University of Tennessee, Knoxville Trace: Tennessee Research and Creative Exchange

Masters Theses Graduate School

5-2008 Development of a Bacteriophage Based Bioluminescent Bioreporter System for the Detection of K12 and O157:H7 Courtney M. Johnson University of Tennessee - Knoxville

Recommended Citation Johnson, Courtney M., "Development of a Bacteriophage Based Bioluminescent Bioreporter System for the Detection of Escherichia coli K12 and O157:H7. " Master's Thesis, University of Tennessee, 2008. https://trace.tennessee.edu/utk_gradthes/388

This Thesis is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Courtney M. Johnson entitled "Development of a Bacteriophage Based Bioluminescent Bioreporter System for the Detection of Escherichia coli K12 and O157:H7." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in . Gary Sayler, Major Professor We have read this thesis and recommend its acceptance: Steve Ripp, Todd Reynolds, David Golden Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) To the Graduate Council:

I am submitting herewith a thesis written by Courtney Marie Johnson entitled “Development of a Bacteriophage Based Bioluminescent Bioreporter System for the Detection of Escherichia coli K12 and O157:H7.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Microbiology.

Gary Sayler, Major Professor

We have read this thesis and recommend its acceptance:

Steve Ripp

Todd Reynolds

David Golden

Accepted for Council:

Carolyn R. Hodges, Vice Provost and

Dean of the Graduate School

(Original signatures are on file with official student records.)

Development of a Bacteriophage Based Bioluminescent

Bioreporter System for the Detection of Escherichia coli K12

and O157:H7

A Thesis presented for the

Master of Science Degree

The University of Tennessee, Knoxville

Courtney Marie Johnson

May 2008 Acknowledgements

I would like to thank Dr. Gary Sayler and Dr. Steve Ripp their guidance. Thanks to all members of CEB for their help and friendship, you are some of the best people I have ever known. I want to acknowledge my committee members, Drs. Todd Reynolds and David Golden. Lastly but most important, I would like to thank Scott and my parents because your love and support kept me going.

ii

Brief Abstract

Detection of pathogenic in the environment and food products is of increasing importance especially in light of recent outbreaks of Escherichia coli

O157:H7. I describe here a bacteriophage based bioluminescent bioreporter method for

E. coli O157:H7 detection that combines the specificity bacteriophage have for their host, quorum sensing, and lux based from Vibrio fischeri. This new method for detection of E. coli utilizes the luxI/luxR quorum sensing present in V. fischeri which uses N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) as an autoinducer (Miller &

Bassler 2001). Once the concentration of OHHL is high enough it binds the LuxR initiating transcription of luxCDABE and additional luxI, leading to the production of light. This bioluminescent bacteriophage bioreporter method uses 2 components, first, the bioreporter cell called E. coli OHHLux which carries the complete lux cassette (luxCDABE) along with the transcriptional regulator luxR (Ripp et al. 2006).

The OHHLux ( resistant) detects the diffusible OHHL produced by the second component, the luxI engineered phage which when infecting target E. coli cells produces autoinducer.

The initial feasibility of the phage based reporter assay was tested using the well characterized lambda phage and E. coli K12 model (Ripp et al. 2006). Once the initial feasibility of this bacteriophage based bioluminescent bioreporter system was confirmed, an E. coli O157:H7 specific phage, PP01, was constructed with a luxI insert. This PP01- luxI phage was used in conjunction with E. coli OHHLux to test for E. coli O157:H7 in pure culture, apple juice, ground beef, tap water, and spinach rinsates. This method has iii the potential to be a sensitive, rapid, and non-invasive method of screening for E. coli

O157:H7 contamination. The system provided rapid and sensitive detection with results in well under 24 h even at E. coli O157:H7 concentrations as low as 1 CFU/ml (Brigati et al. 2007, Ripp Submitted 2007). Our system is self-sufficient and could be adapted to be fully-automated and capable of high throughput screening in a production line setting.

iv

Abstract

Detection of in the environment and food products is of increasing importance especially in light of recent outbreaks of Escherichia coli

O157:H7. Current detection methods for this bacterium can be sensitive and specific although they are usually costly, time consuming, and require skilled lab personnel to carry them out. I describe here a sensitive, rapid, and non-invasive method for E. coli

O157:H7 detection that combines the specificity bacteriophage have for their host, quorum sensing, and lux based bioluminescence from Vibrio fischeri.

The lux produce multiple that carry out a complex set of reactions leading to the emission of visible light at approximately 490 nm. The lux genes include a cassette of five genes (luxCDABE) whose products are necessary for the generation of light, and two genes (luxI and luxR) that produce the components of a quorum sensing system that activates the transcription of the luxCDABE cassette (Meighen 1991). The luciferase genes (luxAB) encode the responsible for generating bioluminescence, while the reductase (luxC), transferase (luxD) and synthetase (luxE) genes encode for proteins that produce an aldehyde substrate required for the bioluminescence reaction.

Previously developed bacteriophage based bioluminescent bioreporters carry the luxAB genes, but lack the luxCDE genes (Ulitzur & Kuhn 1987, Stewart et al. 1989, Kodikara et al. 1991, Chen & Griffiths 1996, Loessner et al. 1996, Waddell & Poppe 2000) . Thus, once the recombinant bacteriophage infects the target cell, detection requires the addition of exogenous substrate (the aldehyde component). Since the entire luxCDABE cassette is

ii too large to be accommodated by most phage , a different approach is necessary to abolish the need for exogenous substrate.

This new method for detection of E. coli utilizes a bacterial quorum sensing system. Quorum sensing is a type of cell-to-cell communication between bacteria based on the synthesis of autoinducer molecules which trigger changes in expression at high concentrations. In this research, luxI/luxR quorum sensing present in V. fischeri was employed which uses N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) as an autoinducer (Miller & Bassler 2001). V. fischeri have low level basal expression of luxI which generates small quantities of this diffusible OHHL. When enough V. fischeri cells are present a threshold is reached where the concentration of OHHL is high enough to bind the LuxR protein initiating transcription of luxCDABE and additional luxI, leading to the production of light. This bioluminescent bacteriophage bioreporter method uses the complete lux cassette (luxCDABE) along with the transcriptional regulator luxR which is carried by the bioreporter cell called E. coli OHHLux (Ripp et al. 2006). OHHLux

(lambda phage (λ) resistant) detects the diffusible OHHL produced by the phage infected

E. coli cells.

The initial feasibility of the phage based reporter assay was tested using the well characterized λ phage and E. coli K12 model (Ripp et al. 2006). A lambda phage was engineered to contain the luxI gene from V. fischeri in its . This reporter phage,

λluxI, was used as a general E. coli detection method. This initial assay proved to be capable of rapidly detecting E. coli at very low population densities. In pure culture, 1

CFU/ml was detected within 10.3 h with an additional preincubation step of 5 h. Lettuce iii washings were also artificially contaminated with E. coli at concentrations ranging from

1 × 108 to 130 CFU/ml yielding bioluminescence within 2.6 to 22.4 h, respectively, including an additional preincubation step required at lower initial cell densities.

Once the initial feasibility of this bacteriophage based bioluminescent bioreporter system was confirmed, an E. coli O157:H7 specific phage, PP01, was similarly constructed with a luxI insert. This PP01-luxI phage was used in conjunction with E. coli

OHHLux to test for E. coli O157:H7 in pure culture, apple juice, ground beef, tap water, and spinach rinsates. This method has the potential to be a viable option of screening for

E. coli O157:H7 contamination. The system provided rapid and sensitive detection with results in well under 24 h even at E. coli O157:H7 concentrations as low as 1 CFU/ml

(Brigati et al. 2007, Ripp Submitted 2007). Our system is self-sufficient and could be adapted to be fully-automated and capable of high throughput screening in a production line setting.

iv

Table of Contents

Chapter I: Introduction ...... 1

Bioluminescence and Vibrio fischeri ...... 2

Bioluminescence reaction ...... 3

Quorum sensing...... 4

Organization of lux genes ...... 6

Regulation of V. fischeri bioluminescence ...... 6

Acyl Homoserine Lactones (AHLs) ...... 7

Structure ...... 8

Synthesis ...... 11

General bacteriophage characteristics ...... 13

E. coli O157:H7...... 14

Traditional detection methods ...... 15

Bioluminescent bioreporters ...... 18

Bacteriophage based bioluminescent bioreporters ...... 20

E. coli O157:H7 bacteriophage based bioluminescent bioreporter system ...... 21

PP01 bacteriophage background ...... 23

Recombinant luxI phage and E. coli OHHLux ...... 27

v

System mechanics ...... 29

Chapter II: Linking bacteriophage infection to quorum sensing signaling and bioluminescent bioreporter monitoring for direct detection of bacterial agents ...... 31

Abstract ...... 32

Introduction ...... 33

Materials and methods ...... 36

Bacterial strains and bacteriophage ...... 36

Genetic construction of the λluxI reporter bacteriophage ...... 37

Genetic construction of E. coli OHHLux ...... 40

Dose response kinetics of the E. coli OHHLux bioreporter to OHHL ...... 43

Specificity of the E. coli OHHLux bioreporter towards OHHL ...... 44

Phage reporter pure culture assay ...... 44

Lettuce leaf wash assays ...... 46

Analytical measurement of OHHL ...... 48

Results ...... 48

OHHL dose response of the E. coli OHHLux bioreporter...... 48

Specificity of the E. coli OHHLux bioreporter towards OHHL ...... 50

Phage reporter assay in pure culture ...... 50

Lettuce leaf rinse assays...... 53 vi

Discussion ...... 55

Chapter III: Bacteriophage based bioluminescent bioreporter for the detection of E. coli

O157:H7 ...... 61

Abstract ...... 62

Introduction ...... 63

Materials and methods ...... 66

Bacterial strains and bacteriophage ...... 66

Construction of the reporter phage ...... 66

Detection of E. coli O157:H7 in pure culture ...... 73

Specificity of assay for E. coli O157:H7 ...... 74

Detection of E. coli O157:H7 in apple juice ...... 75

Results ...... 76

PP01-luxI reporter phage assay in pure culture ...... 76

Specificity of PP01-luxI reporter phage assay ...... 76

PP01-luxI reporter phage assay to detect E. coli O157:H7 in apple juice ...... 79

Discussion ...... 79

Part IV: Bacteriophage-amplified bioluminescent sensing of E. coli O157:H7 ...... 85

Abstract ...... 86

Introduction ...... 87 vii

Materials and methods ...... 89

Bacterial strains and bacteriophage ...... 89

96-well microtiter plate sampling formats for pure culture, apple juice, tap water,

and ground beef detection assays ...... 90

Real-time bioluminescent imaging of E. coli O157:H7 in spinach leaf rinsate ...... 93

Results ...... 94

Determination of E. coli O157:H7 inoculum concentrations ...... 94

Detection of E. coli O157:H7 in pure culture ...... 95

Detection of E. coli O157:H7 in apple juice ...... 97

Detection of E. coli O157:H7 in tap water ...... 97

Detection of E. coli O157:H7 in ground beef ...... 100

CCD imaging of E. coli O157:H7 in spinach leaf rinsates ...... 100

Discussion ...... 101

Chapter V: Conclusions ...... 106

References ...... 110

Vita ...... 130

viii

List of Tables

Table 1. AHL examples and their host organism ...... 9

Table 2. luxCDABE bioreporters ...... 19

Table 3. Bacteriophage based bioluminescent bioreporters for the detection of target ...... 22

Table 4. Bacterial strains used to test PP01 phage‟s specificity and host range ...... 25

Table 5. Strains tested for interference against the OHHLux bioluminescent bioreporter45

Table 6. Detection of E. coli XL1-Blue in leaf lettuce wash assays ...... 54

Table 7. LCPS in pure culture averaged across all five E. coli O157:H7 strains tested ... 78

Table 8. Detection of E. coli O157:H7 ATCC 43888 in apple juice ...... 81

ix

List of Figures

Figure 1. lux gene organization ...... 6

Figure 2. AHL general structure ...... 8

Figure 3. Bacteriophage based bioluminescent bioreporter for detection ...... 30

Figure 4. Reporter phage assay for targeted detection of bacterial pathogens ...... 35

Figure 5. Genetic construction scheme for the λluxI reporter bacteriophage ...... 38

Figure 6. Cloning format for construction of an OHHL specific bioluminescent bioreporter E. coli OHHLux ...... 41

Figure 7. OHHL dose response curve of E. coli OHHLux...... 49

Figure 8. Bioluminescent response of E. coli OHHLux when co-cultured with each bacterium listed in Table 5 ...... 51

Figure 9. Detection limits of the phage bioreporter assay in a pure culture of E. coli XL1-

Blue target cells...... 52

Figure 10. Bacteriophage based bioluminescent bioreporter for pathogen detection ...... 67

Figure 11. Genetic construction of pCR2.1-soc2soc1-PL-luxI-rrnB-soc ...... 70

Figure 12. between plasmid pCR2.1-soc2soc1-PL-luxI-rrnB- soc and phage PP01 ...... 72

Figure 13. Detection of E. coli O157:H7 without pre-incubation ...... 77

Figure 14. Cross reactivity of the PP01-luxI detection system ...... 80

Figure 15. Establishment of the bioluminescent response in a pure culture 1:10 dilution series of E. coli O157:.H7 ...... 96

x

Figure 16. Bioluminescent detection of E. coli O157:H7 in artificially contaminated apple juice ...... 98

Figure 17. Response time profile of bioluminescence emission in 25 ml sample volumes of E. coli O157:H7 contaminated tap water ...... 99

Figure 18. Real-time imaging of bioluminescence emission from rinsates from E. coli

O157:H7 contaminated spinach leaves ...... 102

xi

Chapter I: Introduction

1

Bioluminescence and Vibrio fischeri symbiosis

Many organisms including bacteria are known to produce light in a process called bioluminescence. Perhaps some of the most well studied bioluminescent bacteria are those of the genus Vibrio. For example, Vibrio fischeri, a bioluminescent bacterium, has long been a focus of research due to its symbiotic relationship with Euprymna scolopes or as it is more commonly known, the Hawaiian bobtailed squid (Ruby 1999, Visick &

McFall-Ngai 2000, Nyholm & McFall-Ngai 2004). V. fischeri, a marine bacterium, exists in a free-living planktonic state in the world‟s oceans. This bacterium is known to colonize the light organ of the squid where they grow to very high densities (1010 to 1011 cells/ml) in this nutrient rich environment (Nealson & Hastings 1979, McFall-Ngai &

Ruby 1991, Ruby & McFall-Ngai 1992, Ruby 1996, Graf & Ruby 1998, Nyholm &

McFall-Ngai 1998).

Interestingly, the bioluminescence of V. fischeri is induced only when the population has reached a certain density. This process, called quorum sensing, was first discovered and described in V. fischeri and V. harveyi (Nealson et al. 1970, Eberhard

1972, Nealson & Hastings 1979). As the Vibrio cells grow they produce an autoinducer that accumulates as the population rises. Once the autoinducer, in the case of V. fischeri, the acyl homoserine lactone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), reaches a critical threshold bioluminescence genes are induced, producing light.

The light organ of E. scolopes provides an ideal environment for V. fischeri to flourish leading to production of OHHL in a confined area (where concentrations can exceed 100 nM) leading to light production (Boettcher & Ruby 1995). Bioluminescence 2 from the light organ has been suggested to help the squid avoid . The squid is nocturnal and the luminescence produced by the bacterium provides a way to project light downward so it does not appear as a dark silhouette against the moonlight when it swims through the water, thereby camouflaging itself and making it less of a target to predators (Ruby & McFall-Ngai 1992, Ruby 1996, Graf & Ruby 1998, Ruby 1999,

Visick et al. 2000, Visick & McFall-Ngai 2000, Nyholm & McFall-Ngai 2004). Each morning the squid expels a large population of V. fischeri from its light organ in a process known as "venting". This is hypothesized to prevent overgrowth of bacteria in the light organ and provide the source of free-living bacteria for newly hatched squids allowing the cycle to continue (Visick & McFall-Ngai 2000).

Bioluminescence reaction

The luminescence produced by V. fischeri is due to a set of genes called lux. The basis of the lux reaction consists of proteins encoded for by the luxCDABE

(Meighen 1994). The luxC, luxD, and luxE genes produce a reductase, thioesterase, and synthetase that form a multienzyme fatty acid reductase complex (Meighen 1993). The complex diverts tetradecanoic acid from the fatty acid biosynthesis pathway generating tetradecanal, a long chain fatty aldehyde (Boylan et al. 1985, Boylan et al. 1989, Meighen

1991, Meighen 1993). The luxA and luxB genes encode for the α and β subunits of a heterodimeric luciferase (Engebrecht & Silverman 1984, Meighen 1991,

Meighen 1993). The luciferase catalyzes the oxidation of reduced flavin mononucleotide

(FMNH2) along with a long chain fatty aldehyde and O2 resulting in emission of blue 3 green light at 490 nm (Dunn et al. 1973, Hastings & Nealson 1977, Hastings 1978, Lee

1991, Meighen 1991, Baldwin 1992, Meighen 1993, 1994b, Tu & Mager 1995). The chemical reaction occurs as follows:

FMNH2 + RCHO + O2 → FMN + RCOOH + H2O + hv (490 nm).

After the reaction LuxCDE regenerates the aldehyde substrate and a gene called luxG is thought to encode a flavin mononucleotide reductase that produces more FMNH2 substrate (Meighen 1993, Zenno & Saigo 1994).

Quorum sensing

The quorum sensing mechanism that alters gene expression leading to bioluminescence in V. fischeri was initially described by Nealson and Eberhard as the production, release, and detection of signal molecules called autoinducers by a population of bacteria as it increases in density (Nealson et al. 1970, Eberhard 1972, Nealson &

Hastings 1979). Once the autoinducer (and by proxy the population) reaches a certain threshold concentration the bacteria respond through a change in gene expression. The functions controlled by quorum sensing are numerous and varied and include , formation, production, motility, sporulation, bioluminescence, even things like competence and conjugation are activated when an adequately large population or quorum is reached (Miller & Bassler 2001).

There are several types of quorum sensing that have been identified, each using a different type of signaling molecule as autoinducer. Both gram positive and gram 4 negative bacteria use furanosyl borate diester as an autoinducer for their AI-2 system.

Gram positive bacteria use oligopeptides in a two component system for quorum sensing.

Lastly, but most pertinent to this paper is the use of acyl homoserine lactones (AHLs) by gram negative bacteria for the LuxI/R quorum sensing mechanism.

A broad range of organisms in subclasses α, β, and γ of Proteobacteria are known to use LuxI/R quorum sensing for intraspecies communication (Camara et al. 2002,

Daniels et al. 2004). The LuxI/R system is composed of two major components: (1) LuxI which synthesizes the autoinducer and (2) LuxR which binds the signaling molecule

(Federle & Bassler 2003, Taga & Bassler 2003, Henke & Bassler 2004, Parsek &

Greenberg 2005, Shiner et al. 2005, Reading & Sperandio 2006). As the bacteria grow they release autoinducers which accumulate as the population rises. Short-chain AHLs, like OHHL from V. fischeri, freely diffuse across the cell membrane while AHLs with longer acyl chains (e.g., the 12-carbon AHL of aeruginosa) must be transported actively with efflux or influx pumps (Kaplan & Greenberg 1985, Evans et al.

1998, Pearson et al. 1999). This is due to the fact that long hydrophobic side chains decrease permeability through cell membranes. Once the autoinducer reaches a threshold level it binds to LuxR and activates the receptor protein which acts as a transcriptional activator or repressor controlling the expression of quorum sensing related genes. In the specific case of V. fischeri, the AHL autoinducer (OHHL) is produced which binds LuxR leading to the transcription of the luxCDABE cassette and producing bioluminescence.

5

Figure 1. lux gene organization

Organization of lux genes

The lux genes in V. fischeri are located on two separate that are transcribed in opposite directions with an intergenic regulatory region called the lux box separating them as shown in Figure 1. The genes encoding the proteins needed for the luminescence reaction, luxCDABEG, and the luxI gene which encodes for the quorum sensing autoinducer OHHL, are located together on the same operon (Engebrecht &

Silverman 1984). The luxR gene that encodes for the transcriptional activator that initiates luminescence after binding OHHL is separate from and in a different orientation than the rest of the lux genes (Engebrecht & Silverman 1987).

Regulation of V. fischeri bioluminescence

The luxICDABEG operon has a low basal level of transcription when V. fischeri cell population is low. This low transcriptional level leads to low concentrations of the

OHHL autoinducer which means very little luminescence is produced (Nealson et al.

1970, Eberhard 1972). As OHHL concentration increases in correlation with cell density a critical threshold of 10 nM is reached and at this threshold OHHL attaches to the N- terminal of the LuxR binding domain causing a conformational change and dimerization

6

(Nealson et al. 1970, Engebrecht & Silverman 1984, Hanzelka & Greenberg 1995,

Egland & Greenberg 2001, Trott & Stevens 2001, Kiratisin et al. 2002, Vannini et al.

2002, Zhang et al. 2002, Lamb et al. 2003). When OHHL reaches significant amounts at high cell densities (>1010 cells/ml) it binds LuxR and in turn the C-terminal domain of

LuxR binds to the lux box, an inverted repeat region upstream of the luxCDABEG operon

(Devine et al. 1989, Choi & Greenberg 1991, Shadel & Baldwin 1992a, Stevens et al.

1994, Egland & Greenberg 1999, Kiratisin et al. 2002, Vannini et al. 2002, Zhang et al.

2002, Lamb et al. 2003). Binding leads to expression of the lux operon concurrently increasing bioluminescence and OHHL synthesis in an exponential fashion leading to maximum induction of bioluminescence (Lupp et al. 2003, Lupp & Ruby 2005),(Nealson et al. 1970, Nealson 1977, Fuqua & Winans 1994). Generation of more OHHL during this process positively affects luxICDABEG while repressing luxR transcription

(Engebrecht et al. 1983, Dunlap & Ray 1989, Shadel & Baldwin 1991, 1992b, Egland &

Greenberg 2000). These effects counterbalance one another to control light output in times of high cell density.

Acyl Homoserine Lactones (AHLs)

LuxI-type proteins synthesize acyl homoserine lactones which are used for intraspecies communication. Homoserine lactones are chemical signals involved in microbiological quorum sensing and lead to changes in gene expression. AHLs are used as autoinducer molecues in the LuxI/R type quorum sensing system of gram negative bacteria as previously discussed. V. fischeri OHHL was the first AHL discovered 7

(Eberhard et al. 1981). Of most importance here is that the presence of high concentrations of AHLs leads to transcription of the lux genes in V. fischeri thereby generating bioluminescence. Although this is our focus, similar systems are found in other bacteria which control expression of a multitude of genes.

Structure

AHLs consist of an acyl side chain linked to a homoserine lactone ring via an amide bond. The homoserine lactone ring is common among all AHLs. However, depending on the bacterial species these molecules differ in the length of their acyl side

chain (4 to 18 carbons), designated in Figure 2 by ( )n (Marketon & Gonzalez 2002).

Interestingly, most AHLs discovered have an even number of carbons in their side chains. Acyl chains may also be saturated or unsaturated as seen in the examples in

Table 1 (Gray et al. 1996, Schripsema et al. 1996, Puskas et al. 1997). In addition, the chain can contain an oxo, hydroxyl or no substitution on the third carbon atom, designated as R in Figure 2. Although many gram negative bacteria produce AHL there are some well known pathogenic ones that do not, for example: Escherichia coli,

Haemophilus influenza, Klebsiella , Salmonella spp., ,

Campylobacter jejuni, and Helicobacter pylori (Swift 1999).

Figure 2. AHL general structure

8

Table 1. AHL examples and their host organism Organism AHL Type AHL Structure Reference

(Eberhard et al. N-(3-oxohexanoyl)-L-homoserine 1981, Engebrecht et V. fischeri lactone al. 1983, Engebrecht & Silverman 1984)

(Cao & Meighen 1989, Martin et al. N-β-(hydroxybutyryl)-L-homoserine 1989, Bassler et al. V. harveyi lactone 1993, Bassler et al. 1994, Sun et al. 1994)

(Piper et al. 1993, Zhang et al. 1993, Fuqua & Winans Agrobacterium 1994, Hwang et al. N-(3-oxooctanoyl)-L-homoserine lactone tumefaciens 1994, Fuqua et al. 1995, Hwang et al. 1995, More et al. 1996) (Bainton et al. 1992a, Bainton et al. 1992b, Jones et al. Erwinia N-(3-oxohexanoyl)-L-homoserine 1993, Pirhonen et al. carotovora lactone 1993, Chatterjee et al. 1995, Cui et al. 1995, McGowan et al. 1995)

9

Table 1. continued

Organism AHL Type AHL Structure Reference

(Pierson et al. 1994, Pseudomonas N-hexanoyl-L-homoserine lactone Wood & Pierson aerofaciens 1996, Wood et al. 1997) (Gambello & Iglewski 1991, Gambello et al. Pseudomonas N-(3-oxododecanoyl)-L-homoserine 1993, Passador et al. aeruginosa lactone 1993, Pearson et al. 1994, Davies et al. 1998, de Kievit & Iglewski 2000)

Ralstonia N-octanoyl-L-homoserine lactone solanacearum (Flavier et al. 1997)

(van Brussel et al. 1985, Cubo et al. Rhizobium N-(3-hydroxy-7-cis-tetradecanoyl)-L- 1992, Gray et al. leguminosarum homoserine lactone 1996, Schripsema et al. 1996, Lithgow et al. 2000)

10

Some bacterial species have been shown to produce more than one type of AHL although only one acts as the main quorum sensing signal (Winson et al. 1995, Schaefer et al. 1996b, Fuqua 1999, Parsek et al. 1999, Marketon et al. 2002). These secondary

AHLs act as competitors against the primary AHL signal by binding the LuxR and homologue proteins in place of the primary AHL without actually activating the protein

(Eberhard et al. 1986, Kuo et al. 1996, Schaefer et al. 1996a, Zhu et al. 1998). Research has suggested that AHL specificity stems from its acyl side chain, thus identification of the correct acyl-acyl carrier protein (acyl-ACP) by the LuxI protein leads to specificity in

AHL production (Fuqua 1999). Structural studies indicate that the size of the LuxI-type protein cavity which interacts with the acyl side chain may be responsible for the selectivity of certain acyl-ACPs, i.e. larger cavity equals longer AHLs and vice versa

(Watson et al. 2002, Gould et al. 2004). Since these molecules can be so similar they may show overlapping signal specificity. Thus a compound closely related to a bacterium‟s native autoinducer can cause weak gene expression and even compete with the native AHL.

