Production, Isolation & Quantification of AI-2, a Small Molecule Involved in Quorum Sensing

Holly Bull

Summer Fellows 2015 Mark Vaughan

ABSTRACT

Bacteria communicate with one another through a biochemical process called

quorum sensing. Quorum sensing is dependent on the production of small molecules

called autoinducers. Once a population of bacteria has reached a certain density, the

extracellular concentration of autoinducers is able to trigger a physiological response.

These responses are dictated by the specific autoinducer system, depending on factors

such as the producing bacteria, the type of autoinducer, and the receiving bacteria. The

signalling molecule Autoinducer-2 (AI-2) has proven to be fundamental for a variety of

processes in bacteria including the formation of biofilm, a multispecies bacterial matrix

that is resistant to immune responses and antibiotics. The aim of my project is to develop

an effective biological method to produce AI-2, and furthermore, isolate the molecule for

future study. This report will discuss the experimental findings generated over the

summer, and suggest alternative approaches for future study.

INTRODUCTION

On Earth, bacteria were the only living presence for three billion years. Although

they are not visible to the human eye, today they still account for the majority of the

Earth’s biomass (Blaser, 2014). Eukaryotes are often hosts to micro-ecosystems

consisting of bacteria and other microorganisms, and therefore, these two life forms have

coevolved for a minimum of one billion years (Blaser, 2010). Consequently, eukaryotes

have had the help of bacteria in carrying out crucial metabolic, defense, and reproductive

functions for at least 500 million years (Cho & Blaser, 2012). In these animal based

micro-ecosystems there are many different forms of small organisms, such as single

celled eukaryotes (fungi, primitive algae, etc.) archaea and bacteria. According to Martin

Blaser, the conservation of microbial ecosystems in animal hosts suggests the presence of

a biological equilibrium (2006). This equilibrium would consist of the balance between

the varying interests of microbial species and the host (Blaser & Kirschner, 2007).

However, little is known about the biochemical mechanisms that maintain these

ecosystems (Blaser, 2010).

In the human body, the influence of resident microorganisms is still poorly

understood, with bacterial cells outnumbering human cells ten to one (Kelly et al., 2012).

At the phylum level, humans are very similar to one another. However, in looking at the

genus, species, and strain population levels, humans differ more in the composition of

their inhabitant bacteria than they do at the genomic level (Blaser, 2010). Again, the

biochemical interactions between bacteria and the human host, bacteria and other

microorganisms, and bacteria themselves are lacking scientific inquiry. My summer

project contributes to the research being done on a specific biochemical mode of

communication between bacteria.

Contrary to our prior conceptions, the majority of bacteria do not reside in their

planktonic (free floating) forms. Instead they favour the membership of tough

multispecies communities, called biofilms. Biofilms are matrices of bacteria held together

by polysaccharide extracellular adhesive. The stages of formation of biofilm include

initial attachment, cell-to-cell adhesion, proliferation, maturation, and detachment (figure

1.) (Kong et al., 2006).

Figure 1. The stages of biofilm formation include (1) Initial attachment, (2) cell to cell

adhesion, (3) proliferation, (4) maturation, and (5) detachment. Figure retrieved from

emerpharmaservices.com.

Once in a biofilm, physiological characteristics of the bacteria change. For

example, metabolism slows down, antibiotic resistance develops, genetic transfer

between bacteria occurs, and on occasion they can become virulent to the host or other

organisms (Vendeville et al., 2005). Current medication for bacterial infections, i.e.

antibiotics, is more effective for bacteria in the planktonic state (Arranga et al., 2013).

Therefore, the ever-increasing medical dilemma of antibiotic resistance is largely tied to

the resilient qualities associated with biofilms (Arranga et al., 2013). Although the

various biological stages of biofilm formation have been identified, the biochemical

interactions that cause the formation and dissipation of the matrices are unknown. The

study of a biochemical system called quorum sensing (small molecule communication

between bacteria) is beginning to provide insight on the biochemical complexity within

these processes.

Bacteria communicate interspecially (within the species) and intraspecially

(between species) through quorum sensing, which relies upon the production of small

molecules to regulate gene expression (figure 2.) (Galloway et al., 2011). These small

molecules are called autoinducers and are synthesized within individual bacteria through

enzymatic processes. As bacterial density within a colony grows there is an increase of

autoinducer abundance within the extracellular space. Once this concentration reaches a

certain threshold, the autoinducers bind to receptor proteins on the cell surface of the

bacteria, triggering the expression or repression of specific genes, and thereby eliciting a

specific physiological response (Kong et al., 2006). These responses include biofilm

formation, development of virulence, the production of antibiotics, swarming, fruiting

body formation, and gene transfer (Thiel et al., 2009)

Figure 2. Quorum sensing. Once the signaling molecules reach a certain concentration, they bind to the receptors on the cell surface and cause a physiological response.

