THE POTENTIAL ROLE OF BIOFILM IN THE PATHOGENICITY

OF ENTEROBACTER CLOACAE, CAUSAL AGENT

OF ENTEROBACTER ONION BULB DECAY

By

SARAH MICHELLE DOSSEY

A thesis submitted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE IN PLANT PATHOLOGY

WASHINGTON STATE UNIVERSITY Department of Plant Pathology

May 2011

ii

To the faculty of Washington State University:

The members of the Committee appointed to examine the thesis of SARAH MICHELLE DOSSEY find it satisfactory and recommend that it be accepted.

_____________________________ Brenda K. Schroeder, Ph.D, Chair

_____________________________

Jack D. Rogers, Ph.D

_____________________________ Linda Thomashow, Ph.D

_____________________________

Lindsey J. du Toit, Ph.D

iii

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my adviser, Dr. Brenda Schroeder, for all of the

support she has given me. I’d like to acknowledge Dr. Jodi Humann for all of her support with

the genetic aspects of my work. I’d like to extend my gratitude to my committee members, Dr.

Rogers, Dr. Thomashow, and Dr. du Toit, for all of their guidance and support. I must also

express sincere gratitude to Dr. Lee Hadwiger for all of his help with my experiments, as well as

the Kahn lab for allowing me the use of their spiral plater.

I would like to express my thanks to my graduate colleagues, who have treated me like

family. I would especially like to acknowledge Austin Bates for singing in lab (especially that

one Wyoming song that was so inspiring).

I would like to also thank Dr. Michael Knoblauch, Dr. Valerie Lynch-Holm, and Dr.

Christine Davitt of the Franceschi Microscopy Center for their patience as well as Pam Brunsfeld

and the University of Idaho Stillinger Herbarium for nurturing my love for science.

I would also like to thank my sister, Nicole Dossey, for commiserating over statistics and

providing intellectual conversation outside of my research topic. I would like to thank my father,

Tim Dossey, for instilling in me the gifts of curiosity and stubbornness. I would like to thank my

mother, Zareen Dossey, for being my role model and best friend (and for being the best stats lab

partner ever). I would also like to acknowledge Caesar, Rajah, Tenchi and Trampie for being

there for me on a daily basis.

My deepest thanks go to Graciela Dinis and Jossie Guevara for putting up with my constant

lameness over the last few years. Also, I would like to extend my gratitude to the Suzuki,

Rossetti, Johanson, Alden, and Carpenter families for sharing their lives with me.

Lastly, I would like to thank Fyoder Dostoyevsky and Koushun Takami.

iv

THE POTENTIAL ROLE OF BIOFILM IN THE PATHOGENICITY

OF ENTEROBACTER CLOACAE, CAUSAL AGENT

OF ENTEROBACTER ONION BULB DECAY

Abstract

by Sarah Michelle Dossey, M.S. Washington State University

May 2011

Chair: Brenda K. Schroeder

Washington State is the third largest producer of storage onions, with a farm-gate value of

greater than $100 million. A current problem in Washington onion bulb production is

Enterobacter bulb decay, which is caused by the bacterium Enterobacter cloacae. This

postharvest disease produces a discoloration of one or two inner scales of onion bulbs in storage

with no tissue maceration, odor, or symptoms. In order to identify genes that have a role in the E.

cloacae-onion interaction, a mini-Tn5 mutagenesis library of E. cloacae strain EcWSU1R was

screened on Luria Bertani agar amended with congo red (CR) to identify polysaccharide mutants.

Forty-six unique CR mutants were assayed for solid-surface-associated biofilm production by

using a crystal violet staining assay. Twenty-two CR mutants produced a solid-surface-

associated biofilm that was significantly less than the rifampicin-resistant strain EcWSU1R

(P<0.0001). Using whole-bulb assays, it was determined that the CR mutants were not reduced

in ability to cause onion bulb rot compared to ECWSU1R (P=0.2395). E. cloacae strains

EcWSU1R, CR42, and CR78 (a rifampicin-resistant strain, a biofilm underproducer and an

overproducer, respectively) were inoculated into the leaves of 17-week-old onion plants to

v

determine movement dynamics in planta. All three strains moved bidirectionally through the

leaves and were detected 0.5 – 2 cm from the point of inoculation in the first 2 weeks and 14 –

26 cm from the point of inoculation in the third through sixth weeks. Populations of all three

strains increased in planta from 106 CFU plant-1 initially to 108 CFU plant-1 after six weeks.

Scanning electron microscopy (SEM) was used to visualize EcWSU1R, CR42 and CR78 in

onion leaves in an attempt to determine which tissues were colonized by the bacteria. Bacterial

cells were not observed in any tissues; however, a matrix was present in the phloem of onion

leaves inoculated with strains EcWSU1R, CR42 and CR78, which was not present in water-

inoculated tissues. This research increased our understanding of the E. cloacae-onion interaction

to assist in developing management strategies that reduce Enterobacter bulb decay of onion.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS…………….…………………………………….….………….……iii

ABSTRACT………………………………………………………….…………….….….……...iv

LIST OF TABLES…………………………………………………….…………..…..…..........vii

LIST OF FIGURES ……………………………………………………………..……..…...….viii

DEDICATION……………………………………………………..……………………….…….x

LITERATURE REVIEW…………………………………….…….………..…………………...1

MATERIALS AND METHODS..…………………………….……….…..……………....….....8

RESULTS………………………………………….……………….…………………......…….16

DISCUSSION………………………………………..……………………………...…...….…..22

FUTURE DIRECTIONS………………………….………………………………...…...……...30

REFERENCES……………………………………………………….…………...……………..32

TABLES…………………………………………………………….……………...….………...40

FIGURES…………..……………………………………………….……………….…………...46

vii

LIST OF TABLES

Page

1. Bacterial strains and plasmids of Enterobacter cloacae or Escherichia coli used in these

studies…………………………………………………………………………………… 40

2. Characteristics of congo red mutants of Enterobacter cloacae.……………………….........42

viii

LIST OF FIGURES

Page

1. Selection of colonies with mini-Tn5 disruptions ……….………………………………….....46

2. Selection of Enterobacter cloacae strain ECWSU1R mini-Tn5 mutants with altered congo red

phenotype………………………………………………………………..….………………….47

3. Production biofilm by Enterobacter cloacae wild-type or mutants in glass tubes………..….48

4. Production of biofilm by Enterobacter cloacae wild-type (EcWSU1) on glass tubes ….......49

5. Biofilm of Enterobacter cloacae wild-type or mutants stained with crystal violet................50

6. Measuring the concentration of crystal violet attached to adhered biofilm of Enterobacter

cloacae wild-type or mutants using spectrophotometer cuvettes.……………….…….....…51

7. Inoculation of onion bulbs with Enterobacter cloacae..…………………...…… ……...…....52

8. of inoculation of Enterobacter cloacae wild-type or mutants for movement assay ……..….53

9. Isolation of tissue from onion leaves inoculated with Enterobacter cloacae wild-type or

mutants..…............................................................................................................................54

10. Disease symptoms resulting from artificial inoculation of strains of Enterobacter cloacae

EcWSU1R …………………………………………………….……………….…….....…..55

11. Biofilm production of Enterobacter cloacae wild-type strain EcWSU1, rifampicin-resistant

strain EcWSU1R and thirty congo red mutants from three separate experiments…..……..56

12. Biofilm production of Enterobacter cloacae of wild-type strain EcWSU1, rifampicin-

resistant strain EcWSU1R and twenty-four congo red mutants from specific gene families

compared to the wild-type strains. ………………………………………………….….…..57

13. Total movement of Enterobacter cloacae through onion leaves over 6 weeks.…...…....…58

ix

14. Net movement of Enterobacter cloacae through onion leaves. ……………….….….…...59

15. Mean total populations of E. cloacae in planta …………………………….………...…...60

16. Onion leaf anatomy………………………………………………...………….……..…..…61

17. Scanning electron microscopy (SEM) of cross sections of onion leaves colonized by strains

of Enterobacter cloacae………………………………………………….…..……..…......62

18. The matrix associated with the colonization of Enterobacter cloacae is present throughout

the phloem of inoculated onion leaves………………………….…………….……….…..63

19. Cells of Enterobacter cloacae…………………………………………………..……….…64

x

DEDICATION

I dedicate this to my parents.

Under the Bell Tree,

The flowers will be blooming

Soft, Dr. Rossetti

1

LITERATURE REVIEW

Allium cepa, more commonly known as the Spanish onion, is a biennial monocot that is a

common crop in the state of Washington. The Spanish onion was first brought to the Columbia

Basin of Washington state in the 1880s (Pelter and Sorenson 2004). Washington State’s net

revenue for storage onion was $81,852,000 in 2009, making it the second most important

vegetable crop in the state behind potato (NASS 2010). Onions are produced in the Columbia

Basin for fresh market as well as storage. Washington is the third largest producer of storage

onion in the US. Storage onions are also produced in California, Colorado, Idaho, Nevada, New

York, Oregon, and Texas (NASS 2010).

Storage onions are grown as an annual crop in the state of Washington. Onions are grown

under irrigation in the Columbia basin, with the bulb production season typically running from

March or April to September. Seeds or onion sets are grown for 6 months or until the bulbs have

fully matured. Once mature, the plants will senesce and the bulbs are harvested. At the onset of

natural senescence, irrigation is discontinued and the leaves are allowed to dry. When

approximately 80% of the onion tops are down, the onion plants are undercut and the bulbs are

cured in the field (Pelter and Sorenson 2004). The bulbs are topped to remove dry leaf tissue and

transferred to storage sheds to be cured further. This postharvest curing process involves air,

either ambient or heated, forced through the storage shed to remove excess moisture from the

onion bulbs, enabling the bulbs to form tight, dry wrapper scales and allowing the neck of the

bulb to fully dry. This process produces bulbs that will not lose a significant amount of moisture

during storage and prevents the movement of fungal and bacterial pathogens through the neck

into the bulb, thereby preventing the potential decay of bulbs in storage (Brewster 2006).