Synthesis

AHL molecules are synthesized by the LuxI protein and other homologues. LuxI proteins produce AHLs using metabolic substrates in the cell. When the luxI genes are transformed into a non-native host they can still produce AHL (Engebrecht et al. 1983,

Pearson et al. 1994, Pearson et al. 1995, Throup et al. 1995, More et al. 1996). This is a requirement for our system since E. coli is being used to produce OHHL for pathogen detection and not the native host for the gene, V. fischeri.

11

LuxI and its homologues vary greatly in gene size and protein sequence (Swift et al. 1996, Swift et al. 2001, Watson et al. 2002). When compared, protein sequences show only ten conserved amino acids mostly in the amino-terminal region of the protein, seven of which have a charge (Parsek et al. 1997, Fuqua 1999). Mutation of the amino- terminus of LuxI resulted in loss of function with five of the amino acids mutated being conserved across all known LuxI-type proteins (Arg25, Glu44, Asp46, Asp49 and Arg70)

(Parsek et al. 1997). This previous research implies that the amino-terminal region and specifically these highly conserved residues may be responsible for the function of LuxI- type proteins while the variable carboxy-terminal regions may be involved in recognition of acyl-ACPs side chains (Parsek et al. 1997).

Research has determined that S-adenosylmethionine (SAM) and acyl-ACPs are the substrates involved in AHL synthesis (Schaefer et al. 1996a, Schaefer et al. 1996b,

Val & Cronan 1998, Hoang et al. 1999, Hoang & Schweizer 1999, Parsek et al. 1999).

SAM, a common cellular amino acid substrate, is used by LuxI to produce the homoserine lactone ring of AHL (Green 1996). The acyl side chain is acquired from acyl-ACP, a fatty acid biosynthesis metabolite. Specifically, 3-oxohexanoyl-ACP is used for synthesis of OHHL. There are several steps involved in LuxI production of AHL.

The first step involves LuxI binding to SAM (Schaefer et al. 1996b, Parsek et al. 1999,

Watson et al. 2002). Next, the acyl-ACP forms an amide bond with SAM yielding the acyl side chain of the AHL. Lastly, The SAM carboxylate oxygen forms a bond with the

γ carbon of SAM causing lactonization resulting in the homoserine lactone ring (Schaefer et al. 1996b, Parsek et al. 1999, Watson et al. 2002).

12

General bacteriophage characteristics

Bacteriophage are that are capable of infecting and replicating in a bacterial host. Since their discovery in the early 20th century, phage have been studied extensively especially those such as lambda. Bacteriophage are ubiquitous in the environment being found in most environments colonized by bacteria such as soil, sewage, and water. The structure of bacteriophage is comprised of a DNA or RNA genome which is contained in a protein coat. Phage are highly diverse and can have host ranges as diverse as a bacterial genera to as specific as a single bacterial species or strain.

This specificity is mediated through the phage tail fibers. A bacteriophage recognizes its host via highly specific binding to receptors found on the cell surface of the bacterium.

These receptor proteins are found on the outer membrane and can include things such as , , flagella, pili and capsules (Heller 1992). Once the phage attaches to the bacterial cell it injects its nucleic acid into the host whose cellular machinery it will use for replicating and forming new progeny. Bacteriophage can follow one of two life cycles, lytic or lysogenic. A lytic phage will inject its genome into the host, use the bacterium‟s genetic machinery to assemble numerous mature phage particles, and then proteins which lyse the cell release the bacteriophage. Alternatively, a lysogenic life cycle consists of integration of the phage genome into the host chromosome producing a . The lysogenic phage will be replicated and passed with the division of the bacterium. Lysogenic phage may switch to a if certain environmental conditions are present such as cell damage.

13

E. coli O157:H7

Recent outbreaks of disease due to pathogenic bacteria have pushed the need for monitoring of food and water supplies to the forefront. Shiga-toxin (Stx) producing E. coli (STEC), specifically the enterohemorrhagic E. coli (EHEC), have been a major culprit in many cases. EHEC E. coli can cause a variety of diseases like diarrhea, hemorrhagic colitis (HC), and in some cases hemolytic uraemic syndrome (HUS) and central nervous system abnormalities. Infection by EHEC can lead to very serious disease and even prove deadly particularly in children, elderly, and immunocompromised individuals. STEC expressing somatic (O) antigen 157 and flagellar (H) antigen 7 is the serotype most frequently isolated from human beings and most commonly associated with severe human disease. E. coli O157:H7 is the major serotype responsible for outbreaks.

The first outbreaks associated with E. coli O157:H7 in the United States causing

HC and HUS were described in 1983 (Riley et al. 1983). Following this it was soon discovered that E. coli O157:H7 produced a toxin related to (Stx) of Shigella dysenteriae (O'Brien et al. 1983). Since then, E. coli O157:H7 has been well documented as an important cause of disease by impacting public health through outbreaks involving ground beef (CDC 1997, Macdonald et al. 2000, CDC 2002, MMWR 2002, Proctor et al.

2002, Jay et al. 2004, Vogt & Dippold 2005), unpasteurized apple juice (CDC 1996b,

Cody et al. 1999), unpasteurized milk (Bielaszewska et al. 1997, Keene et al. 1997), lettuce (Ackers et al. 1998, Hilborn et al. 1999), recreational/drinking water (Swerdlow et al. 1992, Akashi et al. 1994, Keene et al. 1994, CDC 1996a, Ackman et al. 1997, Levy et

14 al. 1998, CDC 1999, Paunio et al. 1999, Olsen et al. 2002, Samadpour et al. 2002, Bopp et al. 2003, Bruce et al. 2003), and recently spinach (CDC 2006). Given the obvious public health importance of this pathogen, a detection method that is rapid, specific, and capable of detection at low concentrations is needed.

Traditional detection methods

A rapid and reliable method of detection for E. coli O157:H7 in food and water is required to quickly detect outbreaks and hopefully limit the number of individuals affected. Currently, there are a variety of methods available for the detection of E. coli

O157:H7 but most are slow and/or require a significant amount of sample manipulation

(Deisingh and Thompson, 2004; Gehring et al., 2004; Lee et al., 2005; Moxley, 2003).

The most common method used for identifying E. coli O157:H7 is culturing on Sorbitol

MacConkey agar (SMAC) which consists of bile salts, the carbohydrate source sorbitol, and a pH indicator (Farmer & Davis 1985). SMAC can differentiate bacteria based on their ability to ferment sorbitol. Fermentation of sorbitol will produce acid, droping the pH of the media and resulting in a color change around the colony. Most E. coli strains

(95%) are sorbitol positive meaning they have the capacity to ferment sorbitol, whereas

O157:H7 strains are not able to use this sugar. E. coli O157:H7 will appear as colorless colonies because large amounts of acid are not being produced while other

Enterobacteriaceae appear as pink colonies due to the color change of the pH indicator.

Plating methods can also involve enrichment steps before plating on SMAC plates which can increase the sensitivity of E. coli O157:H7 detection (Chapman et al. 1994, 15

Sanderson et al. 1995). Overall, plating methods can be effective in identifying E. coli

O157:H7 but require 24-48 h of incubation, not including enrichment steps, for results to be obtained (Moxley 2003, Deisingh & Thompson 2004).

Immunological methods and Enzyme-Linked ImmunoSorbent Assays (ELISAs) have also been employed for the detection of E. coli O157:H7 (Moxley 2003, Deisingh &

Thompson 2004, Gehring et al. 2004). These methods focus on the E. coli O157 and H7 antigens. Commercially available ELISAs for O157 and H7 antigens are widespread and are fairly sensitive and rapid. A Direct Immunoflourcscence (DIF) assay for the detection of E. coli O157 was developed by Park et al. (1994). DIF consists of using human fecal samples stained with flourescein conjugated anti-E. coli O157 antibodies which are examined microscopically. Immunological methods do have several drawbacks: (1) numerous staining and washing steps (Moxley 2003, Deisingh &

Thompson 2004, Gehring et al. 2004) (2) false negatives for H7 because of culturing methods and (3) O157 false positives seen with Citrobacter freundii, E. hermannii, and

Salmonella urbana (Park et al. 1994, Dylla et al. 1995). Immunogenic separation of target cells can however prove useful for lowering detection limits/times and is now being integrated into assays (Goodridge et al. 1999, Favrin et al. 2001, Su & Li 2004, Fu et al. 2005, Wang et al. 2007, Ripp Submitted 2007).

Detection by Polymerase Chain Reaction (PCR) has also been used for E. coli

O157:H7 although there are several hurdles. Due to the fact that there are many inhibitors found in feces and food samples, PCR is most commonly used to confirm isolates as E. coli O157:H7 but it can also be used to directly detect the organism. To 16 ensure there are no false positives and that E. coli O157:H7 is the only organism that will produce the desired amplification, multiplex PCR is often employed. Multiplex PCR is a variation of normal PCR, in which several targets of interest are simultaneously amplified in one reaction, using more than one pair of primers. Hu et al. (1999) used the first successful multiplex PCR to specifically detect E. coli O157:H7 serotype. This multiplex

PCR amplified several targets: (1) eaeAO157 , the gene encoding for intimin which is involved in the attaching and effacing adherence phenotype (2) stx1 and stx2, the genes responsible for Shiga toxin production, (3) filC, the H7 flagellar antigen gene and (4) rfbE, which encodes the E. coli O157 serotype. Hu et al. (1999) successfully detected target organisms in isolated colonies, however, attempts directly from feces failed without enrichment. PCR is rapid and sensitive considering that theoretically DNA from a single bacterial cell can be amplified in about 1 h for a positive result if no enrichment is necessary. Problems with the PCR method include its complexity, possible contamination, inability to distinguish between live and dead bacterial cells, and the fact that food and environmental samples often contain PCR inhibitors (Deisingh &

Thompson 2004).

Quantitative PCR (Q-PCR) which allows simultaneous amplification, via PCR, and quantification of targeted DNA or RNA molecules, via flourescnt probes, has also been applied as an E. coli O157:H7 assay. Target sequences ranged from eaeA, stx, to rfbE either alone or in a multiplex format and concentrations down to 1 CFU/ml or g were detected in relatively short periods of time (< 8 h including enrichment) (Meng et al.

1996, Fortin et al. 2001, Jothikumar & Griffiths 2002, Sharma & Dean-Nystrom 2003, Fu

17 et al. 2005, Hsu et al. 2005, Holicka et al. 2006, Yoshitomi et al. 2006). This is a viable method for sensitive, rapid and quantitative way to screen for E. coli O157:H7.

Although, just like all other methods Q-PCR has its limitations, the necessary equipment and reagents are costly. As with basic PCR, Q-PCR is complex, subject to contamination, detects viable but non-culturable cells (VBNC) and free DNA, and inhibitors present in the complex samples may affect assay reliability.

Bioluminescent bioreporters

Bioluminescent bioreporters are organisms genetically engineered to react to the presence of a specific analyte by generation of light. Bioreporters are constructed by placing a promoter which is specifically induced by a particular analyte upstream of the reporter genes, in our case the lux bioluminescence genes. This construct can be contained in the host bacterium as a plasmid or can be integrated into the chromosome.

They are often a rapid, non-invasive, sensitive, and specific alternative to other detection methods. Bioluminescent bioreporters can be used to determine bioavailability along with possible toxic effects produced by chemicals on organisms.

There is a long history of bioluminescent bioreporters with detected analytes including environmental pollutants, toxic chemicals in wastewaters, bacterial nutrients, and pathogens. Examples of these bioreporters and their detection limits are shown in

Table 2. These bioreporter systems all use the V. fischeri luxCDABE genes although other common bioluminescence reporter genes from Photorhabdus luminescens and V. harveyi

18

Table 2. luxCDABE bioreporters Analyte Concentration Reference 2,3 Dichlorophenol 50 mg/ L (Hay et al. 2000) 3-Xylene 3 μM (Burlage 1998) 4-Chlorobenzoate 380 μM – 6.5 mM (Rozen et al. 1999) Alginate production 50 – 150 mM NaCl (Wallace et al. 1994) Ammonia 20 μM (Simpson 2000) Antibiotic effectiveness against 100 CFU (Francis et al. 2000) in mice BTEX (benzene, toluene, 0.03 – 50 mg/L (Applegate et al. 1998) ethylbenzene, xylene) Cadmium 19 mg/kg (Corbisier et al. 1996) Chromate 10 μM (Peitzsch et al. 1998) Cobalt 2.0 mM (Tibazarwa et al. 2000) (Holmes et al. 1994, Copper 1 μM – 1 mM Daunert et al. 2000) DNA damage (mitomycin) 0.032 μg/ml (Vollmer et al. 1997) Gamma-irradiation 1.5 – 200 Gy (Min et al. 2000) (Van Dyk et al. 1995, Heat shock Various Rupani et al. 1996) Hemolysin production 5 mM cAMP (Bang et al. 1999) in vivo monitoring of Salmonella 100 CFU (Contag et al. 1995) typhimurium infections in living mice Iron 10 nM – 1 μM (Khang et al. 1997) Isopropyl benzene 1 – 100 μM (Selifonova & Eaton 1996) Lead 4036 mg/kg (Corbisier et al. 1996) Mercury 0.025 nM (Selifonova et al. 1993) N-acyl homoserine lactones Not specified (Winson et al. 1998) Naphthalene 12 – 120 μM (King et al. 1990) Nickel 0.3 mM (Tibazarwa et al. 2000) Nitrate 0.05 – 50 μM (Prest et al. 1997) Organic peroxides Not specified (Belkin et al. 1996) PCBs 0.8 μM (Layton et al. 1998) p-chlorobenzoic acid 0.06 g/l (Rozen et al. 1999) Salicylate 36 μM (King et al. 1990) Tetracycline 5 ng/ml (Hansen & Sorensen 2000) Trichloroethylene 5 – 80 μM (Shingleton et al. 1998) Ultrasound 500 W/cm2 (Vollmer et al. 1998) Ultraviolet light 2.5 – 20 J/m2 (Elasri & Miller 1998) Zinc 0.5 – 4 μM (Erbe et al. 1996)

19 can be used as well. In addition, many other bioreporter systems have been constructed using only the luxAB genes. The disadvantage of just using luxAB is that only the luciferase is self generated and exogenous aldehyde substrate must be added to the assay to generate bioluminescence. The bioreporter gene product, luminescence, is easily detected and measured by light-measuring devices, such as photomultiplier tubes, photodiodes, microchannel plates or charge-coupled devices. In fact, bioreporters can be integrated into biosensors, forming devices that are a blend of biological and electrical components so that the light produced can be measured to detect and quantify the target.

The properties of the bioluminescence signal produced make it ideal for construction of high throughput, continuous and real-time monitoring systems.

Bacteriophage based bioluminescent bioreporters

Bioluminescent bioreporters can also be adapted to be used with bacteriophage to detect bacterial pathogens. The combination of the phage based detection with bioluminescence genes provides a viable detection system (Billard & DuBow 1998,

Galindo et al. 2002, Hazbon 2004, Petty et al. 2007). Phage can exhibit a high specificity for their hosts making them ideal to use for specific bacterial pathogen detection. These bacteriophage based systems can be specific, sensitive, rapid, and cost effective. A phage based system can detect living cells and those in a viable but non-culturable (VBNC) state unlike PCR and antibiotic based methods. Phage are relatively inexpensive and easy to produce in large quantities and detection of target at concentrations as low as 1

CFU/ml is possible with limited preincubation. There have been phage based 20 bioluminescent bioreporters created to detect bacteria such as ,

Mycobacterium tuberculosis, M. smegmatis, Salmonella spp., E. coli and other enteric bacteria (Table 2). In classical phage based detection systems, the recombinant phage usually carries the bacterial luxAB or eukaryotic lucAB genes. When they infect the host organism the lux genes are transferred into the host cell and expressed producing the luciferase enzyme. In the presence of exogenous aldehyde substrate, this results in generation of bioluminescence which can be readily measured, determining if the target organism is present.

E. coli O157:H7 bacteriophage based bioluminescent bioreporter system

There are various methods available for the detection of E. coli O157:H7 all have similar limitations including slow results, false positives, complex procedures, and significant sample manipulations. The phage reporter method described here differs from the aforementioned methods in that it exploits the specificity that bacteriophage exhibit for their host to produce a new technique for identifying this important pathogen. The combination of lux bioluminescence and bacterial quorum sensing genes with a specific

21

Table 3. Bacteriophage based bioluminescent bioreporters for the detection of target pathogens Phage Detection Target Pathogen Genes Used Sample Tested Detection Limit Reference Used Time

V. harveyi Culture 2 h 100 - 103 cells/ml (Loessner et al. L. monocytogenes A511 luxAB Spiked salad 22-24 h 1 cell/g 1996)

Spiked V. harveyi pudding,cabbage 1 cell/g (Loessner et al. L. monocytogenes A511 20 h luxAB Spiked 10-100 cells/g 1997) meat,cheese Firefly (Banaiee et al. Clinical isolates M. tuberculosis TM4 Luciferase 2-3 days ND 2001, Bardarov and sputum (Fflux) et al. 2003) (Carriere et al. 1997, Riska et al. 1997, Riska M. tuberculosis TM4 Fflux Sputum 24 - 48 h ND & Jacobs 1998, Riska et al. 1999) M. smegmatis and (Jacobs et al. TM4 Fflux Culture 24 h 104-105 cells tuberculosis 1993) 20 h 10 3 cells/g (Sarkis et al. M. smegmatis L5 Fflux Culture 40 h 100 cells/g 1995) Soil, water, and (Turpin et al. S. typhimurium P22 luxAB 24 h 1 cell/ml sewage sludge 1993)

V. fischeri Culture 6 h 10 cells/ml (Chen & Salmonella sp. P22 luxAB Eggs 24 h 10 cells/egg Griffiths 1996)

V. fischeri 50 min 10 4 cells/g (Kodikara et al. Enteric bacteria ФV10 Meat luxAB 4 h 10 cells/g 1991)

V. harveyi (Waddell & E. coli O157:H7 ФV10 Culture 1 h ND luxAB Poppe 2000)

Vibrio (Kuhn et al. Salmonella sp. Felix 01 Culture ND ND luxAB 2002)

λCharo n V. fischeri (Ulitzur & E. coli Milk and urine 1 h 10 - 100 cells/ml 30 luxAB Kuhn 1987)

22

E. coli O157:H7 phage creates a bioreporter system for sensing O157:H7 in samples.

This provides a novel detection technique that is rapid and amenable to real-time monitoring.

Our method utilizes a bacteriophage that specifically infects E. coli O157:H7 in combination with quorum sensing and lux based bioluminescence to create a phage based bioreporter system for this pathogen. Genetic manipulation of bacteriophage using reporter genes such as bacterial luciferase (luxAB), β-galactosidase (lacZ) and green fluorescent protein (gfp) to create “reporter phage” has been well documented (Ulitzur &

Kuhn 1987, Stewart et al. 1989, Kodikara et al. 1991, Turpin et al. 1993, Chen &

Griffiths 1996, Loessner et al. 1996, Waddell & Poppe 2000, Goodridge & Griffiths

2002). Since phage are metabolically inactive outside of their host, the reporter proteins encoded in these phage are not produced until the specific host bacterium is infected. In previously described lux based phage reporter systems, bacteria are detected after phage infection, an incubation/growth step, and the addition of an exogenous substrate

(Kodikara et al. 1991, Chen & Griffiths 1996, Loessner et al. 1996). Although these current methods are able to specifically and sensitively detect bacteria, they are not amenable to continuous monitoring because of the requirement for downstream sample manipulation.

PP01 bacteriophage background

The phage used to develop our reporter system was a virulent bacteriophage referred to as PP01 that is specific for E. coli O157:H7. It was isolated and characterized

23 in 2002 from swine stool sample (Morita et al. 2002b). The PP01 phage infects E. coli

O157:H7 but fails to infect other O-serogroups and the strain K-12 (Table 4) (Morita et al. 2002b). Analysis of the amino acid sequence from the tail fiber proteins Gp37 and

Gp38, the end proteins in each tail fiber which are involved in attachment and determining host range, determined PP01 was related to T2 phage (Morita et al. 2002b).

Numerous phage were isolated from the stool samples tested but Morita et al. (2002a) found that PP01 phage was the only isolate to decrease the OD600 of an E. coli O157:H7 culture, evidence that PP01 is a lytic phage. One PP01 phage resistant E. coli mutant was found in the process. Analysis of the mutant showed it had lost the outer membrane protein OmpC, a protein which forms a large diameter transmembrane channel or pore in the E. coli cell wall but had instead increased production of an alternative porin OmpF

(Schindler & Rosenbusch 1978, Nikaido & Rosenberg 1983). A 14 kb deletion upstream of and including a portion of OmpC caused the loss of the protein (Morita et al. 2002a).

Complementation of OmpC from an E. coli O157:H7 strain (but not a K-12 strain) restored the mutant‟s susceptibility to PP01 causing Morita et al. (2002a) to suggest that the OmpC protein serves as the receptor for this bacteriophage.

Recently, a group has modified this PP01 bacteriophage by labeling it with green fluorescent protein (GFP) to detect E. coli O157:H7 (Oda et al. 2004). The bacteriophage‟s small outer (SOC) protein, which plays an important role in phage capsid stability (Ishii & Yanagida 1977), was used as an insertion point for a GFP marker. The process involved creating through PCR and digest that have the GFP marker inserted at either the C or N terminal region of the soc

24

Table 4. Bacterial strains used to test PP01 phage’s specificity and host range Host serotype or species Strain Susceptibility E. coli O6:H16 941-88 - E. coli O7 ATCC23503 - E. coli O55 992-97 - E. coli O116:H10 ATCC23541 - E. coli O119:H6 997-97 - E. coli O126:H12 947-88 - E. coli O157:H7 980013 + E. coli O157:H7 9800012 + E. coli O157:H7 EDL933 + E. coli O157:H7 ATCC43888 + E. coli O157:H7 CR-3 + E. coli BE - E. coli K-12 12017 - E. coli K-12 RK4784 - E. coli K-12 W3110 - Citrobacter freundii KTY-1 - Erwinia cartovora MAFF301614 - PAO1 - Yersinia enterocolitica O9 ZT23 - (taken from Morita et al. 2002b)

25 gene (pUC-GFP/SOC and pUC-SOC/GFP). A bacterium carrying this plasmid was then infected with wild type PP01. Homologous recombination between the plasmid and the host genome produced recombinant GFP phage which were detected through plaque hybridization assays. The fusion did not change PP01‟s natural host range nor did it decrease the bacteriophage‟s binding affinity for its host bacterium. The fusion did however decrease the stability of the phage in alkaline solution (Oda et al. 2004).

Oda et al. (2004) reported that this recombinant phage allows for specific detection of culturable and VBNC E. coli O157:H7 (2004). Detection is achieved through mixing the recombinant phage with a culture or sample. Attachment of the GFP- phage to the E. coli cell surface allows visualization of the bacterial pathogen even in a

VBNC state under a fluorescent microscope. In addition, an increase in fluorescence intensity of the culture over time, indicating replication and increase of recombinant phage numbers, signifies a culturable form of the pathogenic bacterium is present. The

PP01-GFP reporter phage does appear, at least in preliminary studies, to be rapid and specific; however like every GFP based assay it needs a high energy light source for excitation of the GFP protein which could limit its potential use in real-time online industrial and environmental monitoring. The PP01 reporter phage presented in this paper uses the bacterial lux reporter genes to create a construct that allows for detection without the addition of substrate and has potential for use in real-time online monitoring.

26

Recombinant luxI phage and E. coli OHHLux

λluxI phage

The recombinant λluxI reporter phage was developed as a proof of concept prior to the final E. coli O157:H7 specific PP01-luxI phage creation. The construction of λluxI is fully described in Chapter II and Figure 5 in detail including primer sequences, vectors, and restriction enzymes used. Briefly, a plasmid carrying luxI was fused with the promoter (PL) of phage lambda upstream and the transcriptional , rrnB T1T2, downstream to create a PL-luxI-rrnB construct. Next, this PL-luxI-rrnB was placed into the Lambda ZAP II . The recombinant lambda phage DNA produced was then packaged into phage heads. Resulting plaques were screened using a plaque hybridization assay. Positive plaques containing the luxI incorporated phage were then isolated and propagated on top agar plates and used in subsequent assays with E. coli

OHHLux bioreporter cells to determine the viability of their use as a screening method for E. coli K12.

