a 6;fotd upregulation of

PAS065 in P. aenrginosa biofilms as compared to planktonic growth. Our experiments

that demonstrated survival of PA01 with an inactivated chromosomal PA5065 in the

presence of a plasmid-borne ubi& confinned that PA5065 was a fûnctional homologue

of u b i k . UbB, which is involved in the respiratory electron transport chain, is required

for the fust monoxygenase step in coenzyme Q (COQ) biosynthesis and thus, is essential

to aerobiosis [79]. The heterogeneity of O2 tension levels in biofilms has been

demonstrated with the aid of rnicroelectrodes [4]. Ubiquinone (Coenzyme-Q, or COQ) is

a prenylated benquinone that functions in the respiratory electron transport chain of the

imer mitochodondrial membranes of eukaryotes and in the plasma membrane of

prokaryotes (921. The functional role of the ubiB gene product in the hydroxylation of

octaprenylphenol is unknown. However, the presence of homologues in E. coii, Pr.

stuartii, Y. choierue and N. meningitidis suggests conservation of this gene product, at

least in Gram negative bacteria. Studies in E. coli biofilms also showed upregulation of

genes in response to reduced O2 levels [35]. Although ubiquinones are primarily involved

in respiration, there is evidence supporthg their role in providing electrons for disulfide

bond formation in the periplasm of bacteria [93]. Interesthgly, it was dernonstrated that

bacteria unable to form disulfide bonds (dsb mutants) were defective in motility,

adhesion and lipopolysaccharide production, al1 factors thought to play significant roles

in biofilm formation [94]. Given that PA5065 mutations were lethal, we were unable to

assess any phenotypes that may be associated with loss of PASO65 fûnction. Comparing

the effect of overexpression of PA5065 in the planktonic envuonment versus a biofilm

mode of growth may provide insight into the importance of this gene in biofilms.

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- - PA3782was homoIogous to fiembei%oftîïë AraCfamiTy oftranscriptional

regulators, particularly adpA of S. griseus and argR of P. aeruginosa. PA3782 has 15 of

the 17 amino acids that are conserved in the C-terminal portion of AraC family proteins

[83]. The AraC family of transcriptional regulators is involved in regulation of carbon

metabolism, production of virulence factors, fimbriae and adhesins, and response to

environmental stressors [83]. Interestingly, AdpA mutants of S. griseus are unable to

produce streptomycin or form aerial mycelium or spores [95]. Streptomyces spp. undergo

complex morphological differentiation to form vertical multicellular hyphae terminating

in resistant spores. Based on recent evidence, biofilm development can also be viewed as

a developmental process that shares some features with other bacterial developmental

processes such as sporulation in Gram positive bacteria (961, hiting body formation in

~ o c o c c u s xanthus [97,98], and stalked-cell formation in CouIobacter crescentus [99].

Therefore PA3782, a putative transcriptional regulator, may play a significant role in

biofilm architectural development.

RNA dot-blots probed with PA3782 were consistently negative. This may reflect

a low copy nurnber of PA3782 message amongst the already small mRNA population

(-1%) that exists in total bacterial RNA. An altemate strategy to assess whether PA3782

is upregulated in biofilms would be use of a multicopy lac2 reporter construct and p-

galactosidase assays to quantitate expression in a biofilrn versus planktonic growth. The

~ ~ 3 7 8 2 : : ~ m ~ mutant was sipificantly impaired in its ability to fom biofilms. in

cornpetition with the wild-type, ~ ~ 3 7 8 2 : : ~ m ~ comprised less than 0.05% of the mixed

biofilm by the fourth day. Furthemore, SEM of 24 h ~ ~ 3 7 8 2 : : ~ m ~ biofilms

demonstrated severe deficiencies in biofilm formation. Given that PA3782 is a

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transcriptional regulator, the expression of several genes may be influenced by its

absence. Further studies are required to detemine the fuaction of this transcriptional

regulator. SELEX technology (Systematic Evolution of Ligands by EXponential

e ~ c h m e n t ) could be used to determine sequences of DNA that bind to the PA3782

protein [100]. Altematively, with the advent of DNA microarrays, cDNA fiom PA0 1

and ~ ~ 3 7 8 2 : : ~ r n ~ grown in a biofilm could be used to hybridize to the array to evaluate

differential expression secondary to the lack of PA3782.