2

While plant pathogens of onion can be a problem during the growing season, postharvest

storage problems can severely impact onion bulbs in storage, decreasing the overall revenue for

growers. The major postharvest storage rots are caused by fungal pathogens, but current research

is exposing the importance of bacterial pathogens in postharvest situations. There are 26

pathogens of storage onions, 11 of which are bacterial (Schwartz and Mohan 2008). Many of the

symptoms produced by bacterial bulb rot pathogens are similar, but there are some symptoms

that are unique and specific to the causal agents. Burkholderia cepacia, gladioli pv. alliicola, and

Pantoea ananatis tend to cause water soaking, discoloration, and maceration of onion bulb tissue

and produce a foul odor (Schwartz and Mohan 2008). P. ananatis is the causal agent of center rot

of onion, which is vectored by thrips (Gitatis et al. 2003). As expected, the control of thrips is the

main means of control in areas affected by P. ananatis. Center rot is characterized by water-

soaked leaves, necks, and bulbs. Symptoms are present in both the field and storage, but P.

ananatis has not been isolated from infected onion bulbs in the state of Washington. B. cepacia

is the causal agent of sour skin, a bulb rot disease found in the field and in storage. Sour skin is

characterized as a maceration of the outer scales of the onion bulb (Schwartz and Mohan 2008).

B. gladioli pv. alliicola is the causal agent of slippery skin, another bulb rot disease that causes

maceration of the outer scales (Schwartz and Mohan 2008). Both Burkholderia species have

been shown to cause disease symptoms in Washington State (Schroeder and Humann

unpublished). In 2009, E. cloacae was described as the causal agent of Enterobacter bulb decay

in Washington state. Enterobacter bulb decay is characterized by discoloration of 1 or 2 inner

scales with no associated odor or scale maceration (Schroeder and du Toit 2009). Enterobacter

bulb decay was previously identified in Colorado (Otto and Schwartz 2000) and California

(Bishop and Davis 1990). The symptoms are latent while the bulbs are still in the field, with

3

symptoms usually only visible during storage. There is one reported case of Enterobacter bulb

decay symptoms observed in onion bulbs removed directly from the field in California; this

incident coincided with unusually high air temperatures (Bishop and Davis 1990).

While E. cloacae is present in Washington, it is challenging to identify the causal agent of

onion bulb rot without the time-consuming process of isolation and identification. Therefore,

there is little information about how much of the bulb rot present in the storage onion crop in

Washington state is caused by this particular bacterial pathogen. Favorable field conditions for

infection of onion by E. cloacae tend to include durations of high temperature (Bishop and Davis

1990). It has been shown that pure cultures of E. cloacae artificially inoculated into healthy

onion bulbs produce symptoms of Enterobacter bulb decay (Otto and Schwartz 2000; Schroeder

et al. 2009). While it is known that E. cloacae is able to cause bulb rot, it is unknown how E.

cloacae infects and colonizes onion plants, how or when the disease symptoms are produced, and

which tissues within onion are colonized by E. cloacae.

E. cloacae is a Gram-negative bacterium in the Enterobacteriaceae family (Richard 1984). It

is commonly isolated from soil, manure, plants, and the guts of mammals and insects

(Clementino et al. 2001; Jang and Nishijima 1990; Marchini et al. 2002; Richard 1984). It is a

facultative anaerobe that has the ability to survive in various environments, including on

substrates such as dry soil, water pipes, and metal and plastic medical equipment (Herson et al.

1987; Rattray et al. 1995; Poilane et al. 1993). E. cloacae can be biochemically differentiated

from other facultative anaerobes in the Enterobacteriaceae that do not produce pectinases by the

production of arginine dehydrolase (Richard 1984). Due to its ubiquitous nature, E. cloacae is a

common catheter contaminant and can be an opportunistic pathogen of immunocompromised

humans (Poilane et al. 1993; Harbarth et al. 1999; Mayhall et al. 1979; Watson et al. 2005). It

4

has been isolated from nosocomial infections in immunocompromised patients as well as animals

in meat production facilities. It has been shown that E. cloacae produces a toxin that is toxic to

Vero cells. However, the genes that code for the production of this toxin have not been

characterized (Paton and Paton 1996).

E. cloacae has been shown to be a pathogen of many plants including papaya, orchid, mung

bean sprout, macadamia, ginger, mulberry, and dragon fruit (Wick et al. 1987; Nishijima et al.

1987; Takahashi et al. 1997; Nishijima et al. 2004; Nishijima et al. 2007; Masyahit et al. 2009;

Wang et al. 2008). The most common symptoms of these diseases are wilts that are produced in

the field. However, in papaya, ginger, macadamia, and onion, E. cloacae is the causal agent of

postharvest diseases (Nishijima et al. 1987; Schwartz and Otto 2000; Nishijima et al. 2004;

Nishijima et al. 2007). Nishijima et al. (2007) showed that E. cloacae is the causal agent of dark

kernel of macadamia. It was demonstrated that in storage, when the macadamia kernel darkens as

the result of infection by E. cloacae, volatile compounds are released that have the ability to

cause the darkening in kernels other than those infected with the bacterium. In this manner, E.

cloacae can cause dark kernel symptoms in whole storage systems in Hawaii, rendering the

macadamia stored within the facility unsellable. Since macadamia is such a high-value crop, this

makes infection of the kernels by E. cloacae an important production problem. Current cultural

practices include increased air circulation in the storage facilities to reduce the concentration of

volatile gases in storage. Ginger crops infected by E. cloacae result in a spongy rot of the

rhizomes, making them unfit for the fresh market. Nishijima et al. (2004) found that E. cloacae is

associated with the rot, and that it is also isolated with healthy rhizomes. E. cloacae infects

papaya, causing an internal yellowing of immature fruit. The infected fruits will not mature,

rendering them unfit for market. It has been suggested that E. cloacae enters the papaya through

5

the floral structures via an insect vector. In this same study, it was demonstrated that hot water

treatment of papaya fruits can decrease the incidence of the disease in postharvest situations

(Nishijima et al. 1987).

Plant pathogenic bacteria utilize various mechanisms and products to colonize and cause

disease in plants. One such product, biofilm, is important for pathogenesis, virulence, and

colonization by many pathogenic bacteria (Kado 2010). Biofilms are sticky matrices produced

by bacteria to aid in colony formation and adhesion to environmental surfaces. Biofilms consist

mainly of lipopolysaccharides and exopolysaccharides, but DNA, RNA, water, and various

proteins have also been found in biofilm (Danhorn and Fuqua 2007). Biofilm production is, in

part, controlled by quorum sensing, as biofilm is used by bacterial communities for cell-to-cell

communication (Waters and Bassler 2005). In addition, it has been suggested that biofilm can

also play a role in pathogenesis. Pantoea stewartii, the causal agent of Stewart's wilt of corn,

utilizes biofilm to clog xylem vessels and disrupt the flow of water and solutes through the corn

plant, resulting in wilt (Koutsoudis et al. 2006). In P. stewartii, biofilm is also important for

quorum sensing, providing a matrix through which molecules travel between bacterial

individuals. It was shown that when genes coding for biofilm components are disrupted, quorum

sensing is severely decreased (Koutsoudis et al. 2006). Biofilm is also known to be important for

epiphytic survival. The majority of phytopathogenic bacteria need wounds or other openings in

the plant epidermis in order to enter and subsequently colonize the plant and cause disease (Kado

2010). If wounds are not present, the bacteria will remain on the plant surface or will be removed

due to environmental factors such as rain. If bacterial colonies remain on the plant surface, it is

important that the bacteria survive other abiotic stressors, such as heat and ultraviolet light.

6

Biofilm not only enables bacteria to attach to the plant surface, but also acts as a buffer from

abiotic conditions such as UV radiation, desiccation, and extreme pH (Beattie and Lindow 1999).

Biofilm was shown to be important for colonization of plant tissues by phytopathogenic

bacteria. The major components of biofilm, lipopolysaccharide (LPS) and exopolysaccharide

(EPS), have both been shown to be important for colonization of plant tissues by bacterial

phytopathogens. Lipopolysaccharide and EPS work independently of each other, but disruptions

in either have been shown to either decrease or completely disrupt the colonization of plant

tissues. Lipopolysaccharide is an important component of biofilm for some phytopathogens, as it

confers virulence (Hendrick and Sequeira 1984; Drigues et al. 1985; Kao and Sequeira 1991;

Dow et al. 1995; Newman et al. 2000). In Dickeya chrysanthemi, spontaneous mutants deficient

in LPS lost virulence in Saintpaulia plants despite maintaining normal extracellular levels of

pectinolytic and cellulolytic enzymes (Schoonejans et al. 1987). Exopolysaccharide is a sticky

saccharide produced by bacteria that aids in the attachment of bacteria to plant surfaces. In

Ralstonia solanacearum, EPS was shown to be necessary for colonization of the xylem vessels

of tomato roots. Mutants deficient in EPS were not able to access the xylem vessels and

exhibited decreased rates of epiphytic survival in comparison to the wild-type strains (Saile et al.

1997).

The impact of E. cloacae on storage onion production is currently unknown, but it has been

shown that this bacterium is frequently isolated from onion bulbs displaying various decay

symptoms (Schroeder and Humann, unpublished). This suggests that E. cloacae may have a

critical role in the development of bacterial bulb rot of onion. While it is known that E. cloacae

is commonly isolated from even symptomless onion bulbs (Cother and Dowling 1986), it is

unknown as to how E. cloacae infects onion, colonizes the bulb scale, and produces bulb rot.

7

Since very little is known about the genetics of E. cloacae and the components necessary for

production of bulb rot in onion, genes that contribute to the E. cloacae-onion interaction need to

be determined. Once these genes are identified, further research can be performed to better

understand the E. cloacae-onion interaction with the expectation that this will lead to the

development of disease management strategies for control of this bacterial bulb rot pathogen.

8

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. Several Enterobacter cloacae and

Escherichia coli strains were used in this study (Table 1). Enterobacter cloacae strains were

cultured at 28°C in Luria Bertani (LB) broth or on LB agar (Sambrook 1989). Escherichia coli

strains were cultured at 37°C in LB broth or on LB agar amended with the appropriate antibiotics

as necessary. Selection of mini-Tn5 mutants of E. cloacae with altered congo red phenotypes

was conducted using LB agar amended with 0.2 g L-1 congo red dye (Thermo Fisher Scientific,

Waltham, MA). For biofilm assays, bacterial strains were grown in super optimal broth amended

with 10ml L-1 glycerol (SOBG; Sambrook 1989). Bacterial strains were stored in 20% dimethyl

sulfoxide (Thermo Fisher Scientific, Waltham, MA, USA) at -80°C in cryogenic tubes and in 96-

well microtiter plates (Cellstar, Grenier Bio-one, Monroe, NC, USA) for long term storage.