Quorum sensing communication systems can be highly specific to certain species of

bacteria, or widespread to include multiple species. For example, gram-positive bacteria

typically release and respond to peptide-based autoinducers on the surface of the cell,

whereas gram negative utilize diffusible homoserine lactones (Kong et al., 2006; Xavier

& Bassler, 2003). However, a furanosylboratediester called autoinducer-2 (AI-2) has

been found in 55 species of gram-positive and gram-negative bacteria, and therefore has

been noted as the only signaling molecule that is not species specific (Figure 3.)

(Vendeville et al., 2005; Thiel et al., 2009). Despite having an identical biosynthetic

pathway and chemical intermediates across all AI-2 producing species, the responding

genes and phenotypes are uniquely dependent on the receiving species (Xavier & Bassler,

2003; Vendeville et al., 2005). My research aims to find an effective method for the

biological production, and isolation of AI-2, therefore allowing me to further study the

complexity of the AI-2 signaling system.

Figure 3. V. harveyi Autoinducer-2 chemical structure was classified as a furanosyl borate diester.

Biological Production

Production of AI-2 occurs as a byproduct of the activated methyl cycle (AMC),

which accounts for the metabolism, or gene expression in all bacteria. Synthesis of the

signaling molecule utilizes the enzymes Pfs and LuxS to recycle S-adenosyl

homocysteine (SAH) into homocysteine and 4,5-dihydroxyl-2,3-pentandione (DPD), the

precursor for AI-2 (Thiel et al., 2009). The cyclization of DPD to AI-2 is spontaneous

(Figure 4.). The high conservation of AI-2 biosynthesis could be contributed to the

importance of the DPD producing enzyme, LuxS, as it is key in the recycling of

homocysteine for the AMC (Vendeville et al., 2005). It is not yet known the full extent of

the role LuxS plays in quorum sensing. Another autoinducer, AI-3, has also been noted as

a byproduct produced by LuxS, but its role in bacterial communication has been sparsely

studied (Sun et al., 2004).

Figure 4. The activated methyl cycle. AI-2 is produced through pathway II of the AMC. Pfs hydrolyzes SAH into S-ribosyl homocysteine (SRH), which is cleaved by LuxS to produce DPD and homocysteine. Homocysteine is recycled back into the AMC. DPD spontaneously goes through cyclization to form AI-2. Figure retrieved from Sun et al., 2004.

Previous production methods for AI-2 have included chemical, in vitro, and

biological synthesis approaches (Keersmaeker, et al., 2005; Winzer et al., 2002;

Thompson et al., 2014). However, chemical and in vitro synthesis methods were

unfeasible for our lab due to the demand for complex equipment and expensive precursor

chemicals. SAH, the necessary precursor for AI-2 in vitro synthesis (using Pfs/LuxS and

various catalysts) is commercially available at approximately $70/10 mg (Sigma-

Aldrich), which makes this approach challenging for larger scale production. The purely

chemical approach also requires expensive precursors, in addition to lab equipment not

currently available at Quest. A genetic engineering approach, using in vivo cloning, offers

a more cost effective and accessible method for producing autoinducers. The

development of an effective in vivo production method would establish a sustainable

source of AI-2 for later studies on quorum sensing at Quest.

For optimum biological production, the plasmid pSB1C3- BBa_K1202110

(Figure 5.), containing the genes LuxS and Pfs (Figure 6.) under the control of a

constitutive promoter, was used to transform E. coli strain BL21. A constitutive promoter

enables a consistent production of proteins, therefore increasing the overall amount of

protein produced. The addition of the pSB1C3- BBa_K1202110 could accelerate

pathway II of the AMC (Figure 4.) by producing larger amounts of the LuxS and Pfs

proteins, and therefore greater quantities of DPD and AI-2. The BL21 E. coli strain is

engineered for maximum protein production, as it is modified to restrict the production of

protein-degrading enzymes. Therefore, it should be the most efficient method for

producing synthetically useful quantities of the LuxS and Pfs proteins.

Figure 5. Plasmid pSB1C3- BBa_K1202110. The plasmid contains a constitutive promoter (pMB1) with chloramphenicol resistance (CamR), and the gene sequence BBa_K1202110 (Figure 4.). Figure retrieved from iGem.org.

Figure 6. BBa_K1202110. The gene sequence contains LuxS and Pfs, which are the genes that transcribe the LuxS and Pfs enzymes responsible for the production of DPD and AI-2. Figure retrieved from iGem.org.