E. coli OHHLux bioreporter cells

The basics of construction for E. coli OHHLux are as follows but see Chapter II and Figure 6 for a more detailed description including details about primer sequences, vectors, and restriction enzymes used. The OHHL-specific bioluminescent bioreporter E. coli OHHLux was constructed by fusing the luxCDABE genes which are responsible for the bioluminescence reaction with the luxR gene, the transcriptional activator. Along with these genes rrnB T1T2, the transcriptional terminator, was added to prevent readthrough resulting in aberrant light production and a kanamycin resistance gene 27 allowing for stability and selection of our construct in our bioreporter cell. The construction resulted in a plasmid consisting of rrnB-luxCDABE-luxR-Kn which was used along with the PP01-luxI phage in our E. coli O157:H7screening assays.

PP01-luxI phage

The methods used for the construction of our recombinant PP01-luxI reporter phage were based on those used by Oda et al. (2004). The construction of PP01-luxI is fully described in Chapter III and Figures 11 and 12 in detail including primer sequences, vectors, and restriction enzymes used. Briefly, a plasmid carrying luxI fused with the promoter (PL) of phage lambda upstream and the transcriptional terminator, rrnB T1T2, downstream creating a PL-luxI -rrnB construct. Next, soc2, soc1 and soc genes from the

PP01 phage were cloned into the PL-luxI -rrnB construct. The soc2 and soc1 genes were inserted upstream of PL while soc was placed downstream of rrnB producing a soc2soc1-

PL-luxI-rrnB-soc plasmid. This plasmid was then used for homologous recombination with PP01.

Homologous recombination was accomplished by producing E. coli O157:H7 cells containing the soc2soc1-PL-luxI-rrnB-soc plasmid which were then infected with

PP01 phage and incubated. After incubation, phage were extracted and PP01-luxI phage were identified with a plaque hybridization assay. Positively identified PP01-luxI phage were propagated and used in subsequent assays with E. coli OHHLux bioreporter cells to determine the viability of their use as a screening method for E. coli O157:H7.

28

System mechanics

As previously stated, the detection method for E. coli O157:H7 combines the specificity bacteriophage have for their host, quorum sensing and lux based bioluminescence from V. fischeri. How all these components function together and the steps involved in the assay follows and are depicted in Figure 3.

1.) If the target pathogen is present, the luxI reporter phage will specifically infect the

E. coli and insert its genome.

2.) After infection, luxI will be transcribed by the host from the phage genome

resulting in production of OHHL from Acyl-ACP and SAM.

3.) The OHHL diffuses into nearby bioreporter cells, E. coli OHHLux, and binds the

transcriptional activator LuxR.

4.) The LuxR-OHHL complex interacts with PL leading to the transcription of

luxCDABE.

5.) LuxCDE form a multienzyme complex that generates aldehydes (by diverting

myristol-ACP away from fatty acid biosynthesis pathway), while the LuxAB

luciferase oxidizes FMNH2 and a long chain fatty aldehyde resulting in a

measurable light signal at 490 nm and a positive assay result.

29

Figure 3. Bacteriophage based bioluminescent bioreporter for pathogen detection

30

Chapter II: Linking bacteriophage infection to quorum sensing signaling and bioluminescent bioreporter monitoring for direct

detection of bacterial agents

31

This is a version of an article found in the Journal of Applied Microbiology entitled

“Linking bacteriophage infection to quorum sensing signaling and bioluminescent bioreporter monitoring for direct detection of bacterial agents”.

Abstract

The aim of this research was to incorporate into the lambda phage genome a luxI based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target specific visible light signals with no requisite addition of extraneous substrate.

This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 × 108 to 1 CFU/ml within 1.5 to

10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 × 108 to 130

CFU/ml was achieved within 2.6 to 22.4 h, respectively. The initial feasibility of binary phage based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. With further

32 modification, binary phage based reporter assays may be capable of rapidly and cost- effectively detecting pathogenic agents at very low population densities.

Introduction

In 1987, Ulitzur and Kuhn (Ulitzur & Kuhn 1987) reported a novel pathogen detection method that coupled the specificity of bacteriophage for their unique bacterial hosts with bioluminescent signalling. They cloned the luxAB encoded luciferase genes from Vibrio fischeri into the phage lambda genome. Upon infection, the luxAB genes were transduced to Escherichia coli, thus endowing these host cells with a bioluminescent phenotype visible upon addition of a requisite aldehyde substrate. This technique has since been applied to other phage for specific, low-level (10 – 1000 cells) detection of Listeria monocytogenes (Loessner et al. 1996), Salmonella typhimurium

(Chen & Griffiths 1996), E. coli O157:H7 (Waddell & Poppe 2000), enteric bacteria

(Kodikara et al. 1991), and Staphylococcus aureus (Pagotto et al. 1996) within a variety of food matrices. The firefly luciferase (luc) (Sarkis et al. 1995), ice nucleation (inaW)

(Wolber & Green 1990), beta-galactosidase (lacZ) (Goodridge & Griffiths 2002), and green fluorescent protein (gfp) (Funatsu et al. 2002, Oda et al. 2004) genes have similarly been incorporated into bacteriophage for the detection of foodborne pathogens such as

Mycobacterium, Salmonella, and E. coli. Reporter phage have also been labeled with a variety of fluorescent for bacterial specific tagging (Mosier-Boss et al. 2003) and combined with immunomagnetic separation for rapid capture, concentration, and identification of bacterial targets (Goodridge et al. 1999, Favrin et al. 2001). In addition,

33 bacteriophage in their unadorned native form have been used for decades in phage typing schemes to identify foodborne as well as clinical bacterial isolates (Stone 2002).

Clearly, the exploitation of phage specificity for bacterial monitoring has potential for foodborne pathogen monitoring. However, current phage assay systems require addition of substrate or specialized monitoring equipment that is not adaptable to the real- time, on-line monitoring format desired by the food industry. To move towards a fully reagentless phage based assay, we have developed a model binary recombinant phage system for the detection of bacterial pathogens that links phage infection events to quorum sensing signal molecule biosynthesis and bioluminescent bioreporter induction

(Fig. 4). The system‟s foundation centers on the luxCDABE operon of V. fischeri and its regulatory genes luxI and luxR (Meighen 1994). The luxAB component of this operon encodes for a bacterial luciferase that generates bioluminescence when provided with oxygen, FMNH2, and an aldehyde substrate synthesized by the luxCDE gene complex.

The luxI encoded LuxI protein is responsible for generation of a specific acyl-homoserine lactone (AHL) signalling molecule referred to as an autoinducer. Freely diffusible autoinducers interact with LuxR proteins to stimulate transcription of luxCDABE and luxI. As the concentration of AHL autoinducer increases, so does the number of LuxR binding episodes, and an autoamplified quorum sensing loop is established that results in the generation of bioluminescence in a cell density dependent manner (Whitehead et al.

2001).

In the present detection system a phage, chosen based on its specificity for the desired target bacterium, is engineered to contain the luxI gene within its chromosome.

Upon infection, luxI is inserted into the host and expressed, thereby creating a cell that

34

λluxI reporter phage AHL Bioluminescence

luxI

luxR PluxI luxCDABE

Bacterial pathogen E. coli OHHLux bioluminescent (E. coli XL1-Blue) bioreporter

Figure 4. Reporter phage assay for targeted detection of bacterial pathogens

The recombinant phage contains a luxI gene that, upon infection of its specific host, is inserted into the host chromosome where it is transcribed, thereby synthesizing acyl- homoserine lactone (AHL) autoinducer. The diffusible AHL molecules interact with the LuxR regulatory protein to trigger luxCDABE transcription in the neighboring bioluminescent bioreporter cell to generate bioluminescence. In these experiments, E. coli XL1-Blue was used as a model bacterial pathogen.

35 actively synthesizes autoinducer. As the autoinducer molecules diffuse into the extracellular environment, they are detected by an AHL specific bioluminescent bioreporter that contains the luxR and luxCDABE genes (see Daunert et al. (Daunert et al.

2000) or Keane et al. (Keane et al. 2002) for a complete review of bioreporter detection systems). The interaction of autoinducer with LuxR stimulates luxCDABE expression and the bioreporter generates a light signal at 490 nm. Thus, the initial phage infection event yields an autoamplified chemical signature that is sensed and communicated through bioluminescent bioreporter signal induction. The assays are fully reagentless and amenable to high throughput, near real-time screening of bacterial pathogens. As a model system, we have incorporated luxI into phage lambda for the general detection of

E. coli and constructed a luxRCDABE bioluminescent bioreporter for specific sensing of the autoinducer N-3-(oxohexanoyl)-L-homoserine lactone (OHHL). The assay directly detects E. coli in pure culture at 1 × 108 CFU/ml within 1.5 h and at 1 CFU/ml, with an additional preincubation step, within 10.3 h. Washings from lettuce leaves inoculated with E. coli at concentrations ranging from 1 × 108 to 130 CFU/ml produced bioluminescence within 2.6 to 22.4 h, respectively, with requisite independent preincubation at lower initial cell densities.

Materials and methods

Bacterial strains and bacteriophage

The phage bioluminescent system consists of three components; the luxI incorporated reporter phage (λluxI), the AHL specific bioluminescent bioreporter (E. coli

OHHLux), and the target bacterium. The λluxI reporter phage was constructed within

36 temperate phage lambda, lambda resistant E. coli XLOLR (Stratagene, La Jolla, CA) was used for construction of the OHHL specific bioluminescent bioreporter E. coli OHHLux, and the E. coli K12 variant XL1-Blue (Stratagene) was used as the model host strain for phage infection. lux genes were derived from V. fischeri or Photorhabdus luminescens

(Gupta et al. 2003). E. coli strains were typically grown in Luria-Bertani media (LB; 10 g tryptone, 5 g yeast extract, 10 g NaCL per l H2O, pH 7.0). NZY top agar (5 g NaCL, 2 g MgSO4 · 7H2O, 5 g yeast extract, 10 g NZ amine, 7 g agarose per l H2O, pH 7.0) was used to propagate and titer bacteriophage.

Genetic construction of the λluxI reporter bacteriophage

The fundamental construction of the λluxI reporter phage consisted of a fusion of the V. fischeri luxI gene (Accession #Y00509) upstream of the left arm promoter (PL) of phage lambda in a pLEX vector (Invitrogen, Carlsbad, CA) (Fig. 5). Upstream to this fusion was ligated an rrnB T1T2 transcriptional terminator from the pKK223-3 cloning vector (Accession #M77749). Each individual gene and step-wise fusions were initially constructed in pCR2.1- or pCR4-TOPO TA cloning vectors (Invitrogen) and then the entire fusion ligated into the Lambda ZAP II cloning vector (Stratagene) and packaged into phage lambda using Gigapack III Gold packaging extract (Stratagene). DNA isolations were performed with Wizard Minipreps, Midipreps, or Lambda Preps

(Promega, Madison, WI) and purified when necessary with the Geneclean Spin Kit (Q-

Biogene, Carlsbad, CA). PCR reactions were carried out in an MJ Research DNA

Engine tetrad (Waltham, MA) using Ready-To-Go PCR beads (Amersham Piscataway,

NJ). DNA was sequenced at all steps with the ABI Big Terminator Cycle

37

luxI rrnB A. NdeI NdeI C. ClaI

pCR2.1-TOPO pCR4-TOPO luxI rrnB

NdeI restriction digest and ligate into NdeI site within pLEX MCS luxI NdeI B. NdeI PL

pLEX-luxI Linearize

with ClaI

PCR amplify PL- luxI with addition of ClaI restriction sites D. luxI E. luxI ClaI rrnB PL PL EcoRI ClaI EcoRI pCR2.1-TOPO pCR4-TOPO Digest with ClaI PL-luxI PL-luxI-rrnB to remove PL-luxI and ligate into linearized pCR4- TOPO rrnB Digest with EcoRI and ligate into EcoRI site within Lambda ZAP II F. MCS luxI

PL rrnB EcoRI EcoRI Lambda ZAP II Package PL-luxI-rrnB into λ phage

Figure 5. Genetic construction scheme for the λluxI reporter bacteriophage

38

Sequencing reaction kit on an ABI 3100 DNA Sequencer (Perkin-Elmer, Foster City,

CA).

The luxI gene was PCR-amplified from V. fischeri using the primer pairs 5'-

CATATGACTATAATGATAAAAAAATCGG-3' and 5'-

CATATGTTAATTTAAGACTGC-3' to introduce the restriction site NdeI at both termini

(underlined) and cloned into a pCR2.1-TOPO vector (Fig. 5A). The luxI gene was then removed from the TOPO vector by NdeI restriction digestion and ligated into the NdeI multicloning site (MCS) of the pLEX vector, thereby placing luxI in frame with the PL promoter (Fig. 5B). Directionality was confirmed by restriction digestion and sequencing.

The rrnB transcriptional terminator was PCR-amplified from pKK223-3 using the primer pairs 5'-ATCGATAAGAGTTTGTAGAAACGC-3' and 5'-

CTGTTTTGGCGGATG-3' to introduce the restriction site ClaI at the 5' end (underlined) and cloned into a pCR4-TOPO vector (Fig. 5C).

The PL-luxI fusion was PCR-amplified out of pLEX with the primer pairs 5'-

ATCGATGTCGACTCTAGAGGATCC-3' and 5'-

ATCGATATTCGAGCTCGGTACCATA-3' containing the restriction sites ClaI

(underlined) and cloned into a pCR2.1-TOPO vector (Fig. 5D). This vector was then digested with ClaI and ligated into the ClaI site of the rrnB TOPO vector described above to create a PL-luxI-rrnB fusion within a pCR4-TOPO vector (Fig. 5E).

Directionality was again confirmed by restriction digestion and sequencing. Unique

EcoRI sites within the multicloning site of the pCR4-TOPO vector now flanked the PL-

39 luxI-rrnB fusion, allowing for its removal via EcoRI digestion with subsequent ligation into the unique EcoRI site of the Lambda ZAP II vector (Fig. 5F). Resulting recombinant lambda phage DNA was then packaged into phage heads using Gigapack III packaging extract per the manufacturer‟s instructions (Stratagene). Resulting plaques were hybridized with an alkaline phosphatase labeled luxI probe to chemifluorescently identify luxI incorporated phage using an Alkphos Direct Labeling and Detection kit (Amersham).

Positive plaques were then isolated and propagated on top agar plates as described in the

Lambda ZAP II instructions to concentrations of approximately 1 × 1010 PFU/ml and stored at 4ºC.

Genetic construction of E. coli OHHLux

The OHHL specific bioluminescent bioreporter E. coli OHHLux was constructed by fusing the luxCDABE genes from P. luminescens with the luxR gene and luxI promoter region (PluxI) from V. fischeri into an EZ::TN pMOD cloning vector (Epicentre

Technologies, Madison, WI) in conjunction with an rrnB T1T2 transcriptional terminator and a kanamycin resistance gene (Fig. 6). The intermixing of the P. luminescens and V. fischeri lux genes was performed solely due to their ready availability in existing cloning vectors. The construct is similar to that of plasmid pSB401 created by Winson et al.

(Winson et al. 1998), except our design incorporates the cloning region on a hyperactive transposon theoretically capable of insertion into virtually any bacterial chromosome.

The rrnB transcriptional terminator was PCR-amplified from pKK223-3 with the primer pairs 5´-ATCGATAAGAGTTTGTAGAAACGC-3´ and 5´-

GAATTCCTGTTTTGGCGGATG-3´ containing the restriction sites ClaI and EcoRI

40

ClaI EcoRI HindIII HindIII rrnB Kn EcoRI luxCDABE EcoRI SalI luxR SalI

A. B. C. D. Plux pCR2.1- pYES2.1/V5- pCR-XL- pGEM-3Z His-TOPO TOPO I TOPO luxR/PluxI rrnB Kn luxCDABE

ClaI rrnB EcoRI MCS E. HindIII EZ::TN pMOD rrnB digestio EcoRI ClaI rrnB n HindIII F. Kn EcoRI HindIII EZ::TN pMOD rrnB- digestio Kn ClaI rrnB EconRI G. luxCDABE SalI EcoRI digestion EZ::TN pMOD rrnB-luxCDABE- HindIII Kn Kn ClaI rrnB EcoRI HindIII H. luxCDABE EcoRI SalI EZ::TN pMOD P rrnB-luxCDABE- luxI luxR-Kn luxR SalI HindIII Kn HindIII Figure 6. Cloning format for construction of an OHHL specific bioluminescent bioreporter E. coli OHHLux

41

(underlined) at the 5´ and 3´ ends, respectively and cloned into a pCR2.1-TOPO vector

(Fig. 6A). The kanamycin gene was PCR-amplified from a pCR2.1-TOPO vector with the primer pairs 5´-AAGCTTTCAGGGCGCAAGGGC-3´ and 5´-

AAGCTTACTCTTCCTTTTTCAATTCAGAAGAAC-3´ containing terminal HindIII restriction sites (underlined) and cloned into a pYES2.1/V5-His-TOPO vector

(Invitrogen) (Fig. 6B). The luxCDABE gene cassette was PCR-amplified from P. luminescens using the primer pairs 5´-ATTAAATGGATGGCAAATAT-3´ and 5´-

AGGATATCAACTATCAAAC-3´ and cloned into a pCR-XL-TOPO vector (Invitrogen)

(Fig. 6C). The luxR gene and its neighboring PluxI region were PCR-amplified from V. fischeri with the primer pairs 5´-GTCGACCCTATAGGTATAAAGCTTTACTTACG-3´ and 5´-GTCGACTACCAACCTCCCTTGCG-3´ containing SalI restriction sites at both termini (underlined) and cloned into a pGEM-3Z vector (Promega) digested with SalI

(Fig. 6D).

The rrnB TOPO clone was digested with ClaI and EcoRI and ligated into compatible ClaI and EcoRI sites within the MCS of the EZ::TN pMOD vector to create

EZ::TN pMOD-rrnB (Fig. 6E). The kanamycin TOPO clone was digested with HindIII and ligated into the compatible HindIII site within the MCS of EZ::TN pMOD-rrnB to create EZ::TN pMOD-rrnB-Kn (Fig. 6F). The luxCDABE TOPO clone was digested with EcoRI (EcoRI sites are within the pCR-XL-TOPO MCS) and ligated into the compatible EcoRI site at the 3´ end of rrnB in the EZ::TN pMOD-rrnB-Kn vector to create EZ::TN pMOD-rrnB-luxCDABE-Kn (Fig. 6G). Directionality of the luxCDABE insert was confirmed by restriction digestion and sequencing. The luxR/PluxI pGEM clone was digested with SalI and ligated into the SalI site of the MCS in EZ::TN pMOD-rrnB-

42 luxCDABE-Kn to create EZ::TN pMOD-rrnB-luxCDABE-luxR-Kn (Fig. 6H).

Directionality of the luxR/PluxI insert was confirmed by restriction digestion and sequencing. The original plan at this point was to remove the rrnB-luxCDABE-luxR-Kn sequence from the EZ::TN pMOD vector using existing flanking PshAI restriction sites to produce an EZ::TN transposon (Epicentre Technologies) for in vitro insertion into the chromosome of the host strain E. coli XLOLR (Stratagene). However, transposition was not successful and the EZ::TN pMOD-rrnB-luxCDABE-luxR-Kn vector resides within E. coli XLOLR as a plasmid.

Dose response kinetics of the E. coli OHHLux bioreporter to OHHL

Synthetic OHHL (Sigma-Aldrich, St. Louis, MO; catalog no. K-3007) was diluted in 5 ml aliquots of M9 minimal media (Sambrook & Russell 2001) to desired concentrations and 100 μl of each dilution aliquot was added to triplicate wells in black

96-well microtiter plates (Dynex Technologies, Chantilly, VA). E. coli OHHLux was

8 grown in LB at 37ºC to an OD600 of 0.6 (~1 × 10 CFU/ml) and 50 μl added to each microtiter plate well containing the diluted OHHL. As well, E. coli OHHLux was added to wells void of OHHL to determine noninduced background levels of bioluminescence.

Plates were sealed with transparent adhesive film (TopSeal-A, Perkin-Elmer, Boston,

MA) and placed in a Perkin-Elmer Victor2 Multilabel counter at 30ºC with shaking

(„normal‟ speed was selected with a 0.5 mm orbital diameter) with light collection programmed for 1 s/well at 20 min intervals. In this and all experiments described below, resulting bioluminescent measurements were given the arbitrary light unit of counts/s (CPS). A bioluminescent response was considered significant if it achieved an intensity 2 standard deviations (2σ) above the negative control sample.

43

Specificity of the E. coli OHHLux bioreporter towards OHHL

Gram negative and Gram positive bacteria participate in quorum sensing communication networks via the production of many different types of AHL autoinducers or oligopeptides, respectively (Miller & Bassler 2001). To demonstrate that the E. coli OHHLux bioreporter responded to OHHL and not to other autoinducers, the bacteria listed in Table 5 were grown in media and at temperatures specified by the

American Type Culture Collection (ATCC) to an OD600 of 0.6 and individually combined

1:1 in 96-well microtiter plates with E. coli OHHLux grown to an OD600 of 0.6 in LB at

37ºC. Wells containing only E. coli OHHLux served as negative controls for monitoring background levels of bioluminescence. Positive control wells contained E. coli OHHLux and 10 nmol/l synthetic OHHL (final volume). Plates were incubated in the Victor2

Multilabel counter at 30ºC with shaking with bioluminescence monitored for 1 s/well at approximate 20 min intervals.

Phage reporter pure culture assay

To determine target cell detection limits, λluxI reporter phage and E. coli OHHLux were combined with a dilution series of E. coli XL1-Blue down to an estimated 1

CFU/ml. λluxI reporter phage were prepared on top agar overlays and stored at stock concentrations of 1 × 1010 PFU/ml. The OHHLux bioluminescent bioreporter was grown

8 in LB at 37ºC to an OD600 of 0.6 (~1 × 10 CFU/ml) and used as is. The E. coli host

9 XL1-Blue was grown at 30ºC in LB to an OD600 of 0.7 (~1 × 10 CFU/ml) then serially diluted 1:10 down to approximately 1 CFU/ml in 50 ml conical centrifuge tubes containing 9 ml LB. One hundred microliter aliquots of each XL1-Blue dilution were

44

Table 5. Strains tested for interference against the OHHLux bioluminescent bioreporter

Organism Autoinducer, oligopeptide, Reference or autoinducer lactonase

Agrobacterium OOHL (Fuqua & Winans 1994) tumefaciens ATCC 33970

Arthrobacter globiformis AHL lactonase (Park et al. 2003) KACC 10580 Bacillus mycoides AHL lactonase (Dong et al. 2002) ATCC 37015 Burkholderia cepacia OHL (Lewenza et al. 1999) ATCC 25416

Erwinia carotovora OHHL (Pirhonen et al. 1993) ATCC 15713 Pseudomonas aeruginosa OdDHL, BHL, AHL (Huang et al. 2003) ATCC BAA-47 lactonase Rhodobacter sphaeroides 7,8-cis-N-(tetradecanoyl)- (Puskas et al. 1997) ATCC 55304 homoserine lactone Serratia liquefaciens HHL, BHL (Eberl et al. 1996) ATCC 11367

Staphylococcus aureus AIP (autoinducing peptide) (Mayville et al. 1999) ATCC 35556 Yersinia enterocolitica HHL, OHHL (Throup et al. 1995) ATCC 23715

OHHL, N-(3-oxohexanoyl)-homoserine lactone; OdDHL, N-(3-oxododecanoyl)- homoserine lactone; OHL, N-octanoyl-homoserine lactone; OOHL, N-(3-oxooctanoyl)- homoserine lactone; HHL, N-hexanoyl-homoserine lactone; BHL, N-butanoyl- homoserine lactone. For specifics on AHL lactonases and acylases, see Roche et al. (Roche et al. 2004).

45 then distributed columnwise (100 μl/well, 8 wells/column) throughout a black 96-well microtiter plate. A control column received 100 μl of LB/well. To all wells was then

9 added 100 μl of λluxI reporter phage stock (~1 × 10 PFU/ml final concentration) and 50

μl of OHHLux bioreporter (~5 × 106 CFU/ml final concentration). This equates to an approximate upper multiplicity of infection (MOI) of 10, and establishes a high infection rate of XL1-Blue cells. Plates were monitored for bioluminescence overnight in the

Victor2 Multilabel counter (30ºC, shaking, 1 s/well, 20 min intervals). Each XL1-Blue dilution tube was additionally incubated in a standard laboratory incubator with shaking

(200 rev/min) at 37ºC to promote better growth than that achievable in the microtiter plate. After 5 h, 100 μl aliquots were removed from the preincubated dilution tubes as well as from a preincubated LB control tube and transferred to a microtiter plate as described above, with λluxI reporter phage and OHHLux bioreporters added to each well also as described above. The microtiter plate was similarly monitored for bioluminescence.

All dilutions of XL1-Blue, both at the beginning and after the 5 h incubation period, were plated in triplicate on LB agar containing tetracycline at 14 mg/l (LBTc) to determine viable cell counts. Each microtiter plate assay was also run with a duplicate control series of dilutions wherein E. coli XL1-Blue was replaced with the lambda resistant strain E. coli SOLR (Stratagene).