With increased biotilm ceIl density one can expect changes in bacterial

metabolism as micronutrients may be more readily available to surface inhabitants as

compared to those embedded in the matrix. PA2247 was highly homologous to bkct41 of

P. pufida. BkdA is the a-El subunit of a branched chain ketoacid dehydrogenase. The

expression of this operon is regulated by carbon and nitrogen sources [10 11. The enzyme

allows bacteria to utilize branched chain amino acids as carbon and energy sources [8 11.

With increased ce11 density there is likely to be limited access to preferred carbon sources

such as glucose and thus, bacteria may have to resort to altemate nutritional strategies.

There is recent evidence of interplay between genes involved in nutrition, the catabolite

repression control gene crc, and those required for twiiching motility, pilA, that are

essential to biofitm formation [102].

PA2247 was determined to be the ORF downstream of the promoter cloned into

9.29. Growth of 9.29 planktonically in DMM demonstrated a sipificant lag time in

reaching log phase that was consistent with that seen in PAK-AR2 This suggested that

9.29 may not necessady contain a biofilm-active promoter and that perhaps it survived

the initial selection process through the unknown mechanism of purine salvage used by

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PAK-ARZ. To test his hypothesis, we coutdutiIize abïofihn competition experirnent

with 9.29 and PAK in DMM to detemine if 9.29 is eliminated by the fourth day as was

PAK-AR2 when it was grown with PAK. Given this uncertainty, PA2247 was not M e r

studied. However, its importance in biofilm formation cannot be excluded. Further study

at the RNA level is required to determine if it is upregulated in a biofilm and what effect

a PA2247 mutation, if any, has on biofilm formation.

Given that b i o f h formation is dependent on several physical properties one must

be cautious in generalizing results from a particular study. The mutants PA37 10:GmR

and ~ ~ 3 7 8 2 : : ~ m ~ did not appear impaired in their ability to form biofilms in 96-well

polysüyrene microtitre plates. The mutant biofilms were equivalent to those formed by

the wild-type as detennined by O.D. 600 nm readings of solubilized crystal violet. Also,

the mutants and PA01 were equally resistant to SDS treatrnent in this assay. This may

reflect the fact that the mutants formed "normal" biofilms in this static assay or that

PA3782 and PA37 10 do not play a role in SDS resistance. Aside from the different

substratum used, the static nature of this assay may have facilitated biofilm formation in

cornparison to a flowing systeni. Given the variability in biofilm formation with

substratum composition, media and static versus flowing conditions, it is prudent to

restrict conclusions to the system under study. Thus, the genes identified by this work

would not have been recognized as important for biofilm formation without the use of a

flowing system and IVET technology

One of the limitations of applying M T to study bacterial geae expression is the

inability to identiw genes that are downregulated in the environment of interest.

Alternate strategies that would identifi genes dom-regulated in a biofilm environment

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iidiube use of DNA microanay s, sutiinictive by6nXiiation-[87JY or differential display

PCR. Al1 of these techniques rely on cornparison of mRNA populations expressed under

different growth conditions. Of note, a gene such as PA3782 would have been difficult

to detect using these technologies. Its level of expression based on the RNA dot blot

results appears to be extremely low, suggesting it would be poorly represented in a cDNA

population prepared fiom total RNA. Therefore, al though IVET has i ts 1 imitations, it

rnay be superior for detection of genes expressed at low levels.

Conclusions

The IVET system was successfûlly applied to the study of gene expression in P.

aemginosa biofilms and helped shed light on the physiology of fully developed P.

aeruginosa biofilms. Gene expression in P. aemginosa biofilms seems to reflect

responses to physiochemical environmental stimuli. The genes identified in this study

appear to be involved in adaptation to increased osmolarity in association with O2 and

nutrient limitations. PA3782 appears particularly important to biofilm formation on a

silicone surface in a flowing system. Understanding the regdatory role of PA3782 may

shed light on its fùnction in biofilm formation. Further characterization of the identified

ORFs through gene inactivation and phenotypic studies may contribute to the present

understanding of P. aenrginoso biotilm ph ysiology .