Selective media were amended with antibiotics (Thermo Fisher Scientific, Waltham, MA) as

required at the following concentrations per liter: 25 µg of ampicillin (amp25), 100 µg of

rifampicin (rif100), and 50 or 100 µg of kanamycin (kan50 or kan100, respectively).

Mutagenesis. Mini-Tn5 mutagenesis was used to produce a mutant library with random gene

disruptions that were screened to identify E. cloacae mutants with differences in polysaccharide

production. Mini-Tn5 mutants were obtained by mating Enterobacter cloacae strain EcWSU1R

with E. coli SM10 (λpir), which houses the pUTKm plasmid (Herrero 1990), using a modified

mating procedure described by Miller and Mekalanos (1988). Briefly, the strains were inoculated

into 5ml aliquots of LB broth and incubated overnight at 28°C with agitation. A 1ml aliquot of

each strain was harvested by centrifugation, resuspended in an equal volume of sterile deionized

water, and the suspensions were mixed together in a 1:1 ratio. A piece of sterile nitrocellulose

9

was placed onto LB agar and a 100 µl aliquot of the 1:1 mating mixture was placed onto the

nitrocellulose. The plate was incubated for 12 hours at 28°C. The nitrocellulose piece was

transferred using sterile forceps into a 15ml Falcon tube (BD, Franklin Lakes, NJ) containing

1ml sterile water. The tube was shaken on a vortexer for 20 seconds to dislodge the cells and a

100 µl aliquot of the cell suspension was spread-plated onto LBrif100kan100 agar and incubated

overnight at 28°C (Fig. 1). Individual colonies were then streaked onto LBrif100kan50 agar and

grown overnight at 28°C. Single colonies were patched onto LBrif100kan50 agar in rectangular

single well OmniTray plates (Nalge NUNC, Rochester, NY, USA) in a 96-well format.

Congo red screen. It is well established that polysaccharide production is important in the

bacterial-plant interaction. Therefore a traditional microbiological dye screen was utilized to

identify polysaccharide mutants (Hahn 1966; Ayers et al. 1979; Teather and Wood 1982).

EcWSU1R mini-Tn5 strains were screened by plating strains on LB medium amended with 0.2%

(w/v) congo red dye. Specifically, mini-Tn5 strains of EcWSU1R were transferred to LB congo red

0.2% agar in single well square OmniTray plates using a 96-prong replicator (Product # V142, V

& P Scientific, Inc., San Diego, CA, USA). Plates were incubated for 2 days at 28°C. Mutants

with altered phenotypes were identified visually by colony color (Table 1; Fig. 2). Colonies that

were lighter or darker red than EcWSU1R were selected, streaked on LB congo red 0.2%, and

incubated as described above to double check the phenotype.

Arbitrary PCR and sequencing. The DNA sequence of regions flanking mini-Tn5 insertions in

the congo red mutants was obtained by a revised arbitrary PCR from Knobloch et al. (2003) and

sequencing. Genomic DNA was extracted using the Promega Wizard genomic DNA purification

10

kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions.

Two PCR reactions were necessary for amplification of the edge of the kanamycin cassette and

the EcWSU1R genomic sequence adjacent to the cassette. Each reaction used one mini-Tn5-

specific primer (Schroeder and Humann, unpublished) and one arbitrary primer (Griffitts and

Long, 2008). The components of the first PCR reaction were as follows: 0.75 µM dNTPs, 50

pmol of the mini-Tn5 primer mini-Tn1A (5’-CAGTCTGTGTGAGCAGGGGAATTG-3’)

(Schroeder and Humann, unpublished), 50 pmol of ARB1-A (Griffitts and Long, 2008), 1 X

PCR reaction buffer, 1.25 units Go Taq polymerase (Promega Corporation, Madison, WI, USA),

and 500 ng of the template DNA in a 50 µl reaction volume. The program of the first arbitrary

PCR was as follows: 94°C for 3 minutes; 6 cycles of 94°C for 20 seconds, 30°C for 20 seconds,

and 70°C for 1 minutes; 28 cycles of 94°C for 20 seconds, 42°C for 20 seconds, and 70°C for 1

minutes 30 seconds; 70°C for 3 minutes, and 15°C forever. The components for the second PCR

reaction were as follows: 1 x PCR buffer, 0.75 µM dNTPs, 50 pmol of the mini-Tn5 primer

mini-Tn2A (5’-GCTTGCTCAATCAATCACCGGATC -3’) (Humann and Schroeder,

unpublished), 50 pmol of ARB2 (Griffitts and Long, 2008), 1.25 units Go Taq polymerase, and

500 ng of the template DNA. The PCR conditions for second round were as follows: 94°C for 3

minutes; 30 cycles of 94°C for 20 seconds, 52°C for 20 seconds, and 70°C for 90 seconds; 70°C

for 3 minutes; and 15°C forever. The products from the second PCR reaction were sent to ELIM

Biopharmaceuticals (Hayward, CA) for GC-rich sequencing. The components of each

sequencing reaction were as follows: 100 ng of template DNA, 8 pmol mini-Tn2A primer, and

8.5 µl of sterile distilled water in a final volume of 15 µl. The mini-Tn5 sequence was found for

each sample and the adjacent nucleotide sequences were matched to the closest bacterial

sequences using nucleotide BLAST (http://www.ncbi.nlm.nih.gov/blast) (Altschul et al. 1990).

11

Biofilm Assays. The Biofilm production by the E. cloacae EcWSU1R mini-Tn5 mutants with

altered congo red phenotypes was quantified in glass tube assays in a modified protocol from

Yap et al. (2005). Bacterial cells were transferred to 5ml of LB broth and incubated at 28°C

overnight with agitation. A 1.25ml aliquot of each strain was harvested by centrifugation in a

1.7ml microcentrifuge tube. The cells were washed once with sterile deionized water and diluted

to an OD600 = 0.3 (~1 x 108 CFU ml-1). A 100µl aliquot of the bacterial suspension was added to

1.9ml SOBG in a sterile glass tube (13 x 100 mm, Thermo Scientific, Waltham, MA, USA).

There were 5 replicate tubes per treatment. The tubes were placed into racks in a randomized

complete block design and the racks were placed into a 14L storage box (Sterilite Corporation,

Townsend, MA, USA), covered, and incubated at 23°C. After 6 days (Fig. 3), the culture was

decanted and 2ml of 0.85% NaCl was added to each tube, shaken lightly, and decanted (Fig. 4).

The biofilm that adhered to the glass tube was then stained by adding 2ml 0.1% crystal violet.

After 15 minutes, the crystal violet solution was aspirated and the tube was rinsed 5 times with

deionized water (Fig. 5). The tube was allowed to sit for 30 minutes and residual water was

aspirated. To release the crystal violet stain from the biofilm, 2ml of 95% ethanol was added to

the tube. After 5 minutes, the tube was shaken lightly and the ethanol and crystal violet solution

was transferred to a nonsterile clear-sided cuvette (USA Scientific, Ocala, FL, USA) (Fig. 6).

The OD570 was read using a Spectramax PLUS384 (Molecular Devices, Silicon Valley, CA, USA)

with 95% ethanol as the reference. The upper limit of detection for the Spectramax PLUS384 was

OD570=4.0. If samples exceeded the limit, they were diluted and concentration calculated

accordingly.

12

Pathogenicity assays. Bacterial cells were transferred to 5ml LB broth and incubated at 28°C

overnight with agitation. A 1.25ml aliquot of each strain was harvested by centrifugation in

1.7ml microcentrifuge tubes. The cells were washed once with sterile deionized water and

diluted to an OD600 = 0.3 (~108 CFU ml-1). The dry scales of cv. ‘Vaquero’ onion bulbs were

removed and the bulbs were soaked in a 10% bleach solution for 2 minutes, rinsed with water,

and dipped into 70% ethanol. The bulbs were allowed to dry on paper towels in a biosafety

cabinet (Nuaire, Inc., Plymouth, MN, USA). Once dry, the bulbs were inoculated at the shoulder

with ~5 x 107 CFU bacteria using a 2-inch 23-gauge hypodermic needle and a 1ml syringe (Tyco

Healthcare Group, Mansfield MA, USA) (Fig. 7). The point of inoculation was circled with a

black permanent marker, each bulb was sealed in 6 in. x 9 in. sampling bag, and the bulbs were

placed into an incubator at 30°C in a randomized complete block design for 2 weeks. Non-

inoculated and water-inoculated bulbs were used as controls. There were 5 replications per

treatment. The bulbs were then destructively harvested by cutting each bulb in half along the

plane of inoculation. The percentage of surface area showing disease symptoms was determined

using a visual rating of 0-100%. Statistical analyses were performed as described below.

Plant growth conditions. Plants were grown under greenhouse conditions at the Plant Growth

Facilities at the Washington State University Pullman campus. The photoperiod was 14.5 hr day-

1, with 400 Watt high pressure sodium lamps supplementing when needed. ‘Vaquero’ onion

seeds (Nunhems, Parma, ID, USA) were planted in LC1 Sunshine mix (Sun Gro Horticulture

Inc., Bellevue, WA) in 72-cell trays (ITML Horticultural Products Inc., Middlefield, OH). After

the seedlings were 8 weeks of age, they were transplanted into 6 in. square pots with LC1

Sunshine mix, placed in plant trays without holes, and watered from below. Plants were amended

13

with fertilizer every 3 days. Peter’s fertilizer (J.R. Peters Inc., Allentown, PA, USA) was used at

a concentration of 200 ppm nitrogen, 100 ppm phosphate, and 200 ppm potash, along with a

soluble trace element mix and 2 ppm of chelated iron (J.R. Peters Inc., Allentown, PA, USA).

Seedlings and transplanted onions received 30ml and 250ml fertilizer, respectively, every three

days.