Lettuce leaf wash assays

8 E. coli XL1-Blue was grown at 30ºC in 200 ml LB to an OD600 of 0.6 (~1 × 10

CFU/ml), centrifuged at 1000 × g for 10 min, and resuspended in 200 ml sterile water. A

46

1:10 dilution series was then prepared in 200 ml volumes of sterile water to form E. coli contaminated water ranging from 108 to approximately 1 CFU/ml. A control tube not receiving an XL1-Blue inoculum was also prepared. Grocery store purchased iceberg head lettuce was rinsed with 1 l sterile water and spun dry in a kitchen salad spinner

(Zyliss Corp., Foothill Ranch, CA). Ten grams of lettuce were placed in each dilution of

E. coli contaminated water for 5 min with shaking (200 rev/min), spun dry in the salad spinner, and individually transferred to 30 ml of sterile saline. After 2 min of shaking

(200 rev/min), saline diluents were transferred to 50 ml conical centrifuge tubes and centrifuged for 10 min at 3000 × g. Resulting pellets were resuspended in 3 ml LB and then assayed in microtiter plates either immediately or after a 16 h preincubation at 37ºC with shaking (200 rev/min). Preincubation was performed under tetracycline selection

(14 mg/l final concentration) to select for XL1-Blue cells. Aliquots of 100 µl were removed from each LB resuspension either immediately or after the 16 h preincubation and transferred columnwise (100 µl/well, 8 wells/column, 1 column /dilution tube) to a

96-well black microtiter plate. Preincubated samples were washed once with LB to remove residual tetracycline just prior to sample transfer to the microtiter plate to prevent destruction of the OHHLux bioreporter. Each well then received 100 μl of λluxI reporter phage stock and 50 μl of OHHLux bioreporter prepared as described above for the pure culture assays. Microtiter plates were sealed with adhesive and monitored for bioluminescence in the Victor2 Multilabel counter (30ºC, shaking, 1 s/well, 20 min intervals). Each LB resuspension was plated in triplicate on LBTc plates both immediately and after the 16 h incubation to determine viable XL1-Blue cell counts.

47

Analytical measurement of OHHL

OHHL concentrations were analytically determined using a ThermoFinnigan

LCQ DecaXPplus liquid chromatograph-mass spectrometer (LCMS) fitted with a 10 cm ×

4.6 mm id C18 column (Advanced Chromatography Technologies, Chadds Ford, PA).

Triplicate culture supernatant aliquots ranging from 1 to 50 ml were extracted twice with equal volumes of ethyl acetate, dried under nitrogen, and redissolved in 1 ml of methanol.

A flow rate of 0.2 ml/min was used, starting with 20% methanol going to 95% methanol in 27 min with a 7 min hold, returning to 20% methanol in 6 min, and equilibrating for 5 min.

Results

OHHL dose response of the E. coli OHHLux bioreporter

The E. coli OHHLux bioreporter was exposed to varying concentrations of synthetic OHHL to determine detection limits (Fig. 7). Significant bioluminescent signals (2σ above background) were produced in response to OHHL at concentrations ranging from 10 nmol/l to 50 μmol/l. Saturation-type behavior was observed at OHHL concentrations exceeding 50 μmol/l. Since the function of this bioreporter is to detect low-level OHHL, we were more concerned with the lower tailed response curve profile.

The inset to Figure 7 demonstrates response linearity at these lower OHHL concentrations ranging from 20 nmol/l to 2 μmol/l (R2 = 0.99).

48

90,000 45,000 40,000 80,000 35,000 30,000 R² = 0.99 25,000 70,000 20,000 15,000 60,000 Normalized CPS 10,000 5,000 0 50,000 0.01 0.1 1 10 40,000 OHHL concentration (uM) 30,000 Normalized CPS Normalized 20,000 10,000 0 0.001 0.01 0.1 1 10 OHHL concentration (uM)

Figure 7. OHHL dose response curve of E. coli OHHLux.

Synthetic OHHL was diluted at indicated concentrations and added in a 1:2 dilution to a culture of E. coli OHHLux, incubated for 1 h at 30ºC, and then monitored for bioluminescence at 490 nm (n = 3). Inset shows linearity of the bioluminescent response within a lower detection range of 20 nmol/l to 2 μmol/l OHHL as measured by LCMS. Counts/s (CPS) were normalized to control CPS.

49

Specificity of the E. coli OHHLux bioreporter towards OHHL

The E. coli OHHLux bioreporter was co-cultured with bacterial strains synthesizing other classes of quorum sensing autoinducers or oligopeptides (Table 5).

Significant bioluminescence was initiated only in response to the OHHL synthesizing strains Erwinia carotovora and Yersinia enterocolitica, which generated bioluminescence at 87% and 64%, respectively, that of control wells containing E. coli OHHLux exposed to 10 nmol/l synthetic OHHL (Fig. 8). The remaining strains produced bioluminescence at less than 1% of the E. coli OHHLux control.

Phage reporter assay in pure culture

To test assay detection limits and response times, a 1:10 dilution series of target

E. coli XL1-Blue cells ranging from approximately 1 × 108 to 1 CFU/ml was added to

λluxI reporter phage and E. coli OHHLux bioreporters in 96-well microtiter plates both with and without a supplementary 5 h preincubation. Without preincubation, the microtiter plate assay was capable of detecting target E. coli XL1-Blue cells at an initial cell concentration, as determined by plate counts, of 1.1 × 108 CFU/ml within 1.5 h, 2.0 ×

107 CFU/ml within 2.2 h, 1.2 × 106 CFU/ml within 3.6 h, and 2.9 × 105 CFU/ml within

4.9 h (Fig. 9). XL1-Blue cell concentrations below 105 CFU/ml did not generate significant bioluminescence. However, preincubating the dilution tubes at 37ºC with shaking for 5 h prior to initiation of the assay permitted better growth of XL1-Blue cells than in the constrained microtiter plate wells, and allowed for detection down to 1 (± 2.5)

CFU/ml within a total assay time of 10.3 h (Fig. 9). Duplicate control microtiter plates were also prepared substituting E. coli XL1-Blue with the lambda resistant strain E. coli

50

Figure 8. Bioluminescent response of E. coli OHHLux when co-cultured with each bacterium listed in Table 5

E. carotovora and Y. enterocolitica synthesize OHHL and induced bioluminescence in the OHHLux bioreporter when co-cultured. Other quorum sensing autoinducers or oligopeptides produced by the remaining strains did not significantly induce bioluminescence from the OHHLux bioreporter (n = 3).

51

6x106

5x106

4x106

3x106

2x106 18

Bioluminescent CPS Bioluminescent 16 1x106 14 12 10 0 Test tube 8 8 preincubation period 10 7 Hours 10 6 6 10 5 10 4 4 10 3 10 2 10 1 2 1010 0 CFU/ml 101 10-1 0 010-2

Figure 9. Detection limits of the phage bioreporter assay in a pure culture of E. coli XL1-Blue target cells

E. coli XL1-Blue was diluted 1:10 from approximately 1 × 108 to 1 CFU/ml and combined with λluxI reporter phage and E. coli OHHLux bioreporters (n = 3). The assay directly detected E. coli XL1-Blue at mean concentrations ranging from 1.1 × 108 to 2.9 × 105 CFU/ml within 1.5 to 4.9 h, respectively. XL1-Blue cultures at concentrations below 105 CFU/ml required a separate 5 h preincubation and yielded bioluminescent signals in as few as 1 (± 2.5) CFU/ml within 10.3 h, inclusive of the 5 h preincubation.

52

SOLR. No significant bioluminescence was observed in these plates (data not shown).

Lettuce leaf rinse assays

Rinsings from iceberg lettuce artificially contaminated with a 1:10 dilution series of E. coli XL1-Blue cells were exposed to the phage reporter assay. At the highest average concentration of XL1-Blue cells (1.4 × 108 CFU/ml), significant bioluminescence occurred within 2.6 h (Table 6). Successive 10-fold dilutions, yielding average cell concentrations of 1.5 × 107, 1.3 × 106, and 1.7 × 105 CFU/ml, generated significant bioluminescence within 3.3, 10.3, and 12.1 h, respectively. Cell concentrations below 105 CFU/ml did not produce significant bioluminescence.

Therefore, these dilutions were preincubated under tetracycline selection for 16 h to increase target cell concentrations, and then tested in the phage reporter assay. After the

16 h preincubation, the control tube, void of an XL1-Blue inoculum, indicated a background concentration of nontarget tetracycline resistant cells of 3.6 × 108 CFU/ml.

Estimated concentrations of tetracycline resistant XL1-Blue cells within comparable background populations were enumerated in each dilution tube based on similar colony morphology and are listed in Table 6. With selective overnight incubation, the original

104 and 103 inoculums of XL1-Blue cells could be detected within a total assay time, including the 16 h preincubation, of 19.1 h. The original 102 inoculum was detectable within 22.4 h. No significant bioluminescence was observed from XL1-Blue dilutions lower than 1 × 102 CFU/ml.

53

Table 6. Detection of E. coli XL1-Blue in leaf lettuce wash assays

Initial E. coli XL1- Estimated E. coli XL1- Time until Peak Blue inoculum Blue concentration after bioluminescence bioluminescence (CFU/ml)* 16 h pre-incubation induction (h) (CPS) (CFU/ml) 1.4 × 108 NA† 2.6 817,000

1.5 × 107 NA 3.3 31,500

1.3 × 106 NA 10.3 8,600

1.7 × 105 NA 12.1 6,800

1.7 × 104 3.8 × 108 19.1‡ 81,800

1.5 × 103 2.0 × 108 19.1‡ 59,750

130 (± 21) 1.1 × 107 22.4‡ 17,300

20 (± 4) 9.6 × 104 ND§ ND

2 (± 3) 6.6 × 104 ND ND

*Iceberg lettuce (10 g) was washed with water artificially contaminated at levels indicated †NA, not assayed ‡Includes a 16 h preincubation prior to initiation of the assay §ND, not detected

54

Discussion

The key technological metrics required by the food industry for effective detection and monitoring of bacterial pathogens are sensitivity, specificity, speed, simplicity, and cost-effectiveness. Portability can also be added to this list as quality control testing begins to move from the centralized laboratory to strategic on-the-spot monitoring within the production line itself. The traditional methods of selective sample enrichment followed by any number of morphological, biochemical, or serological tests offered accuracy and reliability but little in the way of rapidity, often requiring several days from initial sampling to final analysis. The introduction of nucleic acid based detection technologies (hybridization and PCR assays) afforded some significant increases in response times as well as improved sensitivity and specificity, but the complexity and costs involved in routine analysis limits their universal application. The binary phage assay described here provides a new direction towards pathogen monitoring. By exploiting phage specificity and the autoamplification power of quorum sensing, a single target pathogen can be manifested not as a single entity but rather as an intensified chemical signature. Detecting the chemical signature can then be accomplished using any variety of techniques ranging from highly sensitive but complex analytical methods (LCMS) to variously available easy-to-use AHL-sensing bioreporters

(lacZ, gfp , or lux based) (Shaw et al. 1997, Camara et al. 1998, Winson et al. 1998,

Andersen et al. 2001). We chose and constructed a luxCDABE based bioreporter (E. coli

OHHLux) due to its capacity for autonomous, near real-time, and nondestructive sensing.

Further, the ability to interface lux based bioreporters onto integrated circuit

55 microluminometers permits creation of miniaturized standalone biosensors capable of meeting the remote on-site monitoring needs of the food industry (Nivens et al. 2004).

E. coli OHHLux was designed to specifically respond to OHHL using the

LuxI/LuxR quorum sensing regulatory system. Thus, infection of a host cell by a luxI recombinant phage leads to OHHL synthesis and subsequent bioluminescent signalling by the OHHLux bioreporter. However, bacteria naturally synthesizing OHHL will induce bioluminescence as well, as was shown in Figure 8 with E. carotovora and Y. enterocolitica. The presence of these and any other OHHL producing in a sample would consequently generate false positive signals. It is therefore imperative that assays include a control consisting of bioreporter alone added to the sample to identify intrinsic OHHL.

E. coli OHHLux was assembled within an EZ::TN pMOD cloning vector

(Epicentre Technologies) to permit chromosomal insertion. This provides advantages such as low reporter gene copy number for reduced background bioluminescence, genetic stability, and no requirements for antibiotic selection. However, chromosomal transposition was not successful and the pMOD-lux vector remained as a plasmid in E. coli. Unfortunately, this created an elevated but acceptable level of background bioluminescence and required initial growth under kanamycin selection, thereby creating an extra step in the assay procedure. We are currently working in conjunction with

Epicentre Technologies to ascertain why transposition failed.

The binary phage reporter assay was developed using phage lambda as a model system for the general detection of E. coli. However, phage typing libraries, the host

56 groupings of over 5,000 bacteriophage, and the advent of phage and phage anti- infectives now provide a considerable assemblage of phage from which to construct reporter systems with unique specificity towards select microbial pathogens (Ackerman

2001, Stone 2002). For example, phage PP01 can be used to specifically detect E. coli

O157:H7, even within a background of E. coli strains of other O-serogroups and K-12 strains (Morita et al. 2002b). Although the of phage resistance in target hosts is often cited as a disadvantage in phage based detection strategies, the use of multi- phage cocktails and the natural reversion of host mutants back to phage sensitivity often negates this perceived obstacle (O'Flynn et al. 2004).

The reporter phage assay was capable of direct, rapid (1.5 – 5 h) detection of E. coli XL1-Blue in pure culture at high cell densities (108 – 105 CFU/ml) using a three-step protocol consisting of the addition of reporter phage, bioreporter, and target cells (Fig. 9).

No other reagents or manipulations were required other than incubation and light measurement, making this assay highly amenable to automated high throughput monitoring using any number of available photomultiplier based luminescent readers.

Phage based sensing also ensures only the detection of live cells, thereby overcoming the false positive detection of dead cells often inherent in other assaying methods such as

ELISA and PCR (Wolffs et al. 2005).

Detection of E. coli XL1-Blue in pure culture at cell densities below 105 CFU/ml could not be achieved directly within the microtiter plate assay. Adequate growth of

XL1-Blue cells at these lower cell densities was not possible due to a combination of several factors (i.e., for growth resources with the OHHLux bioreporter cell population, coexistence with the λluxI reporter phage population, and a poorly oxygenated

57 environment within the sealed microtiter plate well). It was therefore necessary to preincubate XL1-Blue cells separately in well-aerated test tubes for 5 h to increase initial cell numbers prior to initiation of the phage reporter assay. This permitted detection at an initial cell concentration of 1 (± 2.5) CFU/ml within a total assay time of 10.3 h (Fig. 9).

When tested against artificially contaminated lettuce, the assay detected original inoculums between 108 – 105 CFU/ml within 2.6 – 12.1 h, respectively (Table 6). This occurred without specific selection for target XL1-Blue cells or additional preincubation steps outside of the normal microtiter plate assay. In these experiments E. coli XL1-Blue was detected within a background of normal microflora, therefore detection times were longer than in the pure culture experiments. This would be expected since contaminating cells nonspecifically bind with phage, thereby reducing effective phage concentrations.

(Similar nonspecific binding would occur in food samples heavily endowed with particulate matter, such as ground beef. Preprocessing of such samples to remove particulates would likely be necessary.) This also caused decreased levels of bioluminescence; as phage titers decline so do phage infection events, and the corresponding reduction in OHHL synthesis resulted in lowered induction rates of the

OHHLux bioreporter. Additionally, growth of XL1-Blue populations was slower due to competition from other cell populations. Collectively, these factors make quantitative assessment of target bacteria difficult, making this assay more of a sentinel first response monitoring system capable of presence/absence detection to be followed with more robust analytical testing.

As with the pure culture assays, detection below 105 XL1-Blue cells/ml was not possible without prior incubation. As well, tetracycline was used to select for XL1-Blue

58 and reduce nontarget background microbial populations. When assaying for an actual pathogen, relevant enrichment methods besides mere antibiotic addition are available and would have to be chosen based on the phage/pathogen combination being used (Sharpe

2001). Although preincubations were carried out under selective conditions, the background concentration of nontarget cells remained high (3.6 × 108 CFU/ml). This prevented optimal growth of E. coli XL1-Blue populations and once more served as a sink for nonspecific phage attachment. As a result, detection times were again higher than those seen with pure culture assays with corresponding XL1-Blue concentrations. In addition, light levels decreased, as observed in Table 6 when comparing, for example, initial inoculums of 1.4 × 108 CFU/ml versus 1.7 × 104 CFU/ml. Although with preincubation the 1.7 × 104 CFU/ml culture eventually grew to an estimated 3.8 × 108

CFU/ml, the presence of a large nontarget microbial population and corresponding reduction in phage titer due to nonspecific binding resulted in bioluminescence levels 10- fold lower than that of the 1.4 × 108 CFU/ml culture (817,000 CPS versus 81,800 CPS).

Detection of XL1-Blue at and below 10 CFU/ml failed due to insufficient enrichment to detectable numbers during the preincubation.

The preliminary testing reported here demonstrates the unique ability of luxI- incorporated bacteriophage to detect target microbes within a simple and cost-effective microtiter plate format. Although detection limits do not yet approach that of more routine monitoring methods such as PCR and ELISA, ongoing genetic enhancements to the reporter phage (i.e., expression of multiple phage based luxI elements rather than a single element) as well as better bacterial target enrichment and isolation techniques (i.e.,

59 immunomagnetic separation coupled with phage based sensing (Goodridge et al. 1999,

Favrin et al. 2001)), may drive detection limits to acceptable levels.

60

Chapter III: Bacteriophage based bioluminescent bioreporter

for the detection of E. coli O157:H7

61

This is a version of an article found in the journal of Food Pathogen Detection entitled

“Bacteriophage Based Bioluminescent Bioreporter for the Detection of Escherichia coli

O157:H7”.

Abstract

Rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this work, the lux reporter genes were used in a new bioassay that allows for pathogen monitoring without multiple sample manipulations or addition of exogenous substrate. A recombinant phage specific for Escherichia coli

O157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3- oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli O157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP01-luxI to detect E. coli O157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PP01-luxI and E. coli O157:H7, and luminescence was monitored. Reporter phage induced light in bioreporter cells within 2 h when exposed to 104 CFU/ml E. coli O157:H7, and were able to detect 10 CFU/ml in pure culture with a pre-incubation step (total detection time 5 h). The detection method was also applied to contaminated apple juice and was able to detect 104 CFU/ml in 2 h after a 6 h pre-incubation.

62

Introduction

Rapid detection methods for E. coli O157:H7 in food and water are necessary to detect both naturally occurring outbreaks and bioterrorist events. A variety of methods exist for detection of E. coli O157:H7, but most are slow or require significant sample manipulation (Moxley 2003, Deisingh & Thompson 2004, Gehring et al. 2004, Lee et al.

2005). Culture methods require skilled personnel and results are not obtained for 24 - 48 h (Moxley 2003, Deisingh & Thompson 2004). ELISAs and other immunological methods require staining and washing steps (Moxley 2003, Deisingh & Thompson 2004,

Gehring et al. 2004). Rapid detection methods based on PCR are fast and reliable but cannot differentiate between live and dead bacterial cells and can be fairly complex

(Deisingh & Thompson 2004). Biosensor based systems can be rapid and amenable to real-time monitoring, but currently cannot detect low concentrations of bacteria, and have difficulty detecting bacteria in food matrices (Radke & Alocilja 2005, Subramamanian et al. 2006).

The specificity of bacteriophage for their bacterial hosts has long been used for the identification of bacterial pathogens. Phage assays are commonly used for identification and typing of bacteria (reviewed in (Marks 2000)). In the last 20 years, genetic manipulation of bacteriophage to make “reporter phage” using reporter genes such as bacterial luciferase (luxAB), β-galactosidase (lacZ) and green fluorescent protein

(gfp) has been documented (Ulitzer & Kuhn 1987, Stewart et al. 1989, Kodikara et al.

1991, Turpin et al. 1993, Chen & Griffiths 1996, Loessner et al. 1996, Waddell & Poppe

2000, Goodridge & Griffiths 2002). Phage are metabolically inert when outside of their host, so the reporter proteins encoded in these phage are not produced until the

63 appropriate bacterium is infected. In the existing lux based phage reporter systems, bacteria are detected following phage infection, an incubation/growth period, and the addition of an exogenous substrate (Kodikara et al. 1991, Chen & Griffiths 1996,

Loessner et al. 1996). While these methods are capable of specific and sensitive detection of bacteria, they are not amenable to continuous monitoring because they require excessive sample manipulation.

Recently, a rapid GFP-reporter phage detection system was described that can detect and differentiate live and dead E. coli O157:H7 cells (Oda et al. 2004, Awais et al.

2006). This GFP reporter was constructed in bacteriophage PP01, which was isolated from swine stools by Morita et al. (Morita et al. 2002b). Phage PP01 was found to infect a variety of E. coli O157:H7 strains (17 out of 19 tested, including toxigenic and non- toxigenic), but none of the 14 non-O157:H7 E. coli strains examined (Morita et al. 2002b,

O'Flynn et al. 2004). Other species of bacteria that have been tested, including

Salmonella enterica serovar Typhimurium, Pseudomonas fluorescens, Pseudomonas aeruginosa, Citrobacter freundii, Erwinia carotovora, and Yersinia enterocolitica, have not shown susceptibility to the phage, (Morita et al. 2002b, O'Flynn et al. 2004). This

PP01-GFP reporter phage is rapid and specific, but like all GFPbased assays it requires a high energy light source for excitation of GFP which may limit its potential for use in real-time online industrial and environmental monitoring. Here, we describe a PP01 reporter phage that uses the lux reporter genes in a unique construct that permits detection without the addition of substrate, and has potential for use in real-time online monitoring.

The lux bioluminescence genes were isolated from the marine bacterium Vibrio fischeri, and includes a cassette of five genes (luxCDABE) whose products are necessary

64 for the generation of light, and two genes (luxI and luxR) that produce the components of a quorum sensing system that activates the transcription of the luxCDABE cassette

(Meighen 1991). The luciferase genes (luxAB) encode the proteins responsible for generating bioluminescence, while the reductase (luxC), transferase (luxD) and synthetase (luxE) genes encode proteins that produce an aldehyde substrate required for the bioluminescence reaction. Bioluminescent bacteriophage based bioreporters developed in the past carry the luxAB genes, but lack the luxCDE genes (Ulitzer & Kuhn

1987, Stewart et al. 1989, Kodikara et al. 1991, Chen & Griffiths 1996, Loessner et al.

1996, Waddell & Poppe 2000). Thus, once these bacteriophage infect their target cells, detection requires the addition of exogenous substrate. The entire luxCDABE cassette is too large to be accommodated by most phage genomes, so to eliminate the need for exogenous substrate an alternative approach is necessary.

The detection system described here takes advantage of the luxI/luxR quorum sensing system. Quorum sensing systems are cell-to-cell communication networks based on the synthesis of diffusible autoinducer molecules (reviewed in (Miller & Bassler

2001)). V. fischeri expresses low levels of luxI and produces small amounts of the diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). When a high concentration of V. fischeri is present the concentration of OHHL becomes high enough to bind to LuxR and activate transcription of luxCDABE and luxI, causing the production of light. In the system used here, the complete lux cassette (luxCDABE) along with the transcriptional regulator luxR are carried by a “bioreporter cell” that detects the diffusible

OHHL produced by a target pathogen infected with a phage carrying the luxI gene (Fig.

10). The initial feasibility of such phage based bioreporters was demonstrated with a

65 phage lambda/E. coli K12 model (Ripp 2006). In this work, luxI was integrated into phage PP01 for detection of its host, E. coli O157:H7, in pure culture and in artificially contaminated apple juice.

Materials and methods

Bacterial strains and bacteriophage

Bacteriophage PP01 was obtained from Hajime Unno (Tokyo Institute of

Technology), and was stored in SM buffer (10 mM MgSO4, 100 mM NaCl, 0.01% gelatin, and 50 mM Tris-HCl, pH 7.5). The bacterial strains used in this work were obtained from ATCC: toxigenic E. coli O157:H7 (43895), non-toxigenic E. coli O157:H7

(43888), E. coli K12 (10798), E. coli DH1 (33849), E. coli MM294 (33625), (14579) and Bacillus mycoides (6462). All strains were grown in Luria Bertani medium (LB; 10 g tryptone, 5 g yeast extract, 10 g NaCl per liter H20, pH 7.0). The bioreporter used in this work was E. coli OHHLux which has been previously described

(Ripp 2006). Briefly, the bioreporter cells carry luxR and luxCDABE under the control of the bidirectional lux promoter. Activation of the lux operon in these cells occurs in response to OHHL at concentrations of 5 nM and higher.

Construction of the reporter phage

The methods used for the construction of the PP01-luxI reporter phage were based on those used by Oda et al. (Oda et al. 2004) to create a PP01-GFP phage. Construction of the luxI-carrying plasmid used for recombination of PP01 entailed the fusion of the V.

66

Figure 10. Bacteriophage based bioluminescent bioreporter for pathogen detection

Bacteriophage based bioluminescent bioreporter for pathogen detection. Phage PP01- luxI, which carries the luxI gene under the control of a strong phage promoter, injects its genome into E. coli O157:H7. Upon infection, luxI is transcribed from the phage genome and as a result N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) is produced. OHHL diffuses out of the host cell and into a nearby bioreporter cell where it binds to the transcriptional activator LuxR. The LuxR-OHHL complex interacts with PL to activate transcription of luxCDABE resulting in the production of a detectable light signal.