Current atternpts to prevent andior treat biofilm-related infections have met with

limited success. Ultirnately, a clearer understanding of gene expression in biofilms

should provide more effective interventional and/or preventative strategies for this

growing problem.

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purEKup

purEKdown

purEKreverse

IVETsequence

aarFup

aarFdown

betAup

betA down

araCup

araCdown

bckcl4up

bckdi4down

rpsAup

rpsAdown

V ~ P

rposdown

aarFK0up

aarFKOdown

araCKOup

araCKOdown

betAKOup

betAKOdown

Y & ~ P yigRd0wn

GmRup

GmRdown

Primer Sequence

5' GAG AGA GGC ACG ACG ATG A

5' ACG AAC TGG GAG AGG AAG G

5' TGC GCT CAT CGT CGT GCC TC

5' GTG CCT CTC TCA AAT GCG

5' GGA AGT GGT GGT GAA GGT G

5' ATT GTG GGC GTG CGG TTG TG

5' GGC GAA TGG CGG GTA GAA

5' GGC AGG GTG GAA GAT GGT GG

5' ACC GCA TCA GTC CGT TCC AC

5' AAA TCC TCC GCC CAC ATC C

5' CCA TCG CCA CCG CCC ACA

5' TTC CTC CGA CCA GAT GCC GA

5' GTC ATC AAC GGC AAG GTC AA

5' TCC ACT TCA TCG CCA ACC T

5' CGG TTT CTC GCC CCT GTT GA

5' CCG ACC TCT TCC AGC GTG C

5' TTT CCG CCA AGG TCA TCG

5' GGC GGA AAT GGG AGT AGT G

5' AGA ATT CGC CTT CGT GGA TCG CCT GG

5' AGA AGC TTG CTC AAC TAC GCC CAG GTC G

5' AGA ATT CGC CTG TGC CGC CTG CTG CT

5' AAA AGC TTC CTC CAC CGC CTC CTT GCC G

5' AGA ATT CCG TGC TGT TGA TGC CCT GA

5' TTA AGC T'TG CCG AGC '][TT TTG GTG CC

5' AAG CCT GTT CGG TTC GTA A

5' TTC TTC CCG TAT GCC CAA

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Probe Bases of ORF lncluded in probe # Bases

purEK 172-956 of 1832 785

aarF 447-1 437 of 1602 99 1

betA 433-1422 of 1674 990

araC 35-401 of 954 367

bckd 341-1056 of 1233 716

rpsA 33 1-986 of 1680 656

~ 0 s 2 10-872 of IOOS 663

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S trainPlamid Strain P. aemcrinosa PA0 1 PAK PAK-AR;! 9.27A 9.278 9.27C 9.27D 9.27E 9.29

E. coli - JM109

Plasmids pCR2. I-TOPO pUCP20 pUCP26 pUCPGM pEX 1 8Ap pUCP26-PAS065

Genotype or relevant characteristics

Wild type, serotype 0 5 Wild type, serotype 06; background for IVET PAK deleted of the purEK gene: spR, smR biofilm-induced IVET strain; TC^, pipR, spR, smR biofilm-induced IVET strain; TC^, pipR, spR, smR biofilm-induced iVET strain; TC^, pipR, spR, smR biofilm-induced WET strain; TC^, pipR, spR, smR biofilm-induced M T strain; TC^, piPR, sPR, smR biofilm-induced IVET strain; TC^, pipR, sPR, smR biofilm-induced IVET snain; TC^, pipR, spR, smR PA0 1 with insertional mutation in PA3 7 10 PA01 with insertional mutation in PA3782 PA0 1 with insertional mutation in PA5065, expressing PAS065 in trans fiom pUCP26 lac promoter PA0 1 with insertional mutation in PA5065, expressing E. coli ubiB in trans fiom pUCP2O lac promoter