Population dynamics in onion leaves. Bacterial cells from E. cloacae strains EcWSU1R, CR42,

and CR78 were transferred to 5ml LB broth and incubated at 28°C overnight with agitation. A

100µl aliquot of each overnight culture was added to 125ml LB broth and incubated at 28°C

overnight with agitation. The cells of each strain were harvested by centrifugation, washed once

in 1ml sterile water, and diluted to an OD600=0.3 (~108CFU ml-1). Bacterial cells were

concentrated by centrifugation and resuspended in 5ml sterile water (~1010CFU ml-1). A 50µl

aliquot was added to each well of a 96-well microtiter plate (Grenier Bio-one, Monroe, NC,

USA). A flame-sterilized probe was inserted into one well perpendicular to the bottom of the

microtiter plate until the tip reached the bottom of the well; a separate well of inoculum was used

for each plant. A mark was made with a permanent marker on the inside of the third youngest

leaf 1 cm above the whorl of the onion plant. The probe that had been dipped into the bacterial

inoculum was then used to wound the marked area by inserting the tip into the plant tissue at the

marked point (Fig. 8). On the day of inoculation and weekly for 6 weeks thereafter, the entire

inoculated leaf was removed from the plant. Tissue from the point of inoculation, as well as from

above and below the point, was destructively harvested in 1cm sections using a flame-sterilized

number 4 cork borer (VWR International Inc., West Chester, PA, USA) (Fig. 9). These samples

were transferred to sterile pre-weighed microcentrifuge tubes using flame-sterilized forceps,

14

weighed, and macerated in 600 µl of 0.125 M phosphate buffer (161ml 0.50 M K2HPO4, 89ml

0.50 M KH2PO4, 750ml sterile water) using sterile pellet pestles (ISC BioExpress, Kaysville,

UT, USA). The suspension was diluted as needed and spiral dilution plated (Model 500A, Spiral

Biotech, Inc., Bethesda, MD, USA). After 2 days, the colonies were counted using a spiral-plate

field and populations were determined.

Fluid velocity measurements. The rate of movement of fluids in onion leaf xylem was

evaluated. Onion plants at 9, 17, and 23 weeks of age were transversely dissected at the neck and

the leaf sections were placed upright in a 1% (w/v) crystal violet solution. There were three

replications per plant age. After 24 and 48 hours, the movement of crystal violet through the

onion tissue was measured using a ruler. This was repeated three times.

Scanning electron microscopy. Leaf tissue was excised from onion plants for visualization by

scanning electron microscopy (SEM). The tissue at and around the point of inoculation was

harvested from 3 water-inoculated and 3 EcWSU1R-inoculated plants and fixed overnight in a

solution of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (1.38 g

KH2PO4, 1.42 g K2HPO4 100ml double deionized water-1). The samples were rinsed twice in 0.1

M phosphate buffer for 10 minutes and twice in deionized water for 5 minutes. The samples

were placed into liquid nitrogen, freeze fractured (Koczan et al. 2009), and freeze dried (Virtis

Co., Gardiner, CA, USA) overnight. The freeze-dried samples were adhered to PELCO tabs that

were fixed to aluminum specimen mounts (Ted Pella, Redding, CA, USA) and gold sputter-

coated using a Hummer V sputter coater (Technics Inc., Alexandria, VA, USA). Freeze-dried

samples that were longitudinally sectioned were not freeze fractured, but placed gently between

15

two pieces of Scotch tape (3M, Inc., St. Paul, MN, USA) and the tape was peeled apart so as to

remove the epidermal layer and reveal the internal tissues. The longitudinal samples were

adhered to PELCO tabs and gold sputter-coated as described above. Overnight cultures of

EcWSU1R, CR42, and CR78 were prepared for microscopy in the same manner as onion leaves.

The sputter-coated samples were viewed with a Hitachi S-570 scanning electron microscope

(Hitachi Ltd., Tokyo, Japan). Four mounts were used per plant and at least 10 views were

observed for each mount. Images were taken that were representative of these views. Bacterial

cells of EcWSU1R, CR42, and CR78 were cultured in LB broth overnight, centrifuged in

microcentrifuge tubes, and mounted as above.

Statistical analysis. Statistical analyses were performed using SAS Version 9.2 (SAS Institute,

Cary, NC, USA). Data were tested for normality using Shapiro-Wilks test using PROC

UNIVARIATE. Bartlett's test was used to determine equal variances and Fisher's Least

Significant Difference (LSD) was run for ANOVA testing using PROC GLM.

16

RESULTS

Mini-Tn5 mutants. Mini-Tn5 mutagenesis yielded E. cloacae mutants with differences in

polysaccharide production (Table 2). A total of 9,600 isolates from the E. cloacae EcWSU1R

mini-Tn5 library were screened with LB agar amended with congo red and 99 mutants were

identified as having a darker or lighter phenotype on LB congo red 0.2% (Table 1). There were 48

isolates that produced a darker colony color compared to the wild-type strain EcWSU1R and 51

isolates that produced lighter colony color than the wild type (Table 2). The number of congo red

EcWSU1R mutants obtained represents approximately 1.03% of the current library. These

isolates were plated onto M9 and NBY agar to visually screen for auxotrophs and unique colony

morphologies, respectively. All 99 isolates grew on M9 agar, suggesting that none of the isolates

were auxotrophs. The mutants CR3, CR6, CR9, CR19, CR56, CR59, and CR66 produced

morphologies that were more mucoid on M9 than the reference strains EcWSU1 and EcWSU1R,

while CR18 produced a drier colony morphology. Mutant CR6 was more mucoid on NBY agar

than EcWSU1R, while CR18 was drier on NBY than EcWSU1 and EcWSU1R (Table 2). The

congo red color of isolates grown on NBY or M9 did not correlate with the colony morphology

(Table 2).

Arbitrary PCR of the mutant strains amplified the edge of the kanamycin cassette from the

mini-Tn5 insertion and the nucleotides from the genome adjacent to the cassette. These

fragments were sequenced from the CR mutants and 24 of the 99 CR mutants either produced no

product in the arbitrary PCR or sequencing of the arbitrary PCR reaction product did not result in

a usable sequence. These 24 mutants were then removed from further studies. Sequence analysis

for the arbitrary PCR reaction products obtained from 75 CR mutants by using nucleotide

BLAST (Altschul et al. 1990) identified the gene found in Genbank

17

(http://www.ncbi.nlm.nih.gov/) with the closest identity to sequence from the arbitrary PCR

reaction products. It was found that 26 unique genes were disrupted, and these genes had close

identity to genes originating from members of the Enterobacteriacae including Enterobacter

spp., Cronobacter spp., Salmonella spp., Escherichia spp., and Citrobacter spp. (Table 2). The

sequences obtained from the arbitrary PCR reaction products amplified from the CR mutants

ranged from 64 to 1036 nt in size. These sequences exhibited 71-91% identity across sequences.

There were nine isolates with disruptions in genes with close identity to peptidase T genes (CR4,

CR28, CR30, CR32, CR33, CR34, CR37, CR39, CR40), five isolates with disruptions in genes

that were similar to aspartokinase/homoserine dehydrogenase I genes (CR9, CR15, CR18, CR24,

CR44), and 19 isolates with disruptions in diguanylate cyclase/phosphodiesterase genes (CR7,

CR22, CR23, CR45, CR46, CR51, CR52, CR53, CR58, CR62, CR63, CR65, CR74, CR72,

CR76, CR78, CR79, CR85, CR90). Twelve of the CR mutants (CR11, CR14, CR15, CR18,

CR24, CR25, CR44, CR48, CR49, CR50, CR54, and CR98) did not have a mini-Tn5 sequence

present, suggesting that these strains were spontaneous mutants (Table 2).

Biofilms and Pathogenicity. Pathogenicity trials were performed with the selected mutants and

reference strains EcWSU1 and EcWSU1R using 5 repetitions. All of the mini-Tn5 isolates of E.

cloacae produced the characteristic discoloration of onion scale tissue associated with

Enterobacter bulb decay. On average, EcWSU1 was able to rot 25.5% of the onion bulb surface

area and EcWSU1R was able to rot 28.5% of the onion bulb surface area. The reference strains

were not found to be significantly different from each other. The average amount of Enterobacter

bulb decay produced by the CR mutants ranged from 10-30% (Table 2, Fig. 10). Due to the

variance within treatments, not one of the CR mutants was statistically different from the wild-

18

type strains (P = 0.2395). The Bartlett’s test showed that the data were equal (P = 0.0017) and

the Shapiro-Wilks test found the data to be normal (P < 0.0001).

Biofilm Quantification. Solid-surface-associated biofilm production by EcWSU1R, EcWSU1,

and the CR mutants was measured in glass tubes. Significant differences between the CR

mutants and EcWSU1 and EcWSU1R were observed (P < 0.0001). EcWSU1 produced an

average solid-surfaceassociated biofilm of OD570 = 5.22 (Table 2, Fig. 5), while EcWSU1R

produced an average biofilm of OD570 = 5.15. There were 22 CR mutants that displayed

statistically lower average solid-surface-associated biofilm production in comparison to

EcWSU1R and EcWSU1, ranging from OD570 = 0.40 to 2.85 (P <0.0001) (Table 2), while CR42

produced an average surface-adhered biofilm of OD570 = 5.18. This production of biofilm was

not statistically different in comparison to the wild-type strain or EcWSU1R (Fig. 11).

Representative strains were chosen because of the disruptions they carried in the respective gene

families mentioned above and were evaluated for the production of solid-surface-associated

biofilm production. It was found that all of the strains with disruptions in genes encoding

peptidase T produced significantly less biofilm than either of the wild-type strains (P < 0.0001).

For the CR mutants carrying disruptions in genes encoding aspartokinase/homoserine

dehydrogenase, 2 of the 3 CR mutants were significantly reduced in their production of solid-

surface-associated biofilm production (P < 0.0001). However, only 5 of the 16 strains of the CR

mutants with disruptions in genes coding for diguanylate cyclase/phosphodiesterase proteins had

significantly decreased biofilm production (P < 0.0001). The data from all trials were found to be

normal (P < 0.0001) and the variances equal (P < 0.0001) (Fig. 12).