67 fischeri luxI gene (Genbank accession #Y00509) upstream of the left arm promoter (PL) of phage lambda in a pLEX vector (Invitrogen, Carlsbad, CA). Downstream to this fusion an rrnB T1T2 transcriptional terminator from the pKK223-3 cloning vector

(Genbank accession #M77749) was ligated. Finally, the soc2, soc1 and soc genes of phage PP01 (Genbank accession #AY247798) were cloned and inserted upstream and downstream of the luxI construct to allow homologous recombination to occur between the plasmid and the phage. Each individual gene and step-wise fusions were initially constructed in pCR2.1- or pCR4-TOPO TA cloning vectors (Invitrogen). DNA isolations were performed with Wizard Minipreps, Midipreps, or Lambda Preps (Promega,

Madison, WI) and purified when necessary with the Geneclean Spin Kit (Q-Biogene,

Carlsbad, CA). PCR reactions were carried out in an MJ Research DNA Engine tetrad

(Waltham, MA) using Ready-To-Go PCR beads (Amersham Biosciences, Piscataway,

NJ). DNA was sequenced at all steps with the ABI Big Dye Terminator Cycle

Sequencing reaction kit on an ABI 3100 DNA Genetic Analyzer (Applied Biosystems,

Foster City, CA).

The luxI gene was PCR-amplified from V. fischeri using forward primer 5'-

CATATGACTATAATGATAAAAAAATCGG-3' and reverse primer 5'-

CATATGTTAATTTAAGACTGC-3' to introduce the unique restriction site NdeI at both termini (underlined) and cloned into a pCR2.1-TOPO vector (Fig. 11A). The luxI gene was excised from the TOPO vector by NdeI restriction digestion and ligated into the NdeI multicloning site (MCS) of the pLEX vector, thereby placing luxI in frame with the

Lambda PL promoter (Fig. 11B). Directionality was confirmed by restriction digestion and sequencing.

68

The rrnB transcriptional terminator was PCR-amplified from pKK223-3 using forward primer 5'-ATCGATAAGAGTTTGTAGAAACGC-3' and reverse primer 5'-

CTGTTTTGGCGGATG-3' to introduce the restriction site ClaI at the 5' end (underlined) and cloned into a pCR2.1-TOPO vector (Fig. 11C).

The PL-luxI fusion was PCR-amplified from pLEX with forward primer 5'-

ATCGATGTCGACTCTAGAGGATCC-3' and reverse primer 5'-

ATCGATATTCGAGCTCGGTACCATA-3' containing the restriction sites ClaI

(underlined) and cloned into a pCR2.1-TOPO vector (Fig. 11D). This vector was then digested with ClaI and ligated into the ClaI site of the rrnB TOPO vector described above to create a PL-luxI-rrnB fusion within a pCR2.1-TOPO vector (Fig. 11E).

Directionality was again confirmed by restriction digestion and sequencing.

The soc2soc1 genes of phage PP01 were amplified using forward primer 5'-

GCGTCGACGAAGAAATCTTTAAACTTTATTATCTG-3' and reverse primer 5'-

CAGTCGACTCTCCTTTTATTTAAATTACATGAC-3' to introduce SalI restriction sites (underlined) and cloned into a pCR2.1-TOPO vector (Fig. 11F). The soc2soc1 genes were excised from the TOPO vector by digestion with SalI and ligated into the SalI

MCS of pCR2.1PL-luxI-rrnB just upstream of PL (Fig. 11G). Directionality was confirmed by restriction digestion and sequencing.

The soc gene of phage PP01 was amplified using forward primer 5'-

AAGATATCCATGGCTAGTACTCGCGGTTA-3' and reverse primer 5'-

TCTCTCGAGGGTTTAATCCAACGATTTAACAT-3' to introduce EcoRV and XhoI restriction sites, respectively (underlined) and inserted into the TA cloning vector

69

Figure 11. Genetic construction of plasmid pCR2.1-soc2soc1-PL-luxI-rrnB-soc

70 pCR4.0-TOPO vector (Fig. 11H). The soc gene was excised from the TOPO vector by digestion with EcoRV and XhoI, and ligated into the EcoRV/XhoI MCS of pCR2.1- soc2soc1-PL-luxI-rrnB just downstream of rrnB (Fig. 11I). Insertion was confirmed by restriction digestion and sequencing.

The completed construct pCR2.1-soc2soc1-PL-luxI-rrnB-soc was electroporated into E. coli O157:H7 (ATCC 43888, non-toxigenic) and transformed cells were selected on LB plates containing 50 µg Kanamycin/ml. Log phase cultures of E. coli O157:H7 containing pCR2.1-soc2soc1-PL-luxI-rrnB-soc were infected with phage PP01 at a multiplicity of infection of 0.01 and 0.1 and incubated 5 h at 37°C to allow homologous recombination between the plasmid and the phage to occur (Fig. 12). Bacteria were lysed by addition of chloroform followed by a 15 min incubation at 37°C. Cell debris was pelleted, and phage were titered in top agar overlays.

Recombinant PP01-luxI phage were detected with a plaque hybridization assay using the Alkphos Direct Labeling and Detection System (Amersham Biosciences). A probe for luxI was obtained by amplifying a 1 kb portion of the luxI gene using the forward primer 5'-GGATCCCAAAAATACACTTGACCATT-3' and the reverse primer

5'-GAGCTCTTATTTGATGCCTGGCAGTT-3', and labeled according to the kit directions. Hybridization was performed according to kit directions. Labeled plaques were visualized with a Storm 840 phosphorescent imager. Phage that were labeled in the hybridization assay were propagated, and the presence of luxI in these phage was confirmed via PCR, using the forward primer 5'-

GCAATTCCATCGGAGGAGTA-3' and the reverse primer 5'-

71

Figure 12. Homologous recombination between plasmid pCR2.1-soc2soc1-PL-luxI-rrnB-soc and phage PP01

Crossover events occur in the soc2soc1 and soc regions, resulting in the integration of PL- luxI-rrnB into the phage PP01 genome.

72

GCACTCTGTTGACCAAGCAA-3'. One confirmed PP01-luxI phage clone was propagated and used for all subsequent experiments.

Detection of E. coli O157:H7 in pure culture

To detect E. coli O157:H7 in pure culture, PP01-luxI reporter phage and E. coli

OHHLux bioreporter cells were combined with a dilution series of E. coli O157:H7.

Phage PP01-luxI was diluted to a concentration of 108 PFU/ml in SM buffer. The E. coli

OHHLux bioreporter cells were grown overnight in LB at 28°C to an OD600 of 0.35 (~1 ×

108 CFU/ml). The volume and concentration of E. coli OHHLux used for detection of

OHHL in the phage bioreporter system was previously optimized (unpublished data).

Host E. coli O157:H7 (ATCC 43888) was grown in LB at 37°C to an OD600 of 0.2 (~1 ×

108 CFU/ml), then serially diluted 1:10 down to 10 CFU/ml in 9 ml LB in 50 ml conical tubes. Samples of each E. coli O157:H7 dilution were then distributed into a black 96- well microtiter plate (Dynex Technologies, Chantilly, VA) (100 µl/well). Next, PP01- luxI reporter phage (100 µl/well, ~1 × 107 PFU/ml final concentration) and E. coli

OHHLux bioreporter cells (100 µl/well, ~1 × 107 CFU/ml final concentration) were added to all wells. Control wells included E. coli OHHLux bioreporter cells (100

µl/well) alone and mixed with LB (100 µl/well), E. coli O157:H7 (100 μl/well, ~1 × 108

CFU/ml) and PP01-luxI (100 µl/well) in separate wells. The plate was sealed with a

Breathe-Easy (Diversified Biotech, Boston, MA) membrane, and monitored for bioluminescence every 30 min over a 20 h period in a Perkin-Elmer 1450 Microbeta Plus liquid scintillation counter at 30°C.

73

To increase the sensitivity of this assay, a pre-incubation step was added. Each tube of the E. coli O157:H7 dilution series was additionally incubated with shaking (200 rev/min) for 3 h at 37°C to promote growth. After 3 h, 100 µl samples were removed from the pre-incubated dilution tubes and transferred to a microtiter plate as described above, with phage PP01-luxI and E. coli OHHLux bioreporters added to each well also as described above. The microtiter plate was sealed and monitored for bioluminescence as described above. A student t-test was performed to determine the point at which luminescence in test wells significantly exceeded luminescence from control wells (P <

0.05).

Specificity of assay for E. coli O157:H7

The specificity of phage PP01 for E. coli O157:H7 is well established (Morita et al. 2002a, Morita et al. 2002b, O'Flynn et al. 2004), but the specificity of the recombinant

PP01-luxI phage needed to be confirmed experimentally. More importantly, the ability of phage PP01-luxI to infect a toxigenic E. coli O157:H7 strain required experimental confirmation. To accomplish this, toxigenic E. coli O157:H7 (43895), along with several control strains (E. coli K12, E. coli DH1, E. coli MM294, B. cereus and B. mycoides) were tested in the reporter assay. The toxigenic strain of E. coli O157:H7 (43895) was

8 grown in LB to an OD600 of 0.2 (~1 × 10 CFU/ml), then serially diluted 1:10 down to 1 ×

105 CFU/ml in LB. Control strains (E. coli K12, E. coli DH1, E. coli MM294, B. cereus and B. mycoides) and the known host strain E. coli O157:H7 (43888) were grown at 37°C to concentrations of ~108 CFU/ml. Phage PP01-luxI was diluted to a concentration of

108 PFU/ml in SM buffer. The E. coli OHHLux bioreporter cells were grown in LB at

28°C to an OD600 of 0.35. One hundred microliter samples of each E. coli O157:H7

74 dilution and each control strain were then distributed into a black 96-well microtiter plate

(100 µl/well). Next, 100 µl of PP01-luxI reporter phage (~1 × 107 PFU/ml final concentration) and 100 µl E. coli OHHLux bioreporter cells (~1 × 107 CFU/ml final concentration) were added to all wells. Controls were as described above. The plate was sealed and monitored for bioluminescence as described above.

Detection of E. coli O157:H7 in apple juice

Phage PP01-luxI was diluted to a concentration of 108 PFU/ml in SM buffer. The

E. coli OHHLux bioreporter cells were grown in LB at 28°C to an OD600 of 0.35. Host

8 E. coli O157:H7 (ATCC 43888) was grown at 37°C in LB to an OD600 of 0.2 (~1 × 10

CFU/ml). The cells were pelleted by centrifugation (10 min at 3000 × g), and suspended in an equal volume of commercial pasteurized apple juice. A 1:10 dilution series of this culture was made in apple juice down to ~1 CFU/ml. For detection, 0.5 ml of the artificially contaminated apple juice was added to 3 ml of LB in a culture tube and pre- incubated in a 40°C shaker-incubator for 1 to 6 h prior to detection. Pure apple juice (0.5 ml) was mixed with 3 ml LB and incubated overnight as a control. One hundred microliter samples of each E. coli O157:H7 dilution were then distributed into a black 96- well microtiter plate (100 µl/well). Next, 100 µl of PP01-luxI reporter phage (~1 × 107

PFU/ml final concentration) and 100 µl E. coli OHHLux bioreporter cells (~1 × 107

CFU/ml final concentration) were added to all wells. Control wells included E. coli

OHHLux bioreporter cells (100 µl/well) alone and mixed with the LB/apple juice mixture

(100 µl/well), E. coli O157:H7 (100 μl/well, ~1 × 108 CFU/ml) and PP01-luxI (100

µl/well) in separate wells. The plate was sealed and monitored for bioluminescence as described above.

75

Results

PP01-luxI reporter phage assay in pure culture

The ability of phage PP01-luxI to detect E. coli O157:H7 at concentrations ranging from 108 CFU/ml to 10 CFU/ml in LB was examined. Phage PP01-luxI was mixed with E. coli OHHLux bioreporter cells and E. coli O157:H7 (ATCC 43888, a non- toxigenic strain) in a 96-well plate and monitored for bioluminescence emission. At a concentrations of 104 - 108 CFU/ml, the assay could rapidly (<2 h) detect E. coli

O157:H7 without a pre-incubation step (Fig. 13). To lower the minimum level of detection, E. coli O157:H7 samples were pre-incubated for 3 h at 37°C prior to the assay, which resulted in a detection limit of ~10 CFU/ml (initial concentration) in a total time

(pre-incubation and assay) of approximately 4 h (Table 7).

Specificity of PP01-luxI reporter phage assay

The specificity of phage PP01 for E. coli O157:H7 is well established (Morita et al. 2002a, Morita et al. 2002b, O'Flynn et al. 2004), and the specificity of the OHHLux bioreporter cells used in this work has been previously described (Ripp 2006). To demonstrate that our assay system did not deviate from the specificities of its components, its ability to detect a toxigenic strain of E. coli O157:H7 (ATCC 43895) was confirmed at concentrations of 105 – 108 CFU/ml. It was also shown to not produce significant light in response to several non-O157:H7 (E. coli K12, E. coli DH1, E. coli

MM294) and non-E. coli (B. cereus, B. mycoides) bacterial strains at concentrations of

108 CFU/ml (Fig. 14). B. cereus and B. mycoides were tested because these bacteria are

76

106

105

LCPS

104

103 0 5 10 15 20

Hours Figure 13. Detection of E. coli O157:H7 without pre-incubation

E. coli OHHLux bioreporters (108 CFU/ml, 100 µl/well) were mixed with E. coli O157:H7 (104 - 108 CFU/ml, 100 µl/well) and phage PP01-luxI (108 PFU/ml, 100 µl/well) in the wells of a 96 well plate (labeled as 108 (♦), 107 (■), 106 (▲), 105 (◊), and 104 (□)). Control wells contained E. coli OHHLux bioreporters alone or mixed with LB, E. coli O157:H7 (108 CFU/ml) or phage PP01-luxI (labeled as OHHLux (×), OHHLux + LB ( ), OHHLux + 108 (○), and OHHLux + phage (●), respectively). Light production was monitored, and is shown as luminescent counts per second (LCPS). Error bars represent the standard deviation of six replicate wells.

77

Table 7. LCPS in pure culture averaged across all five E. coli O157:H7 strains tested Initial concentration Pre-incubation Total assay LCPS at time of of E. coli O157:H7 time (h) time including detection (CFU/ml) pre-incubation (h) 108 0 0.5 6,993 ± 130

107 0 1.0 16,889 ± 2,065

106 0 1.0 11,914 ± 718

105 0 1.0 11,417 ± 627

104 0 1.0 11,255 ± 599

103 3 4.0 14,011 ± 412

102 3 4.0 13,882 ± 357

10 3 4.0 13,796 ± 463

0 (control) n/a 0.5 – 4.0 3,771 ± 763 –

4,286 ± 119

aData shown for first reading in which the averaged luminescent counts per second (LCPS) of the contaminated samples significantly exceeded the LCPS of the controls. Background luminescence slowly increases over time. Control values shown are from the control wells giving the highest luminescence at the time of detection.

78 commonly found in the environment but the inability of phage PP01 to infect these bacteria was not previously demonstrated.

PP01-luxI reporter phage assay to detect E. coli O157:H7 in apple juice

Outbreaks of E. coli O157:H7 infection have been linked to products such as unpasteurized apple juice (CDC 1996b, Cody et al. 1999). The ability to detect E. coli

O157:H7 in apple juice was tested as a potential application for the PP01-luxI phage detection system. Apple juice was artificially contaminated with E. coli O157:H7 at concentrations from approximately 1 CFU/ml to 108 CFU/ml. An initial experiment revealed that detection in undiluted apple juice was not possible, as no light was produced even at the highest concentration of bacteria, even if the samples were brought to a neutral pH (data not shown). To circumvent this problem, samples were diluted into

LB (0.5 ml inoculated apple juice to 3 ml LB) and grown at 40°C for 1 to 6 h prior to detection. The higher incubation temperature was used to favor growth of E. coli

O157:H7 over background microflora (Vimont et al. 2006). This method allowed detection of E. coli O157:H7 at 108 CFU/ml in under 3 h (1 h preincubation, 1.5 h assay), and down to 104 CFU/ml in under 8 h (6 h preincubation, 1.5 h assay) (Table 8). No significant bioluminescence was observed when E. coli concentrations were below 104

CFU/ml.

Discussion

E. coli O157:H7 is an ongoing threat because of naturally occurring foodborne outbreaks and its potential for use in bioterrorism. Rapid methods for detection of E. coli

79

106

105

LCPS

104

NR NR NR NR NR NR 103

E. coli O7E. coli O55 E. coli K12E. coli DH1 E. coli MM294 E. coli O157:H7 E. coli O126:K71 Y. enterocolitica

Figure 14. Cross reactivity of the PP01-luxI detection system

Potential host cells (108 CFU/ml, 100 µl/well) were mixed with phage PP01-luxI (108 PFU/ml, 100 µl/well) and E. coli OHHLux bioreporter cells (108 CFU/ml, 100 µl/well) in the wells of a 96 well plate. Light production was monitored, and peak luminescence is shown as luminescent counts per second (LCPS). Data were normalized by subtracting the background luminescence of E. coli OHHLux bioreporter cells. Error bars represent the standard deviation of three replicate wells.

80

Table 8. Detection of E. coli O157:H7 ATCC 43888 in apple juice Initial concentration Pre-incubation Total assay time LCPS at time of detection of E. coli O157:H7 time (h) including pre- (CFU/ml) incubation (h) 108 1 2.4 125,110 ± 12,448

107 2 4.5 203,026 ± 16,778

106 4 5.9 152,606 ± 14,281

105 5 5.9 337,780 ± 22,202

104 6 7.6 236,900 ± 64,701

0 (control) n/a 2.4 – 7.6 12,836 ± 1739 – 44,843 ±

10,861 aData shown for first reading in which the luminescent counts per second (LCPS) of the contaminated sample significantly exceeded the LCPS of the controls. Background luminescence slowly increases over time. Control values shown are from the control wells giving the highest luminescence at the time of detection.

81

O157:H7 in the product production line and in drinking and recreational water sources are needed to minimize the human health impact of this pathogen. While there are a wide variety of assays available for the detection of E. coli O157:H7, very few are amenable to for real-time use in production line and environmental monitoring. The GFP-

PP01 phage assay introduced by Oda et al. (Oda et al. 2004) was a step toward this, but the required light source to excite GFP restricts its potential for use in miniaturized remote sensing devices. Detection methods based on the bioluminescent lux system can easily be integrated into an existing technology, the bioluminescent bioreporter integrated circuit (BBIC) (Nivens et al. 2004), for use in real-time remote monitoring.

As a first step toward developing a remote monitoring system, we have constructed a PP01-luxI phage that in conjunction with bioreporter cells is capable of specifically detecting E. coli O157:H7. Control assays demonstrated that E. coli

OHHLux bioreporters do not produce significant light when alone or mixed with LB, phage PP01 or E. coli O157:H7. In pure culture, the phage-reporter system is able to detect mid-level (>104) concentrations of non-toxigenic E. coli O157:H7 in 2 h or less

(Fig. 13, Table 7). The luminescence produced by E. coli OHHLux bioreporter cells increases rapidly for the first 2 h, then levels off but remains steady for over 20 h (Fig.

13). To detect lower concentrations of E. coli O157:H7 (10 – 103 CFU/ml), a pre- incubation step is necessary increasing the total assay time to 5 h. We expect that our system will work on most E. coli O157:H7 strains, as PP01 is known to infect a broad range of toxigenic and non-toxigenic strains (Morita et al. 2002b, O'Flynn et al. 2004).

We confirmed that our phage-bioreporter system does not cross react with selected non-

O157:H7 E. coli strains and other tested bacterial species. Only a limited number of

82 strains were examined because the specificity of both components of this system have already been demonstrated (Morita et al. 2002b, O'Flynn et al. 2004, Ripp 2006).

To demonstrate the feasibility of using this detection system in a food sample matrix, E. coli O157:H7 was detected in apple juice. Detection of bacteria directly in apple juice was not possible, even when the pH of the apple juice was adjusted from its initial 3.75 to ~7.0. It appeared that the apple juice interfered with the growth and function of the bioreporter cells, as background luminescence was decreased in these experiments. To avoid this problem without going through lengthy bacterial purification steps, a sample of the contaminated apple juice was diluted into LB prior to the detection assay. Dilution of the sample made a preincubation step necessary, but still allowed for the detection of high concentrations (>108) of E. coli O157:H7 in under 3 h. The detection limit in apple juice was higher than in LB, with no detectable luminescence produced in response to concentrations of E. coli below 104 CFU/ml. At 104 CFU/ml, detection was possible in less than 8 h (6 h pre-incubation, 1.5 h assay). Detection limits of 10 CFU/ml or lower are desirable in food matrix assays because the infectious dose of

E. coli O157:H7 is believed to be 10 – 100 cells (LeBlanc 2003). It is possible that a longer pre-incubation period could decrease the detection limit of this assay. We are now focusing on ways to improve the sensitivity and rapidity of this assay.

The bacteriophage based bioluminescent bioreporter described here represents a unique detection system that couples the specificity of the host-bacteriophage interaction with the lux quorum sensing and luminescence machinery. This bioreporter system has been shown in initial testing to have the capability to detect E. coli O157:H7 from pure culture at mid-level concentrations (104 CFU/ml) without sample preincubation, and at

83 lower concentrations (down to 10 CFU/ml) after sample preincubation in a 96-well plate assay format. While this assay falls short of the desired 10 CFU/ml detection limit in food matrices, its limit of detection is close to that of existing immunoassays and fluorescent-phage based assays (Goodridge et al. 1999, Deisingh & Thompson 2004,

Gehring et al. 2004), and the sensitivity of this assay may be increased through additional genetic modifications of the phage. The assay is demonstrated here in a benchtop format, but the significance of this assay is that it has the potential to be developed into a rapid, low-cost, low-energy, real-time online monitoring system for detection of E. coli

O157:H7 in industrial and environmental settings.

84

Part IV: Bacteriophage-amplified bioluminescent sensing of E.

coli O157:H7

85

This is a version of a journal article submitted to Analytical and Bioanalytical Chemistry entitled “Bacteriophage-amplified bioluminescent sensing of Escherichia coli O157:H7”.

Abstract

Escherichia coli O157:H7 remains a continuous public health threat, appearing in meats, water, fruit juices, milk, cheese, and vegetables where its ingestion at concentrations perhaps as low as 10 to 100 organisms can result in potent toxin exposure and severe damage to the lining of the intestine. Abdominal pain and diarrhea develop, which in the very young or elderly can progress towards hemolytic uremic syndrome and kidney failure. To assist in the detection of E. coli O157:H7, a recombinant bacteriophage reporter was developed that uses quorum sensing (luxI/luxR) signaling and luxCDABE based bioluminescent bioreporter sensing to specifically and autonomously respond to O157:H7 serotype E. coli. The bacteriophage reporter, derived from phage

PP01, was tested in artificially contaminated foodstuffs including apple juice, tap water, ground beef, and spinach leaf rinsates. In apple juice, detection of E. coli O157:H7 at original inoculums of 1 CFU/mL occurred within approximately 16 h after a 6 h pre- incubation, detection of 1 CFU/mL in tap water occurred within approximately 6.5 h after a 6 h pre-incubation, and detection in spinach leaf rinsates using a real-time Xenogen

IVIS imaging system resulted in detection of 1 CFU/mL within approximately 4 h after a

2 h pre-incubation. Detection in ground beef was not successful, however, due to the natural occurrence of quorum sensing autoinducer (N-3-(oxohexanoyl)-L-homoserine lactone; OHHL) generating false positive bioreporter signals in the ground beef samples.

86

Introduction

Bacteriophage, or phage for short, are suggested to be the most numerous organisms on Earth, estimated to reside at population levels approaching 1 × 1031. With a life cycle that requires a bacterial host for survival and proliferation, these extensive numbers of phage have cultivated diverse host ranges, extending from very narrow (down to the genus or species level) to very broad (infection across strains, species, and genera).

The narrowness of phage host ranges has long been realized as a unique mechanism for identifying bacteria. Phage typing schemes, for example, have been applied since the

1940s for specific identification of numerous bacterial pathogens. Newer detection assays that similarly exploit phage/host specificity have since evolved, typically using the phage as a carrier for a reporter signal that indicates when the phage has attached to and/or infected its unique host bacterium. Firefly luciferase (luc) (Sarkis et al. 1995), bacterial luciferase (luxAB) (Chen & Griffiths 1996), beta-galactosidase (lacZ)

(Goodridge & Griffiths 2002), ice nucleation (inaW) (Wolber & Green 1990), green fluorescent protein (gfp) (Funatsu et al. 2002, Oda et al. 2004), quantum dots (Edgar et al.

2006), and nucleic acid stains (Mosier-Boss et al. 2003) have each been used in this regard for the detection of pathogens including Listeria, Mycobacterium, Salmonella,

Staphylococcus, and E. coli in a variety of environmental and foodborne matrices. The ease of manufacturing large quantities of phage, their long shelf-life, and associated low cost further merits their practical application for biological sensing. However, integrated phage signaling mechanisms so far described are tied to assay conditions that require the addition of ancillary substrate(s) and specialized, bulky monitoring equipment for signal observation. To move towards a fully reagentless assay amenable to miniaturized lab-on-

87 a-chip detection schemes (Islam et al. 2007), we engineered a phage reporter system for

E. coli O157:H7 that operates autonomously in its sensory capacity. The reporter system centers on the luxRICDABE genetic operon derived from the bioluminescent marine bacterium Vibrio fischeri (Meighen 1994). The luxAB component of this operon encodes for a bacterial luciferase that generates 490 nm bioluminescent light signals when provided with oxygen, FMNH2, and an aldehyde substrate synthesized by the luxCDE gene complex. The luxI and luxR genes are involved in a cell-to-cell communication network referred to as quorum sensing (Miller & Bassler 2001). The luxI gene synthesizes the autoinducer N-3-(oxohexanoyl)-L-homoserine lactone (OHHL). Freely diffusible OHHL interacts in a cell density dependent manner within the population of V. fischeri cells to activate the regulatory protein LuxR. Activated LuxR subsequently enhances transcription of the lux genes, thereby generating an autoamplified bioluminescent signaling event.