Reference

WI this study this study this study this study this study this study this study this study this study this study

this study

en& 1. gyrA96, hsdR17(rk- mk+). mcrB+recA 1, relA 1, supE44, thi- 1, A (lac-proAB). F '(truD36. proA B. IacIZA M 1 5 ) thi-1 thr leu t o d lacY supE recA RP4-2-Tc::Mu, [IO41 mcrA A(mm-hsdRMS-mcrBC) @OIaoY74 recA l deoR Invitrogen araD 13 9 A (ara-leu) 769 7 galU

Topoisomerase-linked T/A cloning vector, A ~ ~ , KmR pUC 18-derived broad-host-range vector, Ap /RPR pUC 1 û-derived broad-host-range vector, TC^ Source of (3mR cassette; CimR Gene-replacement vector, or ip , soc^+, Consûuct used for in trans expression of PAS065 in ~ ~ 5 0 6 5 : : ~ m ~ insertional mutant Construct used for in trans expression of E. coli ubie in ~ A 5 0 6 5 : : ~ r n ~ insertional mutant Construct used for generation of ~ ~ 5 0 6 5 : : G m ~ insertional mutation Construct used for generation of ~ ~ 3 7 1 0 : : ~ r n ~ insertional mutation Construct used for generation of pA3782::GmR

this study

this study

this study

this study insertional mutation

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1

Table 4. Putative biofilm-induced proteins and their homologues

ORF (nt) Size Homologous Protein8 Function % Identityc % similarityd GenBank '

(a.a.) Accession #

9.27A PA024 1 ( 1 326) 44 1 Hexuronate transporter,

xantoryaeb MFS tansporter, C. crescentusb ExuT, i?. coli

PA0240 (1 266) 42 1 Phak-like protein, P. putida

PA4898, P. aenrginosab PA22 13, P. aeruginosob PhaK, P. putida

Hexuronate transporter

MFS transporter Hexuronate transporter Phenylacetic acid transporter-like Probable porin channel Probable porh chamel Phenylacetic acid transporter Outer membrane porin Outer membrane porin

9.29 PA2247 (1 233) 4 10 BkdA 1, P. putida Branched-chah ketoacid

dehydrogenase

9.278 PA37 10 (1 674) 557 GMC-oxidoreductase, GMC-oxidoreduc tase

Deinococcus radioduransb AlkJ, P. putida Alcohol dehyârogenase BetA, Halamonas elongatab Choline de h ydrogenase Be t A, Escherichia coli Choline dehydrogenase

CABS 105 1 CAB77 176

S10901

Page 104: OF TO A --. UGNOS~ BIOFEM · -A-- IDENTIFICATION OF GENES IMPORTANT TO PSEZllrDOMONRS AERUGllY(3Ls;a (P-AaIj BTO~FO~TION Degree of Master of Science Antonio FineIli lnstitute of Medical

9.27D and E PAS065 (1 602)

3 17 Transcriptional regulator, E. AraC-like banscriptional coli O 1 5 7 : ~ 7 ~ regulator

Transcriptional regulator. Ar&-like transcriptional Sinorhizobium melilot? regulator AdpA, Sfreptomyces griseus AraC-like transcrip t ional

regulator; streptomycin and aerial mycelium production

AdpA Si. coelicolo~ Ad- l ike transcriptional regulator

542 UbiB, E, coli Ubiquinone biosynthesis

UbiB. Vibrio choleraeb Ubiquinone biosynthesis UbiB (AarF), Providericia Ubiquinone biosynthesis stuartii UbiB, Neisseria meningitidis Ubiquinone biosynthesis

' Homologous proteins, identity and similanty scores were detemined by the BLASTX program at NCBI 7

Hypothetical protein " Identical amino acids

Identicai plus conservai amino acids

Page 105: OF TO A --. UGNOS~ BIOFEM · -A-- IDENTIFICATION OF GENES IMPORTANT TO PSEZllrDOMONRS AERUGllY(3Ls;a (P-AaIj BTO~FO~TION Degree of Master of Science Antonio FineIli lnstitute of Medical

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