19

Population dynamics. When EcWSU1R was inoculated into the leaves of onions, the bacteria

progressed from the point of inoculation both toward the leaf tip as well as toward the bulb. The

same result was found for both CR42 and CR78. The furthest point out from the point of

inoculation in which bacterial colonies were quantifiable for plants inoculated with EcWSU1R

was determined to be an average of 0.0, 0.5, 1.0, 12.5, 9.0, 16.5, and 24.0 cm from the point of

inoculation weekly for 6 weeks. For plants inoculated with CR42, the point at which bacterial

colonies were quantifiable was determined to be 0.0, 1.0, 3.0, 15.0, 13.5, 17.0, and 24.0 cm from

the point of inoculation weekly for 6 weeks, respectively. Finally, for plants inoculated with

CR78, the point of at which bacterial colonies were quantifiable was determined to be 0.5, 2.0,

2.0, 16.5, 14.5, 17.0, and 27.0 cm from the point of inoculation for 0, 1, 2, 3, 4, 5, and 6 weeks,

respectively. In all of the treatments, a greater distance of movement was recorded in the third

week in comparison to the distances from the point of inoculation observed in the first and

second weeks after inoculation (Fig. 13). While the distance at which the bacteria were detected

did not increase in the fourth week, the distance did increase 4-7 cm in the fifth and sixth weeks

(P < 0.0001, α = 0.05) (fig. 14). However, by the sixth week, it was possible to recover bacterial

populations from all three strains. The populations for all three treatments increased at least 3-

fold from the second week to the third week. For EcWSU1R, 5.81 log CFU plant-1 were

recovered on the day of inoculation, while 6.45 log CFU plant-1 was recovered in the first week,

5.69 log CFU plant-1 in the second week, 5.97 log CFU plant-1 in the third week, 6.77 log CFU

plant-1 in the fourth week, 7.17 log CFU plant-1 in the fifth week, and 8.03 log CFU plant-1 in the

sixth week. For CR42, the total log CFU plant-1 recovered on day of inoculation was 5.22, while

6.59 log CFU plant-1 was recovered in the first week, 6.01 log CFU plant-1 in the second week,

6.12 log CFU plant-1 on the third week, 6.35 log CFU plant-1 on the fourth week, 6.57 log CFU

20

plant-1 on the fifth week, and 7.26 log CFU plant-1 in the sixth week. For CR78, the total log

CFU plant-1 recovered on the day of inoculation was 5.26 log CFU per plant, while 5.43 log CFU

plant-1 was recovered on the first week, 6.07 log CFU plant-1 in the second week, 7.27 log CFU

plant-1 in the third week, 6.68 log CFU plant-1 in the fourth week, 7.79 log CFU plant-1 in the

fifth week, and 8.40 log CFU plant-1 in the sixth week (Fig. 15). However, there were no

significant differences among the populations of the three different treatments (P=0.289, α

=0.05).

Colonized tissues. While movement both towards the tip of the leaf as well as toward the bulb

was noted for E. cloacae, it was unclear which tissues were being colonized. The rate of fluid

movement through xylem was assayed so that it could be compared to the rate of movement of

E. cloacae. The rate of fluid movement of crystal violet in onion plants did not differ with age (P

= 0.0688). The data were normal (P < 0.0001) and the variances equal (P < 0.0001); however,

there was a trend for the rate of movement to be greater in older onion plants compared to

younger. The average rate of movement of crystal violet dye was 5 cm day-1in an onion plant that

was 9 weeks of age. At 17 weeks, the average rate of movement was 14 cm day-1, and at 23

weeks, the average rate was 19 cm day-1. The rate of movement of crystal violet was faster than

the 4 cm/week average rate observed for bacterial colonization from the assay measuring

movement of E. cloacae in planta (Dossey, data not shown).

Scanning electron microscopy was used to view onion leaf tissues that were inoculated with

EcWSU1R, CR42, and CR78. Freeze fracturing was used so as not to disturb cell contents and to

preserve bacterial colonies. Freeze drying was also used, since it was found that

hexamethyldisilazane, a reagent commonly used in SEM for plant fixation, produced more

21

artifacts in onion and would also remove cell contents (Dossey, data unpublished). Plant tissue

of water-inoculated onion leaves were used to identify the various tissue types present in onion

leaves (Fig. 16). A matrix was present in the phloem of onion leaves inoculated with E. cloacae

that was not present in water-inoculated samples (Fig. 17). This matrix was continuous through

the phloem of green leaf sections that were shown to have recoverable bacterial colonies (Fig.

18). While this matrix was present in the phloem of plants inoculated with EcWSU1R and CR42,

plants that were inoculated with CR78 appeared to have a decreased amount of matrix in the

phloem, but this was not quantified (Fig. 17). For all inoculated plants, however, it appeared that

there was more matrix observed at the point of inoculation than in the area of the leaf that was

newly colonized. While the matrix was present in inoculated samples, there were no bacterial

cells observed in the matrix or in onion leaf tissues (Fig.17, Fig. 18). Pure bacterial colonies of

the strains were visualized to confirm the size and structure of the cells. EcWSU1R, CR42, and

CR78 all had rod-shaped cells that were approximately 1 µm in length. These cells tended to stay

connected to each other and were commonly viewed in a biofilm matrix (Fig. 19). However, no

bacterial cells were observed in any of the onion tissue samples inoculated with EcWSU1R,

CR42, or CR78 using SEM.

22

DISCUSSION

The effect of Enterobacter cloacae on Enterobacter bulb decay and its impact on onion

production in the State of Washington is unclear. E. cloacae is frequently isolated from decaying

onion bulbs (Schroeder and Humann, unpublished) suggesting that the bacterium plays a role in

the development of bulb rot. Screening the 9,600-member E. cloacae EcWSU1R mini-Tn5

mutant library with congo red dye identified 99 mutants (1.03% of the whole library) with

potential disruptions in polysaccharide production, cell-membrane components, or biofilm

production. Sequencing the nucleotide regions around the insertion determined that 73 of these

mutants (0.76% of the total library) were unique gene disruptions. These data indicate that 0.5-

1.5 % of the mutants from the library are unique in one aspect of biology, suggesting that there

are few repeated gene disruptions (de Lorenzo, 1990).

A biofilm assay was developed to measure the amount of biofilm produced by the wild-type

E. cloacae (EcWSU1), the rifampicin resistant strain of E. cloacae (EcWSU1R) and the CR

mutants. Previous assays by Rinaudi et al. (2006) used sterile 96-well polystyrene microtiter

plates to quantify biofilm production in Sinorhizobium meliloti. This protocol was unsuccessful

for quantification of biofilm production by E. cloacae. An alternate protocol by Yap et al. (2005)

using glass tubes was modified and, using this protocol, it was possible to quantify the solid-

surface-associated biofilms produced by EcWSU1, EcWSU1R, and the CR mutants. In addition

to producing a biofilm that adhered to glass surfaces, all of the E. cloacae strains analyzed

produced pellicles on the surface of the media that would detach and fall to the bottom of the

tubes after 4-5 days of static culturing. It was not possible to measure the mass of the pellicle, as

it was so loosely associated that it would disintegrate when the culture medium was decanted. Of

23

the 73 CR mutants, 22 were determined to be biofilm mutants producing 30-80% less solid-

surface-adhered biofilm than did the reference strain EcWSU1R.

The ability of bacteria to colonize plant tissues can be impacted by the ability to produce a

biofilm. A functional pigB gene, which codes for a potential regulatory protein that facilitates the

production of xanthomonadin and EPS in Xanthomonas campestris pv. campestris, has been

shown to be necessary for epiphytic survival and infection of corn (Poplawsky and Chun 1997;

1998). In addition, strains of Ralstonia solanacearum with splice overlap extension PCR

disruptions in the genes pehA, pehB, pehC, pme, egl, cbhA, and aphA-3, which code for EPS

production, were unable to colonize xylem tissues of tomato (Saile et al. 1997).

Lipopolysaccharide has also been implicated as an important part of the pathogenic interaction of

bacteria and plants. While other pathogens have been shown to utilize EPS and biofilm for

pathogenicity, it is currently unknown as to whether it is necessary for the E. cloacae-onion

interaction. To determine if EPS or biofilm is an important aspect of pathogenicity in this

specific interaction, the CR mutants and wild-type strains were inoculated into onion bulbs. After

2 weeks at 30°C, the bulbs were evaluated for the percent of cut onion bulb surface exhibiting

symptoms. It was found that the CR mutants produced the same amount of disease compared to

the reference strains EcWSU1 and EcWSU1R. While there was a variation in disease symptoms

of 5-10% of mutant treatments in comparison to the reference strains, there was too much

variability within treatments for significant differences among treatments to be observed. The

onions used for these experiments had been in storage for 4 months, which may have contributed

to this variation, as onions tend to age in storage systems, impacting their postharvest health

(Brewster, 2006).

24

Multiple genes contribute to biofilm production, EPS, and LPS in various genera of bacteria

(Ausmees et al. 2001). The majority of CR mutants with a decreased ability to produce biofilm

had mini-Tn5 insertion in genes encoding for diguanylate cyclase, aspartokinase, or peptidase T

proteins. Diguanylate cyclase is a regulatory protein with multiple functions. It has been shown

that genes coding for diguanylate cyclase production are important for multicellular behavior

(Ausmees et al. 2001) as well as regulating genes involved in virulence (Tamayo et al. 2007).

Aspartokinase is another enzyme that has been shown to be important for virulence in

bacterial pathogens. When the gene metL, which codes for the aspartokinase protein, was

disrupted in Salmonella typhimurium, the virulence in mouse subjects showed a marked decrease

(De Groote et al. 1997). It has also been shown that metL is actively used by Salmonella sp.

during intestinal infections in pigs (Huang et al. 2007) and is important for the infection to occur.

Since aspartokinase genes have been shown to be important in animal pathogenesis, homologous

genes in plant pathogens may also be important in plant pathogenesis. De Groote et al. (1996)

noted that, when the metL gene in Salmonella typhimurium was disrupted, virulence was

attenuated in mice and complementation of the gene restored full virulence. To date,

aspartokinase research has only occurred in animal systems; however, this research suggests that

aspartokinase may be important in the E. cloacae-onion interaction.