For the specific detection of E. coli O157:H7, a phage, referred to as PP01, was engineered to contain the luxI gene within its genome (thus referred to as PP01-luxI)

(Brigati et al. 2007). Upon host infection, the luxI gene is inserted into and expressed by the E. coli cell which then begins synthesizing OHHL. As the OHHL autoinducer molecules diffuse into the extracellular environment, they interact with an OHHL specific bioluminescent bioreporter cell containing the luxR and luxCDABE genes (Ripp et al.

2006). The LuxR protein is activated, transcription of the luxCDABE genes are enhanced, and the bioluminescent bioreporter cell emits bioluminescent light. Thus, the initial phage infection event leads to an autoamplified chemical response that is detected by a luxRCDABE based bioluminescent bioreporter (Fig. 1, Chapter I). To direct this

88 assay towards foodborne pathogen monitoring applications, we have here demonstrated the utility of PP01-luxI reporter phage in the detection of E. coli O157:H7 in artificially contaminated apple juice, tap water, and ground beef using a high throughput 96-well microtiter plate format. A real-time bioluminescent imaging assay using a Xenogen IVIS

Lumina CCD camera was additionally demonstrated for the detection of E. coli O157:H7 in spinach leaf rinsates.

Materials and methods

Bacterial strains and bacteriophage

Nontoxigenic E. coli O157:H7 (ATCC 43888) was used in all experimental procedures and enumerated on Sorbitol MacConkey agar plates when necessary (17 g peptone, 10 g sorbitol, 5 g NaCl, 3 g polypeptone, 1.5 g bile salts, 0.03 g neutral red, 1.0 mg crystal violet, 13.5 g agar per l H20, pH 7.0). The reporter phage, PP01-luxI, has been previously described (Brigati et al. 2007). Its parent phage, PP01, is highly specific towards strains of E. coli O157:H7 and has been shown to not infect other E. coli serotypes or other bacterial genera (Morita et al. 2002a, O'Flynn et al. 2004). The bioluminescent bioreporter strain used in this study was E. coli OHHLux, which has also been previously described (Ripp et al. 2006). Briefly, OHHLux bioreporters contain the luxR and luxCDABE genes under the control of the lux operon promoter. They bioluminescently respond to OHHL autoinducer within a concentration range from 10 nmol/l to 50 µmol/l.

89

96-well microtiter plate sampling formats for pure culture, apple juice, tap water, and ground beef detection assays

E. coli O157:H7 was tested in a high throughput 96-well microtiter plate format in pure culture and in artificially contaminated apple juice and tap water. For pure culture tests, an overnight culture of E. coli O157:H7 was inoculated 1:10 in Luria-Bertani broth

(LB; 10 g tryptone, 5 g yeast extract, 10 g NaCl per L H20, pH 7.0) and grown to an

8 optical density at 600 nm (OD600) of 0.26 (~1 × 10 CFU/ml) at 37 ºC. A 1:10 dilution series, providing cell concentrations ranging from 1 × 107 to 1 CFU/ml, and a control consisting only of LB, was then prepared in triplicate in 2 mL volumes of LB in a 96- deep well microtiter plate (Abgene 2.2 ml, Thermo Fisher Scientific, Waltham, MA).

The use of 96-deep well and standard well microtiter plates throughout these experiments permitted assimilation of the assay with a Beckman Coulter Biomek FX (Fullerton, CA) automated liquid handing workstation for accommodation of high throughput sample processing. Starting concentrations were always verified by dilution plating in triplicate on Sorbitol MacConkey agar plates. The deep well plate was sealed with a Breath-Easy membrane (Diversified Biotech, Boston, MA) and incubated at 40 ºC at 180 rpm for 6 h.

The plate was then centrifuged (3750 rpm), the supernatant discarded, the pellets resuspended in 100 µl LB, and then transferred to a 96-well microtiter plate (Microfluor white flat bottom, Thermo Fisher Scientific). To each well of the microtiter plate was then additionally added the PP01-luxI reporter phage and the OHHLux bioluminescent bioreporters. The PP01-luxI reporter phage were prepared on top agar overlays (Silhavy et al. 1984) and stored refrigerated at stock concentrations of 1 × 1010 PFU/ml. Each microtiter plate well received 100 µl of this PP01-luxI reporter phage stock (~1 × 109

90

PFU/ml final concentration). The OHHLux bioluminescent bioreporter cells were grown

8 in LB at 37 ºC to an OD600 of 0.35 (~1 × 10 CFU/ml) and 50 µL transferred to each microtiter plate well (~5 × 106 CFU/ml final concentration). The plate was then sealed with a Breath-Easy membrane and placed in a BioTek Synergy2 Microplate reader

(Winooski, VT) programmed to collect bioluminescence every 20 min at an integration time of 1 sec/well with incubation set at 27 ºC and shaking set at „fast‟ speed.

For contaminated apple juice experiments, E. coli O157:H7 was grown in LB at

37 ºC to an OD600 of 0.26, centrifuged at 6000 rpm for 10 min, and washed and resuspended in an equal volume of store purchased pasteurized apple juice. A 1:10 dilution series was then prepared in triplicate 25 mL volumes of apple juice down to 1

CFU/ml and placed at 4 ºC overnight. A control of apple juice without an E. coli inoculum was similarly placed at 4 ºC. For experiments in which there was no pre- incubation, samples were centrifuged the next day, resuspended in 100 µl LB, and combined in a 96-well microtiter plate with 100 µl PP01-luxI reporter phage and 50 µl

OHHLux bioluminescent bioreporter cells. Plates were monitored for bioluminescence in the Synergy2 as described above. For experiments in which there was a pre-incubation step, samples were centrifuged the next day, resuspended in 2 ml LB in a 96-deep well plate, and incubated with shaking (180 rpm) for 6 h at 40 ºC. The deep well plate was then centrifuged and resulting pellets resuspended in 100 µl LB. After transferring to a standard 96-well plate, 100 µl PP01-luxI reporter phage and 50 µL OHHLux bioluminescent bioreporters were added and the assay performed as described above.

Unadulterated tap water was artificially contaminated with E. coli O157:H7 exactly as described for the apple juice and allowed to sit overnight at 4 ºC. Subsequent

91 dilutions, incubations, plate transfers, and bioluminescent monitoring were unchanged from the apple juice experiments.

Grocery store purchased 93% lean ground beef was weighed in 25 g portions and placed in triplicate sterile 50 ml tubes. A dilution series (1 × 107 to 1 CFU/ml) of E. coli

O157:H7 was prepared in LB and 2.5 ml added to each tube. Tubes were then vortexed and allowed to sit overnight at 4 ºC. The next day, the contents of each tube were deposited in separate stomacher bags (1650 ml Filtra Bags, Thermo Fisher Scientific) along with an additional 100 ml LB and stomached (Seward Stomacher 400) for 1 min.

Resulting liquid was removed, placed in sterile 250 ml flasks, and incubated at 40 ºC with shaking (180 rpm) for 6 h. A 10 ml aliquot was then removed from each flask, centrifuged for 10 min at 6000 rpm, and resulting pellets resuspended in 2 ml phosphate buffered saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per l H20, pH 7.4). To isolate E. coli O157:H7 from the media, 20 µl of anti-E. coli O157:H7 immunomagnetic beads (Dynal Dynabeads, Invitrogen, Carlsbad, CA) were added to these 2 ml volumes, gently mixed for 15 min at room temperature, magnetized for 10 min, washed twice in PBS with subsequent magnetic separation, and finally resuspended in 100 µl of LB. Each 100 µl volume was then transferred to a 96-well microtiter plate along with 100 µl of PP01-luxI reporter phage and 50 µl OHHLux bioluminescent bioreporters, as explained above. The plate was then sealed and monitored for bioluminescence in the BioTek Synergy2 (20 min intervals, 1 sec/well integration time,

27 ºC, „fast‟ shaking).

All experiments included triplicate control wells consisting of pairwise combinations of each component of the assay (i.e., E. coli + PP01-luxI reporter phage, E.

92 coli + OHHLux bioluminescent bioreporters, PP01-luxI reporter phage + OHHLux, etc.) as well as each component individually. The highest background was consistently generated in control wells containing only the OHHLux bioluminescent bioreporters, thus, rather than graphing multiple control lines, these data were always used as the representative control. These data were also always used for statistical determination of significant differences. A bioluminescent measurement was considered significant if it achieved an intensity two standard deviations (2σ) above these control values.

Real-time bioluminescent imaging of E. coli O157:H7 in spinach leaf rinsate

Real-time imaging and quantification of bioluminescence in artificially contaminated spinach was performed with a Xenogen IVIS Lumina CCD imaging system

(Alameda, CA). E. coli O157:H7 was grown in LB at 37 ºC to an OD600 of 0.26, centrifuged at 6000 rpm for 10 min, washed, and resuspended in an equal volume of sterile distilled water. A 1:10 dilution series was then prepared to produce a range of E. coli concentrations from 1 × 107 to 1 CFU/ml in 100 mL volumes of sterile distilled water. Store purchased ready-to-eat spinach leaves were weighed in 4 g portions and each portion submerged in a dilution tube and allowed to sit overnight at 4 ºC. A control tube contained spinach leaves in sterile distilled water void of an E. coli O157:H7 inoculum. The next day, spinach leaves were removed from each dilution tube, placed in sterile petri dishes, and dried for 30 min with covers removed in a sterile fume hood.

Each 4 g portion of spinach was then transferred to sterile 100 mL flasks containing 20 ml LB and incubated for 3 h at 40 ºC with shaking at 180 rpm. The entire contents of each flask was then individually placed in stomacher bags and stomached for 2 min.

Each liquid portion was removed, centrifuged for 10 min at 6000 rpm, and the resulting

93 pellets resuspended in 1.5 ml of LB. Anti-E. coli O157:H7 immunomagnetic beads (20

µL) were added to these 1.5 ml volumes, gently mixed for 10 min at room temperature, and magnetized for 3 min. Captured magnetic bead/E. coli complexes were subsequently resuspended in 1.1 ml of LB and 100 µl removed, diluted, and plated on Sorbitol

MacConkey agar plates in triplicate to determine actual E. coli O157:H7 concentrations.

The remaining 1 mL from each tube was transferred to separate 35 mm petri dishes to which was added 1 ml of PP01-luxI reporter phage and 0.5 mL OHHLux bioluminescent bioreporters (prepared as described above). Petri plates were placed in the light-tight

Lumina imaging chamber set at 27 ºC and monitored for bioluminescence emission every

5 min at an integration time of 1 min.

Results

Determination of E. coli O157:H7 inoculum concentrations

In all experiments described below, the initial inoculum of E. coli O157:H7 was obtained from a culture grown to an OD600 of 0.26. This OD yielded an average E. coli concentration across all experiments performed of 1.3 (± 0.03) × 108 CFU/ml.

Consequent 1:10 dilutions prepared from these cultures formed the 1 × 107 to 1 CFU/ml dilution series used in all experiments, and plating of these cultures showed similarly tight standard deviations except at 1 CFU/ml where viable plating often failed to yield colonies, as would be expected at this low of a concentration. Thus, rather than noting each specific E. coli concentration for each experiment, we have simply stated the concentrations as a range that ultimately approximates to 1 × 107 to 1 CFU/ml. Although

94 it was never known from plate counts if the 1 CFU/ml samples actually contained cells, their bioluminescence emission above controls void of cells clearly established that cells were indeed present.

Detection of E. coli O157:H7 in pure culture

When inoculated into LB as a pure culture, E. coli O157:H7 was detectable after

2 (± 0.3) h at a concentration of 1 × 107 CFU/ml (Fig. 15). The lowest detectable concentration not requiring a pre-incubation step was 1 × 103 CFU/ml, where detection occurred within 5 (± 0.3) h of assay initiation. Below this concentration, direct detection of E. coli populations was not possible, and therefore a pre-incubation step was added to increase bacterial levels. Our aim was to detect 1 CFU/ml, and the smallest amount of pre-incubation time necessary to do so was determined to be 6 h (data not shown). Since in real-world samples the starting concentration of cells would be unknown, and one would always need to direct the assay towards the detection of 1 CFU/ml, a 6 h pre- incubation became a standard component of the assay. These additional 6 h of pre- incubation then permitted detection of 100 CFU/ml within 1 (± 0.3) h (~7 h total), 10

CFU/ml within 2.5 (± 0.3) h (~8.5 h total), and 1 CFU/ml within 4.5 (± 0.7) h (~10.5 h total). To account for preparation of cultures and loading of the 96-well microtiter plate, an additional 0.5 h can be added to all detection times to establish the total assay time.

Again, once this 0.5 h preparation time is complete and the microtiter plate has been placed in the light reader, no other user interaction is required, except for data analysis at the termination of the assay run. Thus, assays can be completed within a normal 8 h work day with results available within 24 h.

95

1 × 107

1 × 106

1 × 105

1 × 104

1 × 103

O157:H7 CFU/mlO157:H7 100 coli 10

E. 1

0 1 2 3 4 5 6 7 8 9 10 11 12 Hours

Figure 15. Establishment of the bioluminescent response in a pure culture 1:10 dilution series of E. coli O157:.H7 E. coli at concentrations ranging from 1 × 107 to 1 × 103 CFU/ml could be detected directly, without pre-incubation, within approximately 2 to 5 h, respectively, of assay initiation. Lesser concentrations (100 to 1 CFU/ml) required a minimum pre-incubation of 6 h at 40 ºC (black bars) to elevate cell numbers to detectable levels. This resulted in total detection times ranging from 7 h (100 CFU/ml) to 10.5 h (1 CFU/ml).

96

Detection of E. coli O157:H7 in apple juice Twenty-five ml volumes of store purchased pasteurized apple juice were artificially inoculated with E. coli O157:H7 from 1 × 107 to 1 CFU/ml and allowed to set at 4 ºC overnight. These samples were then centrifuged, resuspended in LB, and either used directly in the assay or pre-incubated for 6 h at 40 ºC prior to assay incorporation.

Without pre-incubation, detection from 1 × 105 to 1 × 107 CFU/ml was possible within less than 5 h (Fig. 16). However, concentrations below 1 × 105 CFU/ml failed to initiate bioluminescent signals unless a pre-incubation step was included to increase threshold E. coli levels. With pre-incubation, it was then possible to detect down to 100 CFU/ml in 5

(± 0.7) h. Detection of 10 or 1 CFU/ml, however, remained unsuccessful. Extending the pre-incubation time by several hours did permit detection at these lowest concentrations, but additionally extended the assay time beyond the 24 h limits that we wished to maintain (data not shown). Therefore, sample size was doubled from 25 ml to 50 ml apple juice while preserving all other assay parameters. This then permitted detection of original 10 and 1 CFU/ml inoculums within less than 24 h (19 (± 0.7) h at 10 CFU/ml and 22 (± 0.7) h at 1 CFU/ml).

Detection of E. coli O157:H7 in tap water

E. coli O157:H7 inoculums ranging from 1 × 107 to 1 CFU/ml were added to unadulterated tap water in 25 ml volumes and processed as for the apple juice. The 1 ×

107 to 1 × 105 CFU/ml cultures were detectable without pre-incubation within 5 (± 0.3) to

7 (± 0.7) h, respectively, of assay initiation (Fig. 17). Lower concentrations again

97

110000000 × 107

6 1 1000000× 10

1 ×100000 105

4 1 × 1000010

3 1 × 101000

O157:H7 CFU/mlO157:H7 100100

E. coli coli E. * 1010 * 1 1

0 2 4 6 8 10 12 14 16 18 20 22 24 Hours

Figure 16. Bioluminescent detection of E. coli O157:H7 in artificially contaminated apple juice

At elevated concentrations (1 × 105 to 1 × 107 CFU/ml), E. coli O157:H7 was detectable in 25 ml sample volumes without pre-incubation within less than 5 h. At all lower concentrations, a 6 h pre-incubation step (black bars) was necessary to bring E. coli to threshold detectable levels. *Detection at 10 and 1 CFU/ml required that sample volumes be increased from 25 ml to 50 ml, thereby achieving detection in approximately 19 and 22 h, respectively.

98

7 1 10000000× 10

6 1 ×1000000 10

5 1 ×100000 10

4 1 × 1000010

3

1 × 101000 O157:H7 O157:H7 CFU/ml

100100 E. coli coli E. 1010

1 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Hours

Figure 17. Response time profile of bioluminescence emission in 25 ml sample volumes of E. coli O157:H7 contaminated tap water

Concentrations below 1 × 105 CFU/ml required a 6 h pre-incubation (black bars) at 40 ºC to elevate cells to detectable levels. This then enabled detection down to CFU/ml in a total assay time of 12.3 (± 0.7) h.

99 required a 6 h pre-incubation, providing total detection times ranging from 8.3 (± 0.7) h at 1 × 104 CFU/ml up to 12.3 (± 0.7) h at 1 CFU/ml.

Detection of E. coli O157:H7 in ground beef

Only the highest concentrations (1 × 107 and 1 × 106 CFU/ml) of E. coli

O157:H7 were detectable in artificially contaminated ground beef samples, with detection occurring within 4 – 6 h after a 6 h pre-incubation (data not shown). At all other concentrations, no significant differences existed between sample and control bioluminescence emissions. Control wells containing OHHLux bioluminescent bioreporter cells often generated background bioluminescence 1000X that seen in LB, apple juice, and tap water experiments. The presence of intrinsic OHHL in ground beef has been reported (Bruhn et al. 2004), and it is likely that this accounts for the extensive background induction of the OHHLux bioluminescent bioreporter and subsequent inability to distinguish between it and actual induction by E. coli O157:H7 presence.

Thus, the assay as it currently operates cannot adequately detect E. coli O157:H7 in ground beef, but could be modified to do so (see Discussion).

CCD imaging of E. coli O157:H7 in spinach leaf rinsates

A Xenogen IVIS Lumina CCD imaging camera was used to monitor E. coli

O157:H7 induced bioluminescence in real time in spinach leaf rinsates. Although these experiments began with a 1 × 107 to 1 CFU/ml dilution series, the absorption of E. coli onto spinach leaves and the subsequent processing and incubation steps was not expected to maintain a 1:10 stepwise concentration of cells throughout the experimental procedure.

Therefore, immediately after immunomagnetic separation of E. coli O157:H7 from the

100 rinsate, each sample was plated to determine actual cell numbers directly prior to placing the samples in the IVIS imaging chamber. This resulted in final cell concentrations, in sequential order corresponding to the original 1 × 107 to 1 CFU/ml dilution series, of 5.6

× 108, 2.7 × 109, 6.9 × 107, 4.2 × 106, 8.8 × 105, 1.5 × 104, 1.3 × 103, and 2.3 × 104

CFU/ml 1. At the highest final E. coli concentration of 2.7 × 109 CFU/ml (derived from the original 1 × 106 CFU/ml inoculum), significant bioluminescence was observed within

1.4 h after the 2 h pre-incubation (Fig. 18). The lowest final E. coli concentration (1.3 ×

103 CFU/ml, derived from the original 1 CFU/ml inoculum) generated measurable bioluminescence 3.6 h after the 2 h pre-incubation.

Discussion

The application of bacteriophage for selective identification of bacterial pathogens presents a powerful diagnostic for microbial detection and control. In addition to being highly specific for their target hosts, phage also infect only metabolically active cells, are self-replicating, are as ecologically diverse as their bacterial counterparts, and can be prepared at very high titer for very low cost with extensive shelf-life. In these studies, the testing of the diagnostic reporter phage PP01-luxI was performed in pure culture, apple juice, tap water, ground beef, and spinach leaf rinsate, with the goal of detecting E. coli O157:H7 at 1 CFU/ml in less than 24 h. Although in these particular studies only the nontoxigenic E. coli ATCC 43888 strain was used, we have previously demonstrated that PP01-luxI likewise infects and bioluminescently identifies other toxic

O157:H7 serotypes, for example ATCC strains 43889, 43894, 43895, and 700927

(Brigati et al. 2007).

101

A B C A B C A B C A B C

D E F D E F D E F D E F

G H I G H I G H I G H I

0 hour 1.4 2 hour 3 hour A B C A B C A B C A B C

D E F D Eh ourF D E F D E F

G H I G H I G H I G H I

4 hour 5 hour 6 hour 7 hour

2.5 × 107

7 A 2.0 × 10

B 1.5 × 107 C

7 1.0 × 10

D 5.0 × 106 E F, 0 G IH Hours (control)

Figure 18. Real-time imaging of bioluminescence emission from rinsates from E. coli O157:H7 contaminated spinach leaves

The upper panel depicts time series images of 35 mm petri dishes containing rinsates of varying E. coli O157:H7 concentrations (A (○), 5.6 × 108; B (●), 2.7 × 109; C (□), 6.9 × 107; D (■), 4.2 × 106; E (), 8.8 × 105; F (solid line), 1.5 × 104; G (), 1.3 × 103; H (gray dotted line), 2.3 × 104 CFU/ml; I (+), control) combined with PP01-luxI reporter phage and OHHLux bioluminescent bioreporters, and the graph in the lower panel displays the average bioluminescence emission over time for each plate. The earliest bioluminescent signal was observed 1.4 h after the 2 h pre-incubation at a final cell density of 2.7 × 109 CFU/ml (plate B). Cell densities corresponding to original inoculums of 100, 10, and 1 CFU/ml (plates F, G, and H, respectively) generated observable bioluminescence within 3 – 4 h following the 2 h pre-incubation.

102

It does not infect other E. coli serotypes, other E. coli strains, or other bacterial genera

(Morita et al. 2002a, O'Flynn et al. 2004, Brigati et al. 2007).

Detection of E. coli O157:H7 was successful in all sample matrices except for ground beef. Pure culture LB broth samples established optimal detection of 1 CFU/ml in approximately 10.5 h, inclusive of a necessary 6 h pre-incubation enrichment to elevate E. coli O157:H7 concentrations (Fig. 15). Apple juice and tap water similarly required 6 h pre-incubations, resulting in total detection times of approximately 22 h and

12.5 h, respectively, at initial E. coli inoculums of 1 CFU/ml in sample volumes of 50 mL apple juice or 25 ml tap water (Figs. 16 and 17). The additional volume and time necessary for detection in apple juice is likely due to the documented effects of its acidity

(pH ~ 4) on slowing E. coli growth (Koodie & Dhople 2001). Detection of elevated E. coli concentrations (≥ 1 × 105 CFU/ml) in apple juice and tap water could be achieved directly without pre-incubation in less than 5 and 7.5 h, respectively.

Detection of E. coli O157:H7 in ground beef was successful at high E. coli inoculums (≥ 1 × 106 CFU/ml), but significant bioluminescence emission, as compared to control ground beef samples void of an artificial E. coli inoculum, could not be established at any lower E. coli concentrations. It is known that ground beef inherently contains OHHL autoinducer, thought to be primarily synthesized by resident

Enterobacteriaceae, principally Hafnia alvei (Bruhn et al. 2004). Since the PP01-luxI assay relies on a bioluminescent bioreporter organism sensitive to OHHL, samples harboring OHHL from secondary sources will erroneously activate the bioreporter resulting in false positive signaling. We have demonstrated this vulnerability in the presence of other OHHL producing bacteria such as Yersinia enterocolitica and Erwinia

103 carotovora, as well (Ripp et al. 2006, Brigati et al. 2007). The need for controls containing the OHHLux bioreporter alone combined with test sample are therefore mandatory to identify these false positives. Alternatively, an effective phage reporter for

E. coli in ground beef can be developed using other quorum sensing regulatory circuits

(Miller & Bassler 2001). For example, the rhlI/rhlR quorum sensing network from

Pseudomonas aeruginosa could be substituted for luxI/luxR to create a phage reporter/bacterial bioreporter system sensitive to butanoyl-L-homoserine lactone (BHL) autoinducer rather than OHHL autoinducer. This additionally should allow for multiplexed bacterial target detection via insertion of different autoinducer signaling cascades within each phage reporter.

Detection of E. coli O157:H7 in spinach leaf rinsates was performed with a

Xenogen IVIS Lumina CCD camera using real-time bioluminescence imaging (BLI) technology. The preponderance of BLI techniques center around in vivo non-invasive imaging of bioluminescent and/or fluorescent signals in animal models, but its extreme sensitivity begs for application in other signal detection practices, of which pathogen monitoring in foodstuffs has seen only modest pursuit (Maoz et al. 2002). In these studies, as few as 1 CFU/ml E. coli could be detected within approximately 6 h, inclusive of a 2 h pre-incubation (Fig. 18).

Although diagnostic phage have previously been exploited for foodborne pathogen detection, their typical reliance on reporter gene systems such as green fluorescent protein or reagent constrained luxAB can be detrimental due to excitation light source requirements or ancillary substrate additions. The use of the complete luxCDABE cassette described here, in conjunction with autoamplified quorum sensing signaling,

104 provides a unique reagentless diagnostic that we have shown requires minimal user set-up

(~ 0.5 h) and downstream interaction, and suitably interfaces with robotic liquid handling workstations such as the Biomek FX when high throughput analysis is required. The additional implementation of these phage reporter systems with real-time BLI further enhances the utility of these assays.

105

Chapter V: Conclusions

106

Recent outbreaks of disease due to E. coli O157:H7 have pushed the need for monitoring of food and water supplies to the forefront. Given the obvious public health importance of this pathogen, a detection method that is rapid, specific, and capable of detection at low concentrations is needed to prevent outbreaks or at least quickly detect and limit the number of individuals affected. There are a variety of methods available for the detection of E. coli O157:H7 but most are slow, costly and/or complex because they require a significant amount of sample manipulation. The bacteriophage based bioreporter system is an alternative to these detection methods.