Although the LuxS gene has now been identified in multiple species of bacteria,

the Lux genes are named based on their importance in the process of bioluminescence in

Vibrio bacteria, the genus in which these genes were originally identified. Vibrio harveyi,

an aquatic pathogen, is a species renowned for the natural production of AI-2. The V.

harveyi wild type signaling system consists of the universal LuxS/Pfs biosynthesis

pathway and a LuxP and LuxQ receptor system. LuxP is a periplasmic protein that binds

AI-2 from the extracellular space, and LuxQ is a hybrid sensor kinase that initiates the

signal transduction for luminescence (figure 7.) (Garcia-Alijaro et al., 2012). Once AI-2

has bound to LuxP, a genetic pathway responsible for luminescence is triggered,

therefore causing the bacteria to produce light (figure 8.). The bacteria also responds to

another autoinducer (AI-1) in a luminescent reaction (figure9.) (Surette et al., 1999). The

chemical structure of AI-2 was determined using the AI-2-LuxP complex (figure 4.)

(Chen et al., 2002). Due to its luminescing quality, V. harveyi has since been used as a

standard tool for studying AI-2. Therefore, as it is known to produce AI-2, it will be used

as a positive control throughout this study.

Figure 7. The Lux system in V. harveyi resulting in luminescence. LuxS and LuxM synthesize AI-2 and AI-1, respectively. LuxP binds extracellular AI-2, while LuxQ starts the signal transduction for luminescence. LuxN binds AI-1. Figured retrieved from

http://jb.asm.org/content/186/12/3794/F1.expansion.html.

Figure 8. V. harveyi wild type culture displaying bioluminescence.

Figure 9. AI-1 (homoserine lactone). The identity of the R-group in AI-1 varies

depending on the producing bacteria.

Isolation & Quantification

DPD spontaneously forms AI-2 in equilibrium. However, DPD can undergo many

different reactions to produce a variety of derivatives, only two of which are considered

AI-2 (Figure 10.). As a result, many compounds exist in equilibrium with DPD through

cyclization, hydration, and borate ester formation (Thiel et al., 2009). DPD and AI-2 are

highly water soluble and polar molecules that are produced in low concentrations. V.

harveyi is known to produce AI-2 at relatively high levels compared to other bacteria, yet

researchers have only detected production levels up to 258 ng per milliliter of culture or

1.95 µM (Thiel et al., 2009). Therefore these compounds are difficult to isolate and

quantify from biological samples. However, only small concentrations of AI-2 are

necessary to have a significant biological effect. The chosen methods have been derived

from various studies, and selected based on the standard methods for AI-2 assays and the

resources available to the project.

Figure 10. DPD (1) can react to form molecules (2,3) through cyclization, hydration (4,5,10,11) and with boron (6,7,8,9), all of which exist in equilibrium. Salmonella typhimurium uses derivative 5 in a signaling system, whereas the majority of other bacteria documented, including V. harveyi, utilize AI-2 6. Image retrieved from Thiel et al., 2009.

Biological Quantification: Vibrio harveyi BB170 Bioassay

Vibrio harveyi BB170 bioassay is the standard for the relative quantification of

AI-2 within a supernatant. V. harveyi BB170 is genetically modified so that it does not

produce AI-2 (LuxS knock out) or AI-1, and is insensitive to AI-1 (Winzer et al., 2002).

Therefore, it can only luminesce once AI-2 has been added to the medium (Winzer et al.

2002). This genetic design makes it a prime indicator for the presence of AI-2, and as

more AI-2 is added the light emissions from the V. harveyi grow stronger (Winzer et al.,

2002).

Chemical Quantification

1. Thin Layer Chromatography

Thin layer chromatography (TLC) can be an effective method for separating out

compounds in a solution. Samples are applied on the base line of TLC silica gel plates,

which are then placed in a non-reactive solvent. Molecules are suspended in the solvent

as it climbs up the plate, and are separated based on their affinity for the stationary phase

on the silica gel. If AI-2 is in high enough concentration within a supernatant, it may be

visible on a TLC plate, and therefore this may be an effective method for detection and

quantification of AI-2. However, since AI-2 is a small molecule and is produced in tiny

concentrations, an extraction method must be developed before TLC can be applied.

Extraction of the AI-2 molecule relies on the chemical characteristics of its

precursor, DPD. As AI-2 and DPD are both highly polar and soluble in water, methods

for extraction will require derivitization of the DPD molecule to make it more affinitive

for an organic solvent, allowing us to separate it from the rest of the aqueous supernatant.

The DPD molecule has a relatively unique structure with two adjacent ketones that can

react with 1,2-phenylenediamine to create the stable organic compound 1-(3-

methylquinoxaline-2yl)ethane-1,2,-diol (figure 11.) (Thiel et al., 2009).