Peptidase T was the last family of genes that was found to be commonly disrupted in the CR

mutants. Peptidase T is a tripeptide enzyme. It cleaves tripeptides into monopeptides. Currently,

there is no known function for pathogenicity in plant-pathogen interactions with peptidase T;

however, further research may be able to explain the decreased biofilm phenotype that was noted

with peptidase T-disrupted mutants and their role in the plant-pathogen interaction.

25

While the CR mutants assayed did not show differences in the pathogenicity in onion, E.

cloacae may still utilize biofilms to facilitate the disease progress in postharvest situations, as

only 22 unique genes were actually assayed. Selecting another set of mini-Tn5 mutants to assay

using another screen or a high-throughput assay may be useful in identifying other genes that

may have important implications for disease progress.

The movement of E. cloacae was found to be bidirectional in onion leaves. Other plant

pathogens, such as Erwinia amylovora, move unidirectionally through hosts. E. amylovora

colonizes xylem vessels of apple and pear, and thus moves with the solutes within the xylem

unidirectionally toward the meristematic region (Koczan et al. 2009). Ralstonia solanacearum

also utilizes xylem to colonize and spread through tomato. Vasse et al. (1995) demonstrated that

R. solanacearum enters into roots of tomato via wounds and colonizes the xylem, where it then is

transported along with solutes. The bidirectional movement of E. cloacae suggests that when it is

colonizing the onion leaf, it is not being transported in the xylem.

While it was evident that E. cloacae moves bi-directionally through onion leaves, it was

unclear as to which tissues it as it moves through leaves to the bulb. Koczan et al. (2009) used

SEM to visualize Erwinia amylovora infections of pear, noting that E. amylovora was localized

in xylem vessels with the aid of biofilm. The test of liquid velocity within xylem vessels showed

that the xylem transports liquids very quickly in comparison to the rate of movement of

detectable E. cloacae populations. Onion leaves that were placed in a crystal violet solution

showed that the xylem of onion leaves transports solutions at an average rate of 5 cm a day,

while the fastest movement of E. cloacae during any week was 1.38 cm a day. The movement of

liquid through xylem is unidirectional, with solutes moving toward the tips of leaves. Erwinia

amylovora colonizes xylem vessels in apple and pear, and it has been shown that E. amylovora

26

will only move in a unidirectional manner. In contrast to E. amylovora, movement of E. cloacae

in onion leaves was shown to be bidirectional. This suggests that it is very unlikely that E.

cloacae is colonizing the xylem vessels. In addition, SEM studies determined that a matrix was

present in the phloem vessels of onion leaves inoculated with E. cloacae. In particular, this

matrix was present in phloem vessels of onion leaves inoculated with EcWSU1R, CR42, and, to

a lesser extent, with the biofilm underproducer CR78. This matrix was not present in phloem

vessels of onion leaves inoculated with sterile water or observed in phloem vessels from onion

tissue that was not infected with E. cloacae. Bacterial cells were not observed in any of the

tissues analyzed by scanning electron microscopy. Bruton et al. (1998) found a similar result

when investigating cucurbit yellow vine disease in watermelon and squash. While a matrix was

observed in the phloem vessels of watermelon and squash when inoculated with Serratia

marcescens, no bacterial cells were observed when the tissues were visualized using SEM.

However, when transmission electron microscopy was used to visualize the inoculated tissues,

cells of Serratia marcesens were present in the sieve elements of symptomatic squash and

watermelon. This difference can be explained because SEM scans the surface of a large fragment

of tissue, whereas TEM visualizes samples in cross-section. It is possible that bacteria are located

within the matrix present in the SEM, and cross-sections of phloem vessels visualized with TEM

would be the next logical step in investigating the E. cloacae-onion interaction. The matrix found

in the phloem vessels could be produced by the plant in response to the presence of the bacteria,

as it was only located in areas of onion leaves that were known to have recoverable bacterial

populations. However, this matrix could also be produced by the bacteria. The biofilms

visualized from overnight shake cultures do not have the same appearance as the matrix found in

27

phloem vessels; however, until further biochemical tests are performed to better understand what

the matrix is, there is a possibility that it is produced by the bacteria.

The populations of EcWSU1R, CR42, and CR78 all increased 100-fold from the day of

inoculation to 6 weeks out. These populations were quantified as an average of 106 CFU per

plant on the day of inoculation to an average of 108 CFU per plant at the sixth week. All three

strains showed similar results. There were no disease symptoms observed on the onion leaf

throughout the duration of the experiments (data not shown). In addition, inoculation of E.

cloacae to tobacco plants did not result in a hypersensitive response (data not shown), which

suggests that E. cloacae does not utilize a type III secretion system (Alfano and Collmer 2004)

during pathogenesis. An incompatible interaction between a bacterium and a plant is usually

noted by an exponential increase in bacterial populations followed by the plant’s defenses

recognizing the bacteria and inducing programmed cell death. In a incompatible reaction of a

Pseudomonas syringae pv. phaseolicola wild type strain inoculated on a non-host, such as

tobacco, exponential growth up to 108-109 CFU g-1 occurred before a hypersensitive response

was initiated. Bacterial populations in a disease interaction will initially be at a low level

followed by a logarithmic increase in bacterial populations and symptom development (Lindgren

et al., 1986). Inoculum levels as low as 102-104 CFU ml-1 P. syringae pv. phaseolicola will

eventually result in disease in red kidney bean, a compatible host (Lindgren, 1986). A latent

interaction in which bacterial pathogens do not cause disease symptoms, but survive within the

plant is characterized by relatively consistent bacterial populations that never reach a population

that has the ability to produce either disease or a hypersensitive response. Pectobacterium

carotovorum ssp. carotovorum and Pectobacterium atrosepticum have a latent stage in potato.

During this dormancy, populations of these pathogens may fluctuate, but never reach over 107

28

CFU g-1 in the lenticils, which would initiate disease symptoms (Pérombelon, 1992; Pérombelon

et al., 1979). Ralstonia solanacearum can be present as a latent infection in potato. During the

latent phase of the infection, R. solanacearum can survive at populations of 108 CFU g plant

tissue-1 (Swanson et al. 2005). There are no symptoms present in the green leaves despite

bacteria being present, which would suggest a latent infection. However, latent infections will

eventually become symptomatic infections (Pérombelon et al., 1979). None of the onion plants

that were inoculated with E. cloacae in the greenhouse produced Enterobacter bulb decay in the

onion bulbs once they were harvested and incubated at 30°C for 1 month (data not shown).

Previous association of the onset of Enterobacter bulb decay symptoms with high temperature

(Bishop and Davis 1990) suggests that this assay needs to be developed further to investigate the

role of temperature in the development of Enterobacter bulb decay.

This research was conducted to increase our understanding of the E. cloacae-onion

interaction in order to develop management strategies for better control of bacterial bulb rot of

onion. A mini-Tn5 library of E. cloacae EcWSU1R was generated so that genes involved in the

E. cloacae-onion interaction could be identified. The selected mutants were assayed for biofilm

production, pathogenicity, and movement in planta. Scanning electron microscopy was also

employed to visualize onion leaves inoculated with E. cloacae and to determine which tissues

were colonized by the bacteria. Twenty-two of the 46 CR mutants produced significantly less of

a solid-surface-associated biofilm than that of the rifampicin-resistant strain EcWSU1R. Using

whole-bulb assays, it was determined that the CR mutants were not reduced in ability to cause

onion-bulb rot. The mutants moved bidirectionally through the leaves and bacterial populations

of the strains increased in planta. Using scanning electron microscopy, bacterial cells were not

observed in any plant tissues; however, a matrix was present in the phloem of onion leaves

29

inoculated with the mutants. Currently, it is unknown as to whether E. cloacae utilizes secretion

systems, effectors, or toxins in its interaction with onion. This research increased our

understanding of the E. cloacae-onion interaction to assist in developing management strategies

that reduce bacterial bulb rot of onion.

30

FUTURE DIRECTIONS

While some aspects of the Enterobacter cloacae-onion interaction have been characterized,

more research is needed to fully understand this interaction. These data show that E. cloacae

moves bidirectionally through the leaves of onions and can reach the bulb if infected in the leaf

area above the whorl. The impact of water stress, heat stress, plant age, or temperature on the

populations and movement of the bacterium is not yet known. Further assays are needed to

determine the impact of various conditions on the movement of E. cloacae in planta and the

development of Enterobacter bulb decay. Also, transmission electron microscopy on infected

leaves may be helpful to determine the tissues that E. cloacae localizes, as well as to further

understand the complex matrix that is produced in the phloem vessels.

It is currently unknown as to how E. cloacae gets to and enters onion plants. Pantoea

ananatis, the causal agent of center rot of onion, is vectored by thrips (Gitatis et al. 2003). In the

state of Washington, thrips are an economically important pest for onion producers (Mayer et al.

1987). As E. cloacae has been isolated from the intestines of thrips (Ullman, unpublished), it

would be prudent to research the ability of thrips to vector E. cloacae.

Since only 99 of the 9,600 mini-Tn5 mutants from the mutant library were selected with the

congo red assay, it would be important to utilize other assays to identify pathogenicity and

colonization mutants. While the congo red screen did show differences in polysaccharides and

allow for a useful way to identify mutants with a potential role in the plant-pathogen interaction,

other mechanisms and aspects of the pathogenic interaction may also be important, and would be

useful to understanding the E. cloacae-onion interaction.

The genome of E. cloacae strain EcWSU1 was sequenced during this research. This

resource will enable genes with a potential role in the plant-pathogen interaction to be identified

31

by data mining and will streamline manipulation of these genes to confirm their role in the plant-

pathogen interaction. Also, the mutant strains identified in this project need to have the disrupted

genes complemented to determine a true association of particular genes with the biofilm

phenotype, and an annotated genome will aid these complementation studies.

Understanding the properties of E. cloacae that contribute to the pathogen’s ability to cause

Enterobacter bulb decay will allow onion growers to develop better management strategies that

reduce the impact of this pathogen on losses due to bacterial storage rot. Finally, if E. cloacae

localizes to phloem vessels, there is a potential for the use of E. cloacae in onion as a model

system for Candidatus Phytoplasma spp. and Candidatus Liberibacter spp., as these pathogens

colonize the phloem but are not culturable (Doi, 1967; Oshima et al., 2001). These results

improve our understanding of the factors affecting E. cloacae growth and survival in onion

storage systems and are part of the continuing effort to reduce Enterobacter bulb decay.