The development of two phage based luxI bioreporters (Ch. II) as a general E. coli detection method and (Ch. III and IV) as a specific detection method for the important bacterial pathogen E. coli O157:H7 were examined in this work (Ripp 2006, Brigati et al.

2007, Ripp Submitted 2007). The luxI gene from V. fischeri was placed into the phage genomes which upon infection of the host led to production of quorum sensing molecules, specifically OHHL. OHHL diffuses extracellularly and activates the E. coli

OHHLux bioreporter cells containing the luxR and luxCDABE genes. OHHL binds luxR leading to transcription of luxCDABE to produce a detectable signal in the form of bioluminescence and yielding a positive assay result.

The system requires a simple three step protocol consisting of the addition of reporter phage, bioreporter, and target cells (or sample). No other reagents or manipulations are required other than incubation and light measurement, making the assay amenable to automated high throughput monitoring. The preliminary testing reported here demonstrates the ability of luxI-incorporated bacteriophage to detect target 107 microbes in a simple and cost-effective microtiter plate format. The general E. coli system with the λluxI phage was applied to pure culture and lettuce rinsates resulting in detection times ranging from less than 3 hours for high cell concentrations (108 CFU/ml) to well under 24 hours for lower cell densities (1-130 CFU/ml). The E. coli O157:H7 specific system with PP01-luxI phage was tested in pure culture, apple juice, tap water, ground beef, and spinach leaf rinsates. Detection of E. coli O157:H7 was successful in all sample matrices except for ground beef. Assay detection times for original inoculums

1 CFU/ml ranged from 10.5 h in pure culture, 12.5 h for tap water and 22 h for apple juice. More recent BLI technology has demonstrated real-time detection of E. coli

O157:H7 at concentrations of 1 CFU/ml in spinach leaf rinsates in under 6 h.

While the bacteriophage based bioreporter system does present a viable option for pathogen detection, it has some issues to overcome. Although the phage reporters have shown unique specificity for their host, synthesis of OHHL by other bacteria can induce bioluminescence by the OHHLux bioreporter. The presence of these OHHL producing microorganisms in a sample would therefore generate false positive signals. Thus it is imperative that assays include a control consisting of bioreporter alone added to the sample to identify if there is intrinsic OHHL production. The emergence of phage resistant target hosts may also occur reducing the system‟s effectiveness, but the use of multi-phage cocktails and the natural reversion back to phage sensitivity often occurs

(O'Flynn et al. 2004). Additionally, OHHL lactonases, AHL inactivating enzymes, could interfere with detection if present in high enough concentrations. Also the assay would likely not detect VBNC cells due to the fact that phage infection is very sensitive to the

108 physiologic status of the host cell, thus the reporter phage would be unable to infect and use the host cell machinery to produce the OHHL required for this system.

The application of bacteriophage for the precise identification of bacterial pathogens is a powerful diagnostic for microbial detection. This technology is versatile and can be applied to a wide variety of sample matrices. The assay could be improved by increasing sensitivity and total detection time through additional genetic modifications to the reporter phage. Furthermore, phage reporters can be developed using other quorum sensing systems other than luxI/luxR from V. fischeri (Miller & Bassler 2001). Creating a phage reporter/bacterial bioreporter system that is responsive to autoinducers other than

OHHL could allow for multiplexed bacterial target detection through insertion of different autoinducer signaling cascades within each phage reporter.

109

References

110

Ackerman HW (2001) Frequency of morphological phage descriptions in the year 2000. Arch. Virol. 146:843-857 Ackers ML, Mahon BE, Leahy E, Goode B, Damrow T, Hayes PS, Bibb WF, Rice DH, Barrett TJ, Hutwagner L, Griffin PM, Slutsker L (1998) An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce consumption. J Infect Dis 177:1588-1593 Ackman D, Marks S, Mack P, Caldwell M, Root T, Birkhead G (1997) Swimming- associated haemorrhagic colitis due to Escherichia coli O157:H7 infection: evidence of prolonged contamination of a fresh water lake. Epidemiol Infect 119:1-8 Akashi S, Joh K, Tsuji A, Ito H, Hoshi H, Hayakawa T, Ihara J, Abe T, Hatori M, Mori T, et al. (1994) A severe outbreak of haemorrhagic colitis and haemolytic uraemic syndrome associated with Escherichia coli O157:H7 in Japan. Eur J Pediatr 153:650-655 Andersen JB, Heydorn A, Hentzer M, Eberl L, Geisenberger O, Christensen BB, Molin S, Givskov M (2001) gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication. Appl. Environ. Microbiol. 67:575-585 Applegate BM, Kehrmeyer SR, Sayler GS (1998) A chromosomally based tod- luxCDABE whole-cell reporter for benzene, toluene, ethylbenzene, and xylene (BTEX) sensing. Applied and environmental microbiology 64:2730-2735 Awais R, Fukudomi H, Miyanaga K, Unno H, Tanji Y (2006) A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7. Biotechnol. Prog. 22:853-859 Bainton NJ, Bycroft BW, Chhabra SR, Stead P, Gledhill L, Hill PJ, Rees CE, Winson MK, Salmond GP, Stewart GS, et al. (1992a) A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene 116:87-91 Bainton NJ, Stead P, Chhabra SR, Bycroft BW, Salmond GP, Stewart GS, Williams P (1992b) N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora. The Biochemical journal 288 ( Pt 3):997-1004 Baldwin TOaMMZ (1992) The biochemistry and molecular of bacterial bioluminescence. In: Muller F (ed) Chemistry and biochemistry of flavoenzymes, Vol 3. CRC Press, Boca Raton, p 467-530 Banaiee N, Bobadilla-Del-Valle M, Bardarov S, Jr., Riska PF, Small PM, Ponce-De-Leon A, Jacobs WR, Jr., Hatfull GF, Sifuentes-Osornio J (2001) Luciferase reporter for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico. J Clin Microbiol 39:3883-3888 Bang YB, Lee SE, Rhee JH, Choi SH (1999) Evidence that expression of the Vibrio vulnificus hemolysin gene is dependent on cyclic AMP and cyclic AMP receptor protein. J Bacteriol 181:7639-7642 Bardarov S, Jr., Dou H, Eisenach K, Banaiee N, Ya S, Chan J, Jacobs WR, Jr., Riska PF (2003) Detection and drug-susceptibility testing of M. tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria

111

Growth Indicator Tube (MGIT) system. Diagnostic microbiology and infectious disease 45:53-61 Bassler BL, Wright M, Showalter RE, Silverman MR (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Molecular microbiology 9:773-786 Bassler BL, Wright M, Silverman MR (1994) Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi. Molecular microbiology 12:403-412 Belkin S, Smulski DR, Vollmer AC, Van Dyk TK, LaRossa RA (1996) Oxidative stress detection with Escherichia coli harboring a katG'::lux fusion. Applied and environmental microbiology 62:2252-2256 Bielaszewska M, Janda J, Blahova K, Minarikova H, Jikova E, Karmali MA, Laubova J, Sikulova J, Preston MA, Khakhria R, Karch H, Klazarova H, Nyc O (1997) Human Escherichia coli O157:H7 infection associated with the consumption of unpasteurized goat's milk. Epidemiol Infect 119:299-305 Billard P, DuBow MS (1998) Bioluminescence-based assays for detection and characterization of bacteria and chemicals in clinical laboratories. Clinical biochemistry 31:1-14 Boettcher KJ, Ruby EG (1995) Detection and quantification of Vibrio fischeri autoinducer from symbiotic squid light organs. J Bacteriol 177:1053-1058 Bopp DJ, Sauders BD, Waring AL, Ackelsberg J, Dumas N, Braun-Howland E, Dziewulski D, Wallace BJ, Kelly M, Halse T, Musser KA, Smith PF, Morse DL, Limberger RJ (2003) Detection, isolation, and molecular subtyping of Escherichia coli O157:H7 and jejuni associated with a large waterborne outbreak. J Clin Microbiol 41:174-180 Boylan M, Graham AF, Meighen EA (1985) Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri. J Bacteriol 163:1186-1190 Boylan M, Miyamoto C, Wall L, Graham A, Meighen E (1989) Lux C, D and E genes of the Vibrio fischeri luminescence operon code for the reductase, transferase, and synthetase enzymes involved in aldehyde biosynthesis. Photochemistry and photobiology 49:681-688 Brigati JR, Ripp S, Johnson CM, Jegier P, Sayler GS (2007) Bacteriophage-based bioluminescent bioreporter for the detection of Escherichia coli O157:H7. J. Food Protect. 70:1386-1392 Bruce MG, Curtis MB, Payne MM, Gautom RK, Thompson EC, Bennett AL, Kobayashi JM (2003) Lake-associated outbreak of Escherichia coli O157:H7 in Clark County, Washington, August 1999. Arch Pediatr Adolesc Med 157:1016-1021 Bruhn JB, Christensen AB, Flodgaard LR, Nielsen KF, Larsen TO, Givskov M, Gram L (2004) Presence of acylated homoserine lactones (AHLs) and AHL-producing bacteria in meat and potential role of AHL in spoilage of meat. Appl. Environ. Microbiol. 70:4293-4302 Burlage RS (1998) Organic contaminant detection and biodegradation characteristics. Methods in Molecular Biology (Totowa, New Jersey) 102:259-268

112

Camara M, Daykin M, Chhabra SR (1998) Detection, purification, and synthesis of N- acylhomoserine lactone quorum sensing signal molecules. Methods Microbiol. 27:319-330 Camara M, Williams P, Hardman A (2002) Controlling infection by tuning in and turning down the volume of bacterial small-talk. The Lancet infectious diseases 2:667- 676 Cao JG, Meighen EA (1989) Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi. The Journal of biological chemistry 264:21670-21676 Carriere C, Riska PF, Zimhony O, Kriakov J, Bardarov S, Burns J, Chan J, Jacobs WR, Jr. (1997) Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis. J Clin Microbiol 35:3232-3239 CDC (1996a) From the Centers for Disease Control and Prevention. Lake-associated outbreak of Escherichia coli O157:H7--Illinois, 1995. Jama 275:1872-1873 CDC (1996b) Outbreak of Escherichia coli O157:H7 infections associated with drinking unpasteurized commercial apple juice -- British Columbia, California, Colorado, and Washington, October 1996. MMWR 45:975 CDC (1997) From the Centers for Disease Control and Prevention. Escherichia coli O157:H7 infections associated with eating a nationally distributed commercial brand of frozen ground beef patties and burgers--Colorado, 1997. Jama 278:891 CDC (1999) Outbreak of Escherichia coli O157:H7 and Campylobacter among attendees of the Washington County Fair-New York, 1999. MMWR Morb Mortal Wkly Rep 48:803-805 CDC (2002) From the Centers for Disease Control and Prevention. Multistate outbreak of Escherichia coli O157:H7 infections associated with eating ground beef--United States, June-July 2002. Jama 288:690-691 CDC (2006) Ongoing multistate outbreak of Escherichia coli serotype O157:H7 infections associated with consumption of fresh spinach--United States, September 2006. MMWR Morb Mortal Wkly Rep 55:1045-1046 Chapman PA, Wright DJ, Siddons CA (1994) A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. J Med Microbiol 40:424-427 Chatterjee A, Cui Y, Liu Y, Dumenyo CK, Chatterjee AK (1995) Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Applied and environmental microbiology 61:1959-1967 Chen J, Griffiths M (1996) Salmonella detection in eggs using lux+ bacteriophages. J. Food Prot. 59:908-914 Choi SH, Greenberg EP (1991) The C-terminal region of the Vibrio fischeri LuxR protein contains an inducer-independent lux gene activating domain. Proc Natl Acad Sci U S A 88:11115-11119

113

Cody SH, Glynn MK, Farrar JA, Cairns KL, Griffin PM, Kobayashi J, Fyfe M, Hoffman R, King AS, Lewis JH, Swaminathan B, Bryant RG, Vugia DJ (1999) An outbreak of Escherichia coli O157:H7 infection from unpasteurized commercial apple juice. Ann. Intern. Med. 130:202-209 Contag CH, Contag PR, Mullins JI, Spilman SD, Stevenson DK, Benaron DA (1995) Photonic detection of bacterial pathogens in living hosts. Molecular microbiology 18:593-603 Corbisier P, Thiry E, Diels L (1996) Bacterial biosensors for the toxicity assessment of solid wastes. Environmental Toxicology and Water Quality 11:171-177 Cubo MT, Economou A, Murphy G, Johnston AW, Downie JA (1992) Molecular characterization and regulation of the rhizosphere-expressed genes rhiABCR that can influence nodulation by Rhizobium leguminosarum biovar viciae. J Bacteriol 174:4026-4035 Cui Y, Chatterjee A, Liu Y, Dumenyo CK, Chatterjee AK (1995) Identification of a global repressor gene, rsmA, of Erwinia carotovora subsp. carotovora that controls extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity in soft-rotting Erwinia spp. J Bacteriol 177:5108-5115 Daniels R, Vanderleyden J, Michiels J (2004) Quorum sensing and swarming migration in bacteria. FEMS microbiology reviews 28:261-289 Daunert S, Barrett G, Feliciano JS, Shetty RS, Shrestha S, Smith-Spencer W (2000) Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem. Rev. 100:2705-2738 Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science (New York, N.Y 280:295-298 de Kievit TR, Iglewski BH (2000) Bacterial quorum sensing in pathogenic relationships. Infection and immunity 68:4839-4849 Deisingh AK, Thompson M (2004) Strategies for the detection of Escherichia coli O157:H7 in foods. J. Appl. Microbiol. 96:419-429 Devine JH, Shadel GS, Baldwin TO (1989) Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744. Proc Natl Acad Sci U S A 86:5688-5692 Dong YH, Gusti AR, Zhang Q, Xu JL, Zhang LH (2002) Identification of quorum- quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68:1754-1759 Dunlap PV, Ray JM (1989) Requirement for autoinducer in transcriptional negative autoregulation of the Vibrio fischeri luxR gene in Escherichia coli. J Bacteriol 171:3549-3552 Dunn DK, Michaliszyn GA, Bogacki IG, Meighen EA (1973) Conversion of aldehyde to acid in the bacterial bioluminescent reaction. Biochemistry 12:4911-4918 Dylla BL, Vetter EA, Hughes JG, Cockerill FR, 3rd (1995) Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens. J Clin Microbiol 33:222-224

114

Eberhard A (1972) Inhibition and activation of bacterial luciferase synthesis. J Bacteriol 109:1101-1105 Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH, Oppenheimer NJ (1981) Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry 20:2444-2449 Eberhard A, Widrig CA, McBath P, Schineller JB (1986) Analogs of the autoinducer of bioluminescence in Vibrio fischeri. Arch Microbiol 146:35-40 Eberl L, Winson MK, Sternberg C, Stewart GSAB, Christiansen G, Chhabra SR, Bycroft BW, Williams P, Molin S, Givskov M (1996) Involvement of N-acyl-L- homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens. Mol. Microbiol. 20:127-136 Edgar R, McKinstry M, Hwang J, Oppenheim AB, Fekete RA, Giulian G, Merril C, Nagashima K, Adhya S (2006) High-sensitivity bacterial detection using biotin- tagged phage and quantum-dot nanocomplexes. Proc. Natl. Acad. Sci. U.S.A. 103:4841-4845 Egland KA, Greenberg EP (1999) Quorum sensing in Vibrio fischeri: elements of the luxl promoter. Molecular microbiology 31:1197-1204 Egland KA, Greenberg EP (2000) Conversion of the Vibrio fischeri transcriptional activator, LuxR, to a repressor. J Bacteriol 182:805-811 Egland KA, Greenberg EP (2001) Quorum sensing in Vibrio fischeri: analysis of the LuxR DNA binding region by alanine-scanning mutagenesis. J Bacteriol 183:382- 386 Elasri MO, Miller RV (1998) A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation. Applied microbiology and biotechnology 50:455-458 Engebrecht J, Nealson K, Silverman M (1983) Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell 32:773-781 Engebrecht J, Silverman M (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc Natl Acad Sci U S A 81:4154-4158 Engebrecht J, Silverman M (1987) Nucleotide sequence of the regulatory locus controlling expression of bacterial genes for bioluminescence. Nucleic Acids Res 15:10455-10467 Erbe JL, Adams AC, Taylor KB, Hall LM (1996) Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations. Journal of Industrial Microbiology 17:80-83 Evans K, Passador L, Srikumar R, Tsang E, Nezezon J, Poole K (1998) Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa. J Bacteriol 180:5443-5447 Farmer JJ, 3rd, Davis BR (1985) H7 antiserum-sorbitol fermentation medium: a single tube screening medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 22:620-625 Favrin SJ, Jassim SA, Griffiths MW (2001) Development and optimization of a novel immunomagnetic separation-bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth. Appl. Environ. Microbiol. 67:217-224

115

Federle MJ, Bassler BL (2003) Interspecies communication in bacteria. The Journal of clinical investigation 112:1291-1299 Flavier AB, Ganova-Raeva LM, Schell MA, Denny TP (1997) Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester. J Bacteriol 179:7089-7097 Fortin NY, Mulchandani A, Chen W (2001) Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7. Analytical biochemistry 289:281-288 Francis KP, Joh D, Bellinger-Kawahara C, Hawkinson MJ, Purchio TF, Contag PR (2000) Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect Immun 68:3594-3600 Fu Z, Rogelj S, Kieft TL (2005) Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. International journal of 99:47-57 Funatsu T, Taniyama T, Tajima T, Tadakuma H, Namiki H (2002) Rapid and sensitive detection method of a bacterium by using a GFP reporter phage. Microbiol. Immunol. 46:365-369 Fuqua C, Burbea M, Winans SC (1995) Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene. J Bacteriol 177:1367-1373 Fuqua C, Eberhard, A. (1999) Signal generation in autoinduction systems: synthesis of acylated homoserine lactone by LuxI-type proteins. In: Cell- in Bacteria. ASM Press, Washington, D.C., p 211-230 Fuqua WC, Winans SC (1994) A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumour metabolite. J. Bacteriol. 176:2796-3806 Galindo LF, Ferrer Mde L, Valdes LS, Iglesias HM, Goodridge IM, Mesa JL (2002) Antiamebic effect of metronidazole proved in a study conducted in Cienfuegos province. Revista cubana de medicina tropical 54:101-105 Gambello MJ, Iglewski BH (1991) Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol 173:3000-3009 Gambello MJ, Kaye S, Iglewski BH (1993) LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infection and immunity 61:1180-1184 Gehring AG, Irwin PL, Reed SA, Tu S, Andreotti PE, Akhavan-Tafti H, Handley RS (2004) Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7. J. Immunol. Methods 293:97-106 Goodridge L, Chen J, Griffiths M (1999) Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7. Appl Environ Microbiol 65:1397-1404 Goodridge L, Griffiths M (2002) Reporter bacteriophage assays as a means to detect foodborne pathogenic bacteria. Food Res. Int. 35:863-870

116

Gould TA, Schweizer HP, Churchill ME (2004) Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI. Molecular microbiology 53:1135-1146 Graf J, Ruby EG (1998) Host-derived amino acids support the proliferation of symbiotic bacteria. Proc Natl Acad Sci U S A 95:1818-1822 Gray KM, Pearson JP, Downie JA, Boboye BE, Greenberg EP (1996) Cell-to-cell signaling in the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum: autoinduction of a stationary phase and rhizosphere-expressed genes. J Bacteriol 178:372-376 Green RC (1996) Biosynthesis of methionine. In: Neidhardt FC (ed) Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, D.C., p 542-560 Gupta RK, Patterson SS, Ripp S, Sayler GS (2003) Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Res. 4:305-313 Hansen LH, Sorensen SJ (2000) Detection and quantification of tetracyclines by whole cell biosensors. FEMS Microbiology Letters 190:273-278 Hanzelka BL, Greenberg EP (1995) Evidence that the N-terminal region of the Vibrio fischeri LuxR protein constitutes an autoinducer-binding domain. J Bacteriol 177:815-817 Hastings JW (1978) Bacterial bioluminescence light emission in the mixed function oxidation of reduced flavin and fatty aldehyde. CRC critical reviews in biochemistry 5:163-184 Hastings JW, Nealson KH (1977) Bacterial bioluminescence. Annual review of microbiology 31:549-595 Hay AG, Rice JF, Applegate BM, Bright NG, Sayler GS (2000) A bioluminescent whole- cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2,4- dichlorophenol in soil. Applied and environmental microbiology 66:4589-4594 Hazbon MH (2004) Recent advances in molecular methods for early diagnosis of tuberculosis and drug-resistant tuberculosis. Biomedica 24 Supp 1:149-162 Heller KJ (1992) Molecular interaction between bacteriophage and the gram-negative cell envelope. Arch Microbiol 158:235-248 Henke JM, Bassler BL (2004) Bacterial social engagements. Trends in cell biology 14:648-656 Hilborn ED, Mermin JH, Mshar PA, Hadler JL, Voetsch A, Wojtkunski C, Swartz M, Mshar R, Lambert-Fair MA, Farrar JA, Glynn MK, Slutsker L (1999) A multistate outbreak of Escherichia coli O157:H7 infections associated with consumption of mesclun lettuce. Arch Intern Med 159:1758-1764 Hoang TT, Ma Y, Stern RJ, McNeil MR, Schweizer HP (1999) Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of Mycobacterium tuberculosis RmlD and Pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of purified RhlI. Gene 237:361- 371

117

Hoang TT, Schweizer HP (1999) Characterization of Pseudomonas aeruginosa enoyl- acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. J Bacteriol 181:5489-5497 Holicka J, Guy RA, Kapoor A, Shepherd D, Horgen PA (2006) A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef. Canadian journal of microbiology 52:992-998 Holmes DS, Dubey SK, Gangolli S (1994) Development of biosensors for the detection of mercury and copper ions. Environmental Geochemistry and Health 16:229-233 Hsu CF, Tsai TY, Pan TM (2005) Use of the duplex TaqMan PCR system for detection of Shiga-like toxin-producing Escherichia coli O157. J Clin Microbiol 43:2668- 2673 Huang JJ, Han JI, Zhang LH, Leadbetter JR (2003) Utilization of acyl-homoserine lactone quorum signals for growth by a soil Pseudomonad and Pseudomonas aeruginosa PAO1. Appl. Environ. Microbiol. 69:5941-5949 Hwang I, Cook DM, Farrand SK (1995) A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer. J Bacteriol 177:449-458 Hwang I, Li PL, Zhang L, Piper KR, Cook DM, Tate ME, Farrand SK (1994) TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer. Proc Natl Acad Sci U S A 91:4639-4643 Ishii T, Yanagida M (1977) The two dispensable structural proteins (soc and hoc) of the T4 phage capsid; their purification and properties, isolation and characterization of the defective mutants, and their binding with the defective heads in vitro. J Mol Biol 109:487-514 Islam SK, Vijayaraghavn R, Zhang M, Ripp S, Caylor S, Weathers B, Moser S, Terry S, Blalock B, Sayler GS (2007) Integrated circuit biosensors using living whole-cell bioreporters. IEEE Transactions 54:89-98 Jacobs WR, Jr., Barletta RG, Udani R, Chan J, Kalkut G, Sosne G, Kieser T, Sarkis GJ, Hatfull GF, Bloom BR (1993) Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science (New York, N.Y 260:819-822 Jay MT, Garrett V, Mohle-Boetani JC, Barros M, Farrar JA, Rios R, Abbott S, Sowadsky R, Komatsu K, Mandrell R, Sobel J, Werner SB (2004) A multistate outbreak of Escherichia coli O157:H7 infection linked to consumption of beef tacos at a fast- food restaurant chain. Clin Infect Dis 39:1-7 Jones S, Yu B, Bainton NJ, Birdsall M, Bycroft BW, Chhabra SR, Cox AJ, Golby P, Reeves PJ, Stephens S, et al. (1993) The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. The EMBO journal 12:2477-2482 Jothikumar N, Griffiths MW (2002) Rapid detection of Escherichia coli O157:H7 with multiplex real-time PCR assays. Applied and environmental microbiology 68:3169-3171

118

Kaplan HB, Greenberg EP (1985) Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system. J Bacteriol 163:1210-1214 Keane A, Phoenix P, Ghoshal S, Lau PCK (2002) Exposing culprit organic pollutants: a review. J. Microbiol. Methods 49:103-119 Keene WE, Hedberg K, Herriott DE, Hancock DD, McKay RW, Barrett TJ, Fleming DW (1997) A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk. J Infect Dis 176:815-818 Keene WE, McAnulty JM, Hoesly FC, Williams LP, Jr., Hedberg K, Oxman GL, Barrett TJ, Pfaller MA, Fleming DW (1994) A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei. N Engl J Med 331:579-584 Khang Y-H, Yang ZK, Burlage RS (1997) Measurement of iron-dependence of pupA promoter activity by a pup-lux bioreporter. Journal of Microbiology and Biotechnology 7:352-355 King JMH, DiGrazia PM, Applegate B, Burlage R, Sanseverino J, Dunbar P, Larimer F, Sayler GS (1990) Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science (Washington, DC, United States) 249:778-781 Kiratisin P, Tucker KD, Passador L (2002) LasR, a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer. J Bacteriol 184:4912-4919 Kodikara CP, Crew HH, Stewart GS (1991) Near on-line detection of enteric bacteria using lux recombinant bacteriophage. FEMS Microbiol. Lett. 67:261-265. Koodie L, Dhople AM (2001) Acid tolerance of Escherichia coli O157 : H7 and its survival in apple juice. Microbios 104:167-175 Kuhn J, Suissa M, Wyse J, Cohen I, Weiser I, Reznick S, Lubinsky-Mink S, Stewart G, Ulitzur S (2002) Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella. International journal of food microbiology 74:229-238 Kuo A, Callahan SM, Dunlap PV (1996) Modulation of luminescence operon expression by N-octanoyl-L-homoserine lactone in ainS mutants of Vibrio fischeri. J Bacteriol 178:971-976 Lamb JR, Patel H, Montminy T, Wagner VE, Iglewski BH (2003) Functional domains of the RhlR transcriptional regulator of Pseudomonas aeruginosa. J Bacteriol 185:7129-7139 Layton AC, Muccini M, Ghosh MM, Sayler GS (1998) Construction of a bioluminescent reporter strain To detect polychlorinated biphenyls. Applied and environmental microbiology 64:5023-5026 LeBlanc JJ (2003) Implication of virulence factors in Escherichia coli O157:H7 pathogenisis. Crit. Rev. Microbiol. 29:277-296 Lee Jea (1991) The mechanism of bacterial bioluminescence. In: Muller F (ed) Chemistry and biochemistry of flavoenzymes, Vol 2. CRC Press, Boca Raton, p 109-151