32

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Table 1. Bacterial strains and plasmids of Enterobacter cloacae or Escherichia coli used in these studies.

Strain or Plasmid Relevant Characteristics Source Enterobacter cloacae EcWSU1 Schroeder et al, 2010 EcWSU1R Spontaneous Rifr mutant of

EcWSU1 Thyren, Legler, and Schroeder

2007, unpublished CR1-CR15, CR17-CR100 Congo red mutant, Rifr Kmr This study

Escherichia coli SM10 λ pir thi thr leu tonA lacY supE

recA::RP4-2-Tc::Mu Kmr λ pir

Herrero, 1990

Plasmids pUT-km Apr; Tn5-based delivery

plasmid with Kmr Herrero, 1990

Table 2: Characteristics of congo red mutants of Enterobacter cloacae.

Strain or plasmid

Congo red Morphology

NBY Morphology

Growth on M9

Pathogenicity (% bulb

surface)†

Biofilm production

(OD 570)

Disrupted Gene

Enterobacter cloacae

ECWSU1 Red Wild type + 25.0 5.02. ECWSU1R Red Wild type + 28.5 5.15.

CR1 Dark red Dry + 30.0 3.14 ENC_45260 (aspartokinase/homoserine dehydrogenase

CR2 Dark red Wild type + 20.0 1.29* ECL_04092 (stationary-phase survival protein, surE)

CR3 White Wild type + 20.0 0.43* Escherichia fergusonii strain EF873 aerobactin synthesis and receptor region

CR4 White Wild type + 8.3 0.81* ENC_11050 (peptidase T)

CR5 White Wild type + 30.0 0.63* Salmonella enterica subsp. enterica serovar Heidelberg str. SL476; putative transcriptional regulator, DeoR family, SeHA_C3263

CR6 White Mucoid + 20.0 3.92 Escherichia coli BEN2908, aec44

CR7 Dark red Wild type + 11.7 3.53 ENC_24040 (diguanylate cyclase/phosphodiesterase)

CR8 Dark Red Wild type + 29 1.92* Ent638_1846

CR9 Dark red Wild type + 16.7 2.73 ENC_45260 (aspartokinase/homoserine dehydrogenase)

CR10 White Wild type + 10.0 2.84 ECL_02041 (hypothetical protein)

CR11 Dark Red Wild type + 16.67 2.86 E. cloacae serine protease (degQ), no Tn sequence located

CR12 Dark Red Wild type + 21.67 1.11* No sequence found

CR13 White Wild type + 14.5 4.54 No sequence found

CR14 Dark Red Dry + 20 3.07 Ent638_2308 (response regulator receiver protein), no Tn sequence

41

located

CR15 White Wild type + 18.5 5.14 E. sakazakii, hypothetical protein, aspartokinase I domain,

ESA_03335, no Tn sequence located

CR17 Dark red Wild type + 20.0 4.92 ECL_01753 (Enoyl-CoA hydratase)

CR18 Dark red Dry + 20.0 1.22* Salmonella, aspartokinase/homoserine dehydrogenase I (thrA), SeD_A0002, no Tn sequence located

CR19 Dark red Wild type + 23.3 2.32* ECL_02799 (gspC, general secretion pathway protein C)

CR20 Dark red Wild type + 20.0 2.38* ECL_01753 (Enoyl-CoA hydratase)

CR21 White Wild type + 18.3 2.25* ECL_03685 (OMPP1/FadL/TodX family outer membrane transporter)

CR22 Dark red Wild type + 20.0 1.55* ENC_24040 (diguanylate cyclase/phosphodiesterase)

CR23 White Dry + 13.5 2.70 ENC_24040 (diguanylate cyclase/phosphodiesterase)

CR24 Dark red Wild type + 16.7 2.98 E. coli, fused aspartokinase I ; homoserine dehydrogenase I, ECED1_0001, no Tn sequence located

CR25 Dark Red Wild type + 20.8 2.77 Ent638_3880 (major facilitator superfamily MFS_1), no Tn sequence

located

CR26 Dark Red Wild type + 12.5 2.98 No sequence found

CR28 Dark red Wild type + 11.7 2.63 ENC_11050 (peptidase T)

CR29 Dark red Wild type + 13.3 1.43* ENC_30470 (bacterial protein of unknown function, DUF903)

CR30 Dark red Wild type + 20 3.59 ENC_11050 (peptidase T)

CR31 Dark Red Wild type + 20 3.71 No sequence found

CR32 Dark Red Wild type + 18.5 3.22 ENC_11050 (peptidase T)

CR33 Dark Red Wild type + 20 3.26 ENC_11050 (peptidase T)

CR34 Dark Red Wild type + 20 2.99 ENC_11050 (peptidase T)

CR35 Dark Red Wild type + 18.5 2.64 No sequence found

CR36 Dark Red Wild type + 20 3.38 No sequence found

42

CR37 Dark Red Wild type + 20 3.56 ENC_11050 (peptidase T)

CR38 Dark Red Wild type + 17.5 3.82 No sequence found

CR40 Dark red Wild type + 10 4.91 ENC_11050 (peptidase T)

CR41 White Wild type + 20.0 0.97* ECL_04939 (cell division protein)

CR42 Dark red Wild type + 15 5.18 E. coli 536, hypothetical protein, ECP_1145 CR43 White Wild type + 10.0 1.40* ECL_01753 (Enoyl-CoA hydratase)

CR44 Dark red Mucoid + 18.3 2.41 E. sakazakii, hypothetical protein, aspartokinase I domain, ESA_03335, no Tn sequence located

CR45 White Wild type + 15.0 2.27* ENC_24040 (diguanylate cyclase/phosphodiesterase) CR46 White Wild type + 20.0 2.65 ENC_24040 (diguanylate cyclase/phosphodiesterase) CR47 Dark Red Wild type + 16.1 3.05 No sequence found CR48 Dark Red Wild type + 20 3.04 E. cloacae serine protease (degQ), no Tn sequence located

CR49 Dark Red Wild type + 15 0.97* E. coli, fused aspartokinase I ; homoserine dehydrogenase I,

ECED1_0001, no Tn sequence located CR50 Dark Red Wild type + 8.3 1.69* Ent638_0656 (alpha-N-arabinofuranosidase), no Tn sequence located CR51 Dark red Dry + 13.3 2.99 ENC_24040 (diguanylate cyclase/phosphodiesterase CR52 Dark red Dry + 26.7 3.57 ENC_24040 (diguanylate cyclase/phosphodiesterase CR53 Dark red Wild type + 21.7 2.82 ENC_24040 (diguanylate cyclase/phosphodiesterase CR54 Dark Red Wild type + 15 1.74* Ent638_3962 (uridine phosphorylase), no Tn sequence located CR55 White Wild type + 15.0 1.80* ECL_03810 (transcriptional regulator, RpiR family) CR56

Dark red Wild type + 11.7 1.38* ECL_02076 (lagre repetitive protein)

CR57 White Wild type + 15.5 2.85 No sequence found CR58 Dark red Wild type + 18.33 2.78 ENC_24040 (diguanylate cyclase/phosphodiesterase CR59 Dark red Wild type + 11.5 1.35* ENC_41210 (uncharacterized conserved protein) CR60 White Wild type + 16.5 1.55* No sequence found CR61 White Wild type + 15.0 2.31* ECL_00814 (Hypothetical protein)

CR62 White Wild type + 13.3 3.74 ECL_03390 (putative sensor protein)

43

CR63 Dark red Wild type + 10.0 2.15* ECL_03390 (putative sensor protein)

CR 64 White Wild type + 33.3 4.11 ECL_00365 (hypothetical protein)

CR 65 White Wild type + 15.0 2.38 ECL_03390 (putative sensor protein)

CR66 Pink Mucoid + 10.0 1.82* ECL_05156 (pstB)

CR67 White Wild type + 20.0 0.70* ECL_00365 (hypothetical protein)

CR 68 Dark red Wild type + 18.3 0.47* ECL_00365 (hypothetical protein)

CR 69 White Wild type + 10.0 2.36* ECL_00365 (hypothetical protein) CR 70 White Wild type + 18.33 0.44 ECL_00365 (hypothetical protein) CR71 White Wild type + 12.5 2.20 ECL_00814 (Hypothetical protein) CR72 White Wild type + 18.33 2.48 ECL_00365 (hypothetical protein) CR 73 White Wild type + 21.7 0.42* ECL_00365 (hypothetical protein)

CR74 White Wild type + 13.3 0.48* ECL_03390 (putative sensor protein)

CR75 White Wild type + 17.5 3.25 ECL_04940 (Putative cytoplasmic protein)

CR76 White Wild type + 13.3 1.09* ECL_03390 (putative sensor protein) CR 77 White Wild type + 10.0 2.17* ECL_03390 (putative sensor protein) CR78 White Wild type + 10 1.75* ECL_03390 (putative sensor protein) CR79 White Wild type + 19.0 1.66* ECL_03390 (putative sensor protein) CR80 Dark Red ND ND 15 2.29* No sequence found CR 81 White ND ND 15.7 2.48 ECL_00365 (hypothetical protein) CR82 White ND ND 10.0 2.29* ENC_21440 (ABC-type enterobactin transport system, permease) CR 83 White ND ND 20 2.65 ECL_00365 (hypothetical protein) CR84 White ND ND 15.7 2.31* ECL_01192 (hypothetical protein) CR85 White ND ND 11.0 2.55 ECL_03390 (putative sensor protein) CR87 White ND ND 20.0 2.33* ECL_00814 (Hypothetical protein) CR88 White ND ND 10.0 2.34* ECL_03685 (OMPP1/FadL/TodX outer membrane transporter CR 89 White ND ND 20 2.85 ECL_01192 (hypothetical protein)

44

CR90 White ND ND 18.3 2.35* ECL_03390 (putative sensor protein)

CR 91 White ND ND 15.7 2.95 ECL_04940 (Putative cytoplasmic protein)

CR 92 White ND ND 18.3 2.34* ECL_01552 (hypothetical protein)

CR93 White ND ND 18.3 3.21 ECL_01552 (hypothetical protein)

CR94 White ND ND 10.0 3.41 ECL_04943 (putative inner membrane protein)

CR96 White ND ND 11.5 2.47 No sequence found

CR 97 White ND ND 20 2.80 ECL_01192 (hypothetical protein)

CR98 White ND ND 15.7 1.60* Ent638_0656 (alpha-N-arabinofuranosidase), no Tn sequence located

CR 99 White ND ND 20 1.58* ECL_00814 (Hypothetical protein)

CR100 White ND ND 11.7 1.58* ENC_45260 (aspartokinase/homoserine dehydrogenase)

An * denotes a statistically significant underproducer. (α = 0.05) A † denotes that none of the treatments listed were significantly different from the reference strains. (α = 0.05) ND denotes that this assay was not performed.

45

46

Fig. 1. Selection of Enterobacter cloacae colonies with mini-Tn5 disruptions. Luria Bertani

medium amended with rifampicin and kanamycin was used to select colonies of Enterobacter cloacae with mini-Tn5 disruptions after biparental mating. The mating mixture was plated onto the medium and the plates were incubated overnight at 28°C. The colonies that were present on the medium were streaked for isolation to ensure purity and then they were saved in the library.

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Fig. 2. Selection of Enterobacter cloacae strain ECWSU1R mini-Tn5 mutants with altered

congo red phenotype. E. cloacae mini-Tn5 mutants were plated onto Luria Bertani agar amended with 0.2 g L-1 congo red dye and the phenotypes were compared to ECWSU1R (A) after 48 hours. Colonies that were lighter (B) or darker (C) were selected for further study.

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Fig. 3. Production biofilm by Enterobacter cloacae reference or mutant strains in glass tubes. A

1.9ml aliquot of super optimal broth with glycerol (SOBG) medium was inoculated with a 0.1ml suspension of E. cloacae at of a 108 CFU ml-1. Cultures of E. cloacae EcWSU1R (middle tube) and the mini-Tn5 mutants (right) with an altered congo red phenotype were allowed to grow in static culture for 6 days at 23°C. Non-inoculated SOBG medium (left) was used as a control.

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Fig. 4. Production of biofilm by Enterobacter cloacae reference strain EcWSU1R on glass tubes.

A ring of light-colored biofilm adhered to the glass surface of tubes of super optimal broth with glycerol medium that were inoculated with E. cloacae . This biofilm was undisturbed by slight vortexing and was measured by staining with crystal violet.

50

Fig. 5. Biofilm of Enterobacter cloacae reference or mutant strains stained with crystal violet. A

2ml aliquot of a .1% solution of crystal violet was added to each of the biofilm tubes that had been washed with a 0.85% solution of NaCl. The crystal violet solution was washed out after 15 minutes of biofilm staining. The non-inoculated tubes (left) showed very little staining, while the wild type (middle) showed moderate staining, usually with firm rings that were not easily disrupted. The potential biofilm overproducer, CR42 (right), showed a large amount of staining on a biofilm that was more fluid.

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Fig. 6. Measuring the concentration of crystal violet attached to adhered biofilm of Enterobacter

cloacae reference or mutant strains using spectrophotometer cuvettes. A 2ml aliquot of 95% ethanol was added to the stained biofilm tubes. After 5 minutes, the tubes were vortexed thoroughly and the contents were placed in spectrophotometer tubes. The OD570 was measured in a SpectramaxPlus 384 spectrophotometer. The concentration of crystal violet was proportional to the amount of biofilm adhered to the tubes.

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Fig. 7. Inoculation of onion bulbs with Enterobacter cloacae. Surface-disinfected onion bulbs

were inoculated at the shoulder with 0.5ml E. cloacae (108 CFU ml-1) using a 23 gauge needle and a 1ml syringe. Bulbs were bagged and incubated at 30°C for 2 weeks. Bulbs were cut in half at the point of inoculation and the cut surface was rated (0-100%) for symptoms. The symptoms of the congo red mutants were compared to those of EcWSU1 and EcWSU1R to see if the pathogenicity and virulence of specific mutants differed from those of the reference strains EcWSU1 and EcWSU1R .

53

Fig. 8. Method of inoculation of Enterobacter cloacae wild-type or mutants for movement assay.

The third youngest leaf of a 15-week-old onion plant was inoculated 1 cm above the whorl using a flame-sterilized dissection probe that had been dipped into a suspension (108 CFU ml-1) of E. cloacae. Approximately 106 CFU bacteria was inoculated into the plant. Movement and populations of E. cloacae in the plant was monitored using plate counts. No symptoms were observed resulting from the inoculation of E. cloacae to 15 week onion plants.

54

Fig. 9. Isolation of tissue from onion leaves inoculated with Enterobacter cloacae reference or

mutant strains. A flame-sterilized number 4 cork borer was used to isolate tissue 1 cm in diameter from the point of inoculation and from above and below the point of inoculation every 7 days for 6 weeks. The samples were weighed so that each measurement could be standardized. This was done so that both the gross movement and the populations in different parts of the leaf could be measured despite leaf mass differences.

55

Fig. 10. Disease symptoms resulting from artificial inoculation of strains of Enterobacter

cloacae EcWSU1R. Onion bulbs that were injected with E. cloacae were harvested after 2 weeks at 30°C. The surface area of the internal scales was rated for disease symptoms. The non-inoculated controls (A) and the water controls (B) did not show any symptoms of Enterobacter bulb decay. E. cloacae strains EcWSU1 (C) and EcWSU1R (D) showed typical symptoms of Enterobacter bulb decay, specifically discoloration of the internal scales. The mini-Tn5 congo red mutants all produced symptoms of Enterobacter bulb decay; however, all of the mutant isolates (E-H) (CR57, CR59, CR20, CR26) produced symptoms that were not different from either EcWSU1 or EcWSU1R (P = 0.24, α = 0.05).

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Fig. 11. Biofilm production by Enterobacter cloacae reference strain EcWSU1, rifampicin-

resistant reference strain EcWSU1R and thirty congo red mutants from three separate experiments. None of the mutants were similar to the non-inoculated control or produced higher concentrations of biofilm than did the wild-type strains. Asterisks denote mutants that had a reduced amount of biofilm in comparison to EcWSU1 and EcWSU1R (P < 0.0001, α = 0.05).

57

Fig. 12. Biofilm production by Enterobacter cloacae reference strain EcWSU1, rifampicin-

resistant strain EcWSU1R and twenty-four congo red mutants from specific gene families compared to the wild-type strains. The congo red mutants that were found to have disruptions in genes with homology to diguanylate cyclase/phosphodiesterase, aspartokinase/homoserine dehydrogenase, and peptidase T genes were assayed for the amount of biofilm produced. It was found that all of the peptidase T mutants, 2 of the 3 aspartokinase/homoserine dehydrogenase mutants, and 5 of the 16 diguanylate cyclase/phosphodiesterase mutants had decreased biofilm production in comparison to the reference strains (P < 0.0001, α = 0.05). Asterisk indicates a significantly decreased average of biofilm.

58

Fig.13. Total movement of Enterobacter cloacae through onion leaves over 6 weeks. The

movement of strains EcWSU1R, CR42, and CR78 through the leaves of onion plants was measured each week for 6 weeks. All of the strains moved at approximately the same rate at each time point. An increase in the rate of movement was observed by all strains in the third week. An asterisk denotes a significant increase in movement from the previous week (P < 0.0001, α = 0.05).

59

Fig.14. Net movement of Enterobacter cloacae through onion leaves. The movement of strains

EcWSU1R, CR42, and CR78 through the leaves of onion plants was measured each week for 6 week. There was a large increase of movement at the third week (P < 0.0001, α = 0.05).

0

2

4

6

8

10

12

14

16

1 2 3 4 5 6

Leng

th (c

m)

Week

Net movement of E. cloacae in planta each week

EcWSU1R

CR42

CR78

60

Fig.15. Mean total populations of E. cloacae in planta. Strains EcWSU1R, CR42, and CR78

were inoculated into onion leaves and the populations were measured over 6 weeks. The populations of the three strains increased over 6 weeks. However, none of the strains were found to be different from the others at any point in time. (P=0.289 α = 0.05)

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Fig. 16. Onion leaf anatomy. There are five main types of tissue in onion leaves (A). The

epidermis forms the dermal layer (E), which protects the internal tissues and is important for transpiration. Lining the inside of the epidermis is palisade parenchyma (PP), which is a support for the internal tissues. Mesophyll cells (M) compose the highest percentage of the internal cells. Vascular bundles (V) are spread irregularly throughout the mesophyll cells, but most are located near the palisade parenchyma. Each vascular bundle (B) consists of both xylem (X) and phloem (P).

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Fig. 17. Scanning electron microscopy (SEM) of cross sections of onion leaves colonized by

strains of Enterobacter cloacae. Onion leaf tissue was harvested from the point of inoculation 6 weeks after the introduction of ~ 106 CFU E. cloacae strains EcWSU1R (A), CR42 (B), CR78 (C), or sterile water (D) and gold sputter-coated cross sections of onion leaves were visualized with SEM. A matrix (M) was observed in the phloem (P) of plants colonized by EcWSU1R, CR42, and CR78 that was not observed in the xylem (X) or mesophyll cells. This matrix was only observed in the phloem (P) of leaves that were inoculated with bacteria. The matrix was not observed in onion leaves inoculated with sterile water (D). It was observed that the tissue inoculated with CR78 (C) exhibited less matrix accumulation in comparison to tissue inoculated with EcWSU1R (A) or CR42 (B). The scale bar for each image is 17.6 µm.

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Fig. 18. The matrix associated with the colonization of Enterobacter cloacae is present

throughout the phloem of inoculated onion leaves. The internal tissues in the onion vasculature were exposed by removing the epidermis and palisade parenchyma with tape. A matrix was localized to the phloem vessels when (P) leaves were inoculated with EcWSU1R (B, C), but no matrix was present in the phloem when inoculated with water (A).

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Fig.19. Cells of Enterobacter cloacae. E. cloacae strains EcWSU1R (A), CR42 (B), and CR78

(C) were visualized using SEM so as to be able to distinguish bacterial cells from structures within onion plants, as bacterial cells were not discernable in infected plant micrographs. All three strains produced biofilm matrices that were associated with the bacterial cells.