119

Lee W, Park K, Kim Y, Lee WH, Choi J (2005) Protein array consisting of sol-gel bioactive platform for detection of E. coli O157:H7. Biosens. Bioelectron. 20:2292-2299 Levy DA, Bens MS, Craun GF, Calderon RL, Herwaldt BL (1998) Surveillance for waterborne-disease outbreaks--United States, 1995-1996. MMWR CDC Surveill Summ 47:1-34 Lewenza S, Conway B, Greenberg EP, Sokol PA (1999) Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI. J. Bacteriol. 181:748-756 Lithgow JK, Wilkinson A, Hardman A, Rodelas B, Wisniewski-Dye F, Williams P, Downie JA (2000) The regulatory locus cinRI in Rhizobium leguminosarum controls a network of quorum-sensing loci. Molecular microbiology 37:81-97 Loessner MJ, Rees CE, Stewart GS, Scherer S (1996) Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells. Appl. Environ. Microbiol. 62:1133-1140 Loessner MJ, Rudolf M, Scherer S (1997) Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods. Applied and environmental microbiology 63:2961-2965 Lupp C, Ruby EG (2005) Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors. J Bacteriol 187:3620-3629 Lupp C, Urbanowski M, Greenberg EP, Ruby EG (2003) The Vibrio fischeri quorum- sensing systems ain and lux sequentially induce luminescence gene expression and are important for persistence in the squid host. Molecular microbiology 50:319-331 Macdonald C, Drew J, Carlson R, Dzogan S, Tataryn S, Macdonald A, Ali A, Amhed R, Easy R, Clark C, Rodgers F (2000) Outbreak of Escherichia coli O157:H7 leading to the recall of retail ground beef--Winnipeg, Manitoba, May 1999. Canada communicable disease report, Releve des maladies transmissibles au Canada 26:109-111 Maoz A, Mayr R, Bresolin G, Neuhaus K, Francis KP, Scherer S (2002) Sensitive in situ monitoring of a recombinant bioluminescent Yersinia enterocolitica reporter mutant in real time on Camembert cheese. Appl. Environ. Microbiol. 68:5737- 5740 Marketon MM, Gonzalez JE (2002) Identification of two quorum-sensing systems in . J Bacteriol 184:3466-3475 Marketon MM, Gronquist MR, Eberhard A, Gonzalez JE (2002) Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones. J Bacteriol 184:5686-5695 Marks T, and R. Sharp (2000) Bacteriophages and biotechnology: a review. J. Chem. Tech. Biotech. 75:6-17 Martin M, Showalter R, Silverman M (1989) Identification of a locus controlling expression of luminescence genes in Vibrio harveyi. J Bacteriol 171:2406-2414 Mayville P, Ji G, Beavis R, Yang H, Goger M, Novick RP, Muir TW (1999) Structure- activity analysis of synthetic autoinducing thiolactone peptides from

120

Staphylococcus aureus responsible for virulence. Proc. Natl. Acad. Sci. USA 96:1218-1223 McFall-Ngai MJ, Ruby EG (1991) Symbiont recognition and subsequent as early events in an animal-bacterial . Science (New York, N.Y 254:1491-1494 McGowan S, Sebaihia M, Jones S, Yu B, Bainton N, Chan PF, Bycroft B, Stewart GS, Williams P, Salmond GP (1995) Carbapenem antibiotic production in Erwinia carotovora is regulated by CarR, a homologue of the LuxR transcriptional activator. Microbiology 141 ( Pt 3):541-550 Meighen EA (1991) Molecular biology of bacterial bioluminescence. Microbiol. Rev. 55:123-142 Meighen EA (1993) Bacterial bioluminescence: organization, regulation, and application of the lux genes. Faseb J 7:1016-1022 Meighen EA (1994) Genetics of bacterial bioluminescence. Annu. Rev. Genet. 28:117- 139 Meng J, Zhao S, Doyle MP, Mitchell SE, Kresovich S (1996) Polymerase chain reaction for detecting Escherichia coli O157: H7. International journal of food microbiology 32:103-113 Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165-199 Min J, Lee CW, Moon SH, LaRossa RA, Gu MB (2000) Detection of radiation effects using recombinant bioluminescent Escherichia coli strains. Radiation and Environmental Biophysics 39:41-45 MMWR (2002) Multistate outbreak of Escherichia coli O157:H7 infections associated with eating ground beef--United States, June-July 2002. MMWR Morb Mortal Wkly Rep 51:637-639 More MI, Finger LD, Stryker JL, Fuqua C, Eberhard A, Winans SC (1996) Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates. Science (New York, N.Y 272:1655-1658 Morita M, Fischer CR, Mizoguchi K, Yoichi M, Oda M, Tanji Y, Unno H (2002a) Amino acid alterations in Gp38 of host range mutants of PP01 and evidence for their infection of an ompC null mutant of Escherichia coli O157:H7. FEMS Microbiol. Lett. 216:243-248 Morita M, Tanji Y, Mizoguchi K, Akitsu T, Kijima N, Unno H (2002b) Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes. FEMS Microbiol. Lett. 211:77-83 Mosier-Boss PA, Lieberman SH, Andrews JM, Rohwer FL, Wegley LE, Breitbart M (2003) Use of fluorescently labeled phage in the detection and identification of bacterial species. Appl. Spectrosc. 57:1138-1144 Moxley RA (2003) Detection and diagnosis of E. coli O157:H7 in food producing animals. In: Torrence ME, Isaacson RE (eds) Microbial food safety in animal agriculture. Iowa State Press, Ames, IA, p 143-154 Nealson KH (1977) Autoinduction of bacterial luciferase. Occurrence, mechanism and significance. Arch Microbiol 112:73-79

121

Nealson KH, Hastings JW (1979) Bacterial bioluminescence: its control and ecological significance. Microbiol Rev 43:496-518 Nealson KH, Platt T, Hastings JW (1970) Cellular control of the synthesis and activity of the bacterial luminescent system. J Bacteriol 104:313-322 Nikaido H, Rosenberg EY (1983) Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J Bacteriol 153:241-252 Nivens DE, McKnight TE, Moser SA, Osbourn SJ, Simpson ML, Sayler GS (2004) Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. J. Appl. Microbiol. 96:33-46 Nyholm SV, McFall-Ngai MJ (1998) Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri. The Biological bulletin 195:89-97 Nyholm SV, McFall-Ngai MJ (2004) The winnowing: establishing the squid-vibrio symbiosis. Nature reviews 2:632-642 O'Brien AO, Lively TA, Chen ME, Rothman SW, Formal SB (1983) Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (SHIGA) like cytotoxin. Lancet 1:702 O'Flynn G, Ross RP, Fitzgerald GF, Coffey A (2004) Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. Appl. Environ. Microbiol. 70:3417-3424 Oda M, Morita M, Unno H, Tanji Y (2004) Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage. Appl. Environ. Microbiol. 70:527-534 Olsen SJ, Miller G, Breuer T, Kennedy M, Higgins C, Walford J, McKee G, Fox K, Bibb W, Mead P (2002) A waterborne outbreak of Escherichia coli O157:H7 infections and hemolytic uremic syndrome: implications for rural water systems. Emerg Infect Dis 8:370-375 Pagotto F, Brovko L, Griffiths MW (1996) Phage-mediated detection of Staphylococcus aureus and Escherichia coli using bioluminescence. Bacteriol. Quality Raw Milk 9601:152-156 Park CH, Hixon DL, Morrison WL, Cook CB (1994) Rapid diagnosis of enterohemorrhagic Escherichia coli O157:H7 directly from fecal specimens using immunofluorescence stain. Am J Clin Pathol 101:91-94 Park SY, Lee SJ, Oh TK, Oh JW, Koo BT, Yum DY, Lee JK (2003) AhlD, an N- acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149:1541-1550 Parsek MR, Greenberg EP (2005) Sociomicrobiology: the connections between quorum sensing and . Trends in microbiology 13:27-33 Parsek MR, Schaefer AL, Greenberg EP (1997) Analysis of random and site-directed mutations in rhII, a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase. Molecular microbiology 26:301-310

122

Parsek MR, Val DL, Hanzelka BL, Cronan JE, Jr., Greenberg EP (1999) Acyl homoserine-lactone quorum-sensing signal generation. Proc Natl Acad Sci U S A 96:4360-4365 Passador L, Cook JM, Gambello MJ, Rust L, Iglewski BH (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science (New York, N.Y 260:1127-1130 Paunio M, Pebody R, Keskimaki M, Kokki M, Ruutu P, Oinonen S, Vuotari V, Siitonen A, Lahti E, Leinikki P (1999) Swimming-associated outbreak of Escherichia coli O157:H7. Epidemiol Infect 122:1-5 Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, Greenberg EP (1994) Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A 91:197-201 Pearson JP, Passador L, Iglewski BH, Greenberg EP (1995) A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92:1490-1494 Pearson JP, Van Delden C, Iglewski BH (1999) Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J Bacteriol 181:1203- 1210 Peitzsch N, Eberz G, Nies DH (1998) Alcaligenes eutrophus as a bacterial chromate sensor. Applied and environmental microbiology 64:453-458 Petty NK, Evans TJ, Fineran PC, Salmond GP (2007) Biotechnological exploitation of bacteriophage research. Trends in biotechnology 25:7-15 Pierson LS, 3rd, Keppenne VD, Wood DW (1994) Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J Bacteriol 176:3966-3974 Piper KR, Beck von Bodman S, Farrand SK (1993) Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448-450 Pirhonen M, Flego D, Heikinheimo R, Palva ET (1993) A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. EMBO J. 12:2467-2476 Prest AG, Winson MK, Hammond JRM, Stewart GSAB (1997) The construction and application of a lux-based nitrate biosensor. Letters in Applied Microbiology 24:355-360 Proctor ME, Kurzynski T, Koschmann C, Archer JR, Davis JP (2002) Four strains of Escherichia coli O157:H7 isolated from patients during an outbreak of disease associated with ground beef: importance of evaluating multiple colonies from an outbreak-associated product. J Clin Microbiol 40:1530-1533 Puskas A, Greenberg E, Kaplan S, Schaefer A (1997) A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides. J. Bacteriol. 179:7530-7537 Radke SM, Alocilja EC (2005) A high density microelectrode array biosensor for detection of E. coli O157:H7. Biosens. Bioelectron. 20:1662-1667 Reading NC, Sperandio V (2006) Quorum sensing: the many languages of bacteria. FEMS Microbiol Lett 254:1-11

123

Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, Hebert RJ, Olcott ES, Johnson LM, Hargrett NT, Blake PA, Cohen ML (1983) Hemorrhagic Colitis Associated with a Rare Escherichia coli Serotype. New England Journal of Medicine 308:681-685 Ripp S, Jegier P, Birmele M, Johnson C, Daumer K, Garland J, Sayler G (2006) Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents. J. Appl. Microbiol. 100:488-499 Ripp S, Jegier, P., Johnson, C., Brigati, J.R., and Sayler, G.S. (Submitted 2007) Bacteriophage-amplified bioluminescent sensing of Escherichia coli O157:H7. Analytical and Bioanalytical Chemistry Riska PF, Jacobs WR, Jr. (1998) The use of luciferase-reporter phage for antibiotic- susceptibility testing of mycobacteria. Methods in molecular biology (Clifton, N.J 101:431-455 Riska PF, Jacobs WR, Jr., Bloom BR, McKitrick J, Chan J (1997) Specific identification of Mycobacterium tuberculosis with the luciferase reporter : use of p-nitro-alpha-acetylamino-beta-hydroxy propiophenone. J Clin Microbiol 35:3225-3231 Riska PF, Su Y, Bardarov S, Freundlich L, Sarkis G, Hatfull G, Carriere C, Kumar V, Chan J, Jacobs WR, Jr. (1999) Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box. J Clin Microbiol 37:1144-1149 Roche DM, Byers JT, Smith DS, Glansdorp FG, Spring DR, Welch M (2004) Communications blackout? Do N-acylhomoserine lactone-degrading enzymes have any role in quorum sensing? Microbiology 150:2023-2028 Rozen Y, Nejidat A, Gartemann KH, Belkin H (1999) Specific detection of p- chlorobenzoic acid by Escherichia coli bearing a plasmid-borne fcbA'::lux fusion. Chemosphere 38:633-641 Ruby EG (1996) Lessons from a cooperative, bacterial-animal association: the Vibrio fischeri-Euprymna scolopes light organ symbiosis. Annual review of microbiology 50:591-624 Ruby EG (1999) The Euprymna scolopes-Vibrio fischeri symbiosis: a biomedical model for the study of bacterial colonization of animal tissue. Journal of molecular microbiology and biotechnology 1:13-21 Ruby EG, McFall-Ngai MJ (1992) A squid that glows in the night: development of an animal-bacterial mutualism. J Bacteriol 174:4865-4870 Rupani SP, Gu MB, Konstantinov KB, Dhurjati PS, Van Dyk TK, LaRossa RA (1996) Characterization of the Stress Response of a Bioluminescent Biological Sensor in Batch and Continuous Cultures. Biotechnol. Prog. 12:387-392 Samadpour M, Stewart J, Steingart K, Addy C, Louderback J, McGinn M, Ellington J, Newman T (2002) Laboratory investigation of an E. coli O157:H7 outbreak associated with swimming in Battle Ground Lake, Vancouver, Washington. J Environ Health 64:16-20, 26, 25

124

Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rdedn.|. Publisher|, City Sanderson MW, Gay JM, Hancock DD, Gay CC, Fox LK, Besser TE (1995) Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces. J Clin Microbiol 33:2616-2619 Sarkis GJ, Jacobs WR, Hatfull GF (1995) L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. Mol. Microbiol. 15:1055-1067 Schaefer AL, Hanzelka BL, Eberhard A, Greenberg EP (1996a) Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J Bacteriol 178:2897-2901 Schaefer AL, Val DL, Hanzelka BL, Cronan JE, Jr., Greenberg EP (1996b) Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein. Proc Natl Acad Sci U S A 93:9505- 9509 Schindler H, Rosenbusch JP (1978) Matrix protein from Escherichia coli outer membranes forms voltage-controlled channels in lipid bilayers. Proc Natl Acad Sci U S A 75:3751-3755 Schripsema J, de Rudder KE, van Vliet TB, Lankhorst PP, de Vroom E, Kijne JW, van Brussel AA (1996) small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors. J Bacteriol 178:366-371 Selifonova O, Burlage R, Barkay T (1993) Bioluminescent sensors for detection of bioavailable mercury(II) in the environment. Applied and environmental microbiology 59:3083-3090 Selifonova OV, Eaton RW (1996) Use of an ipb-lux fusion to study regulation of the isopropylbenzene catabolism operon of Pseudomonas putida RE204 and to detect hydrophobic pollutants in the environment. Applied and environmental microbiology 62:778-783 Shadel GS, Baldwin TO (1991) The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene. J Bacteriol 173:568-574 Shadel GS, Baldwin TO (1992a) Identification of a distantly located regulatory element in the luxD gene required for negative autoregulation of the Vibrio fischeri luxR gene. The Journal of biological chemistry 267:7690-7695 Shadel GS, Baldwin TO (1992b) Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex- independent and -dependent luxR transcription. The Journal of biological chemistry 267:7696-7702 Sharma VK, Dean-Nystrom EA (2003) Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins. Veterinary microbiology 93:247-260 Sharpe AN (2001) Food sample preparation and enrichment for rapid detection. Royal Society Chem. 272:129-137

125

Shaw PD, Ping G, Daly SL, Cha C, Cronan JE, Rinehart KL, Farrand SK (1997) Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin- layer chromatography. Proc. Natl. Acad. Sci. USA 94:6036-6041 Shiner EK, Rumbaugh KP, Williams SC (2005) Inter-kingdom signaling: deciphering the language of acyl homoserine lactones. FEMS microbiology reviews 29:935-947 Shingleton JT, Applegate BM, Nagel AC, Bienkowski PR, Sayler GS (1998) Induction of the tod operon by trichloroethylene in Pseudomonas putida TVA8. Applied and environmental microbiology 64:5049-5052 Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions Simpson ML, Paulus, M.J., Jellison, G.E., Sayler, G.S. Nivens, D.E., Dionisi, H.M., Ripp, S., Applegate, B. (2000) Proceedings of the 30th International Conference on Environmental Systems, Toulouse, France Stevens AM, Dolan KM, Greenberg EP (1994) Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region. Proc Natl Acad Sci U S A 91:12619-12623 Stewart G, Smith T, Denyer S (1989) Genetic engineering for bioluminescent bacteria. Food Sci. Technol. 3:19-22 Stone R (2002) Stalin's forgotten cure. Science 298:728-731 Su XL, Li Y (2004) Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7. Analytical chemistry 76:4806-4810 Subramamanian A, Irudayaraj J, Ryan T (2006) A mixed self-assembled monolayer- based surface plasmon immunosensor for detection of E. coli O157:H7. Biosens. Bioelectron. 21:998-1006 Sun W, Cao JG, Teng K, Meighen EA (1994) Biosynthesis of poly-3-hydroxybutyrate in the luminescent bacterium, Vibrio harveyi, and regulation by the lux autoinducer, N-(3-hydroxybutanoyl) homoserine lactone. The Journal of biological chemistry 269:20785-20790 Swerdlow DL, Woodruff BA, Brady RC, Griffin PM, Tippen S, Donnell HD, Jr., Geldreich E, Payne BJ, Meyer A, Jr., Wells JG, et al. (1992) A waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann Intern Med 117:812-819 Swift S, Downie JA, Whitehead NA, Barnard AM, Salmond GP, Williams P (2001) Quorum sensing as a population-density-dependent determinant of bacterial physiology. Advances in microbial physiology 45:199-270 Swift S, Throup JP, Williams P, Salmond GP, Stewart GS (1996) Quorum sensing: a population-density component in the determination of bacterial phenotype. Trends in biochemical sciences 21:214-219 Swift S, Williams, P., Stewart, G.S.A.B (1999) N-acylhomoserine lactones and quorum sensing in the proteobacteria. In: Winans GMDaSC (ed) Cell-Cell Signaling in Bacteria. ASM Press, Washington, D.C., p 291-313 Taga ME, Bassler BL (2003) Chemical communication among bacteria. Proc Natl Acad Sci U S A 100 Suppl 2:14549-14554 Throup JP, Camara M, Briggs GS, Winson MK, Chhabra SR, Bycroft BW, Williams P, Stewart GS (1995) Characterisation of the yenI/yenR locus from Yersinia

126

enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules. Molecular microbiology 17:345-356 Tibazarwa C, Wuertz S, Mergeay M, Wyns L, Van der Lelie D (2000) Regulation of the cnr cobalt and nickel resistance determinant of Ralstonia eutropha (Alcaligenes eutrophus) CH34. Journal of Bacteriology 182:1399-1409 Trott AE, Stevens AM (2001) Amino acid residues in LuxR critical for its mechanism of transcriptional activation during quorum sensing in Vibrio fischeri. J Bacteriol 183:387-392 Tu SC, Mager HI (1995) Biochemistry of bacterial bioluminescence. Photochemistry and photobiology 62:615-624 Turpin P, Maycroft K, Bedford J, Rowlands C, Wellington E (1993) A rapid luminescent-phage based MPN method for the enumeration of Salmonella typhimurium in environmental samples. Lett. Appl. Microbiol. 16:24-27 Ulitzer S, Kuhn J (1987) Introduction of lux genes into bacteria, a new approach for specific determination of bacteria and their antibiotic susceptibility. In: J. Scholmerich RA, A. Kapp, M. Ernst, & W. Woods (ed) Bioluminescence New Perspectives. John Wiley, New York, p 463-472 Ulitzur S, Kuhn J (1987) Introduction of lux genes into bacteria, a new approach for specific determination of bacteria and their antibiotic susceptibility. In: Scholmerich J, Andreesen R, Kapp A, Ernst M, Woods WG (eds) Bioluminescence and Chemiluminescence: New Perspectives. John Wiley & Sons, New York, p 463-472 Val DL, Cronan JE, Jr. (1998) In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases. J Bacteriol 180:2644-2651 van Brussel AA, Zaat SA, Wijffelman CA, Pees E, Lugtenberg BJ (1985) Bacteriocin small of fast-growing rhizobia is chloroform soluble and is not required for effective nodulation. J Bacteriol 162:1079-1082 Van Dyk TK, Reed TR, Vollmer AC, LaRossa RA (1995) Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment with chemical inducers. Journal of Bacteriology 177:6001-6004 Vannini A, Volpari C, Gargioli C, Muraglia E, Cortese R, De Francesco R, Neddermann P, Marco SD (2002) The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA. The EMBO journal 21:4393-4401 Vimont A, Vernozy-Rozand C, Montet MP, Lazizzaer C, Bavai C, Delignette-Muller ML (2006) Modeling and predicting the simultaneous growth of Escherichia coli O157:H7 and ground beef background microflora for various enrichment protocols. Appl. Environ. Microbiol. 72:261-268 Visick KL, Foster J, Doino J, McFall-Ngai M, Ruby EG (2000) Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J Bacteriol 182:4578-4586 Visick KL, McFall-Ngai MJ (2000) An exclusive contract: specificity in the Vibrio fischeri-Euprymna scolopes partnership. J Bacteriol 182:1779-1787

127

Vogt RL, Dippold L (2005) Escherichia coli O157:H7 outbreak associated with consumption of ground beef, June-July 2002. Public Health Rep 120:174-178 Vollmer AC, Belkin S, Smulski DR, Van Dyk TK, LaRossa RA (1997) Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids. Applied and environmental microbiology 63:2566- 2571 Vollmer AC, Kwakye S, Halpern M, Everbach EC (1998) Bacterial stress responses to 1- megahertz pulsed ultrasound in the presence of microbubbles. Applied and environmental microbiology 64:3927-3931 Waddell TE, Poppe C (2000) Construction of mini-Tn10luxABcam/Ptac-ATS and its use for developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7. FEMS Microbiol. Lett. 182:285-289 Wallace WH, Fleming JT, White DC, Sayler GS (1994) An algD-bioluminescent reporter plasmid to monitor alginate production in biofilms. Microbial 27:225- 239 Wang L, Li Y, Mustaphai A (2007) Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation. J Food Prot 70:1366-1372 Watson WT, Minogue TD, Val DL, von Bodman SB, Churchill ME (2002) Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing. Molecular cell 9:685-694 Whitehead NA, Barnard AML, Slater H, Simpson NJL, Salmond GPC (2001) Quorum- sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25:365-404 Winson MK, Camara M, Latifi A, Foglino M, Chhabra SR, Daykin M, Bally M, Chapon V, Salmond GP, Bycroft BW, et al. (1995) Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 92:9427-9431 Winson MK, Swift S, Fish L, Throup JP, Jorgensen F, Chhabra SR, Bycroft BW, Williams P, Stewart GS (1998) Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163:185-192 Wolber PK, Green RL (1990) Detection of bacteria by of ice nucleation genes. Trends Biotechnol. 8:276-279 Wolffs P, Norling B, Radstrom P (2005) Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells. J. Microbiol. Methods 60:315-323 Wood DW, Gong F, Daykin MM, Williams P, Pierson LS, 3rd (1997) N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere. J Bacteriol 179:7663-7670 Wood DW, Pierson LS, 3rd (1996) The phzI gene of Pseudomonas aureofaciens 30-84 is responsible for the production of a diffusible signal required for phenazine antibiotic production. Gene 168:49-53

128

Yoshitomi KJ, Jinneman KC, Weagant SD (2006) Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR. Molecular and cellular probes 20:31-41 Zenno S, Saigo K (1994) Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis. J Bacteriol 176:3544-3551 Zhang L, Murphy PJ, Kerr A, Tate ME (1993) Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362:446-448 Zhang RG, Pappas T, Brace JL, Miller PC, Oulmassov T, Molyneaux JM, Anderson JC, Bashkin JK, Winans SC, Joachimiak A (2002) Structure of a bacterial quorum- sensing transcription factor complexed with pheromone and DNA. Nature 417:971-974 Zhu J, Beaber JW, More MI, Fuqua C, Eberhard A, Winans SC (1998) Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J Bacteriol 180:5398-5405

129

Vita

Courtney was born on January 6, 1982 in Ypsilanti, MI. She received her B.S. in

Biological Sciences with a concentration in Microbiology from the University of

Tennessee, Knoxville. Then she pursued her Master of Science degree in Microbiology at the Center for Environmental Biotechnology at the University of Tennessee under Dr.

Gary Sayler and Dr. Steven Ripp.

130