Figure 11. The derivitization of DPD with 1,2-phenylenediamine to create a stable water-insoluble compound. 2. Gas Chromatography

Similar to TLC, gas chromatography (GC) separates molecules based on their

affinity for stationary versus mobile phases. GC uses an inert gas as a solvent, which

pushes the evaporated sample through a long coiled column. The presence of each sample

is detected upon exit of the column, and using the chromatograph the abundance of each

compound can be calculated. GC can be paired with mass spectrometry (MS) to

determine the mass of each compound in the sample. Although we do not have access to

a mass spectrometer, Thiel et al.’s GC-MS reading can be used as a point of reference

(2009)(Figure 12.).

Figure 12. GC-MS in conjunction allows for the identification of the peak corresponding to the DPD derivative. The arrow points to the peak representing the DPD derivative. Figure derived from Thiel et al., 2009. In order to prepare the samples for GC so that they can evaporate,

chlorotrimethylsilane can be added so that the hydroxyl groups on1-(3-

methylquinoxaline-2yl)ethane-1,2,-diol are protected, therefore lowering the boiling point

(figure 13.). GC is a more sensitive technique than TLC and may be a superior method

for quantifying the DPD complexes in the samples. The replacement of

chlorotrimethylsilane for MSTFA results in the same complex but loses the fluorescent

quality of the product. Although MSTFA may be a useful visual indicator for the success

of the reaction, chlorotrimethylsilane is far more accessible and the GC will pick up the

same compound.

Figure 13. The complete reaction from DPD to the trimethylsilylated quinoxaline. This figure was retrieved from Thiel et al., 2009. Contrarily to this figure, chlorotrimethylsilane was used in replace of MSTFA [N-methyl-N-trimethylsilyl)trifluoroacetamide].

METHODS

Biological Production

A plasmid, pSB1C3, containing the BBa_K1202110 gene sequence under the

control of a constitutive promoter, was cloned into the BL21 E. coli strain. The plasmid

was received from iGem (www.igem.org) in E. coli DH10β, and the plasmid was isolated

using the Qiagen miniprep procedure (http://www.ncbi.nlm.nih.gov/pubmed/18997895)

DH10β is used for plasmid replication, and is not an optimal strain for protein production

(ecoliwiki.net, 2012). V. harveyi wild type was used a positive control, and V. harveyi

BB170 was used as a negative control, as it is engineered not to produce AI-2. V. harveyi

was cultured using Marine Broth 2216 (attc.org). All bacteria were cultured overnight at

30°C, diluted (10x), and cultured again for 24 hours. Aliquots were taken from the

diluted culture at various time intervals and absorbency was measured using a

spectrophotometer at OD600. Analysis of protein production was performed using Sodium

dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, 8% polyacrylamide

gel), a technique that separates proteins based on their size using electrophoresis, on

BL21- pSB1C3- BBa_K1202110 and DH10β-pSB1C3- BBa_K1202110 to determine the

strain and culture density with the most protein production.

Quantification and Isolation Methods

Vibrio harveyi BB170 was provided by Dr. John McCormick, Department of

Microbiology and Immunology at the University of Western Ontario. Wild type Vibrio

harveyi was provided by Dr. David Byers, Department of Biochemistry at Dalhousie

University.

Vibrio harveyi BB170 was cultured overnight in Marine Broth 2216 (attc.org) on

a shaker at room temperature. A 10x dilution of this culture was grown to log phase and

180 µL of culture (OD600 .304), along with 20 µL of supernatant from V. harveyi wild

type or BL21, were added to each well (Winzer et al., 2002). The light emission was

photographed using an alpha imager (41s exposure), and the relative light absorbance

was visually noted. The alpha imager was tested using wild type Vibrio harveyi as a

positive control, and V. harveyi BB170 as a negative control. Vibrio harveyi wild type

culture (OD600 .205) was added in various volumes to 96 well plates and the

luminescence was photographed with alpha imager to use as a standard.

TLC Methods

Methods for the 1,2-phenylenediamine extraction were derived from Thiel et al.,

2009 (see for further details). Each sample of bacteria was spun down and the supernatant

was filter sterilized. 1,2-phenylenediamine was dissolved in a potassium phosphate buffer

and added to each supernatant to sit for 2 hours at room temperature. A 2:1 ratio of the

1,2-phenylenediamine/supernatant mixture and the organic solvent dichloromethane were

mixed thoroughly. The aqueous layer was then removed and TLC was performed on the

remaining organic solvent. 10 drops of each sample were spotted at the starting point.

BL21 (9h, 24h) and V. harveyi wild type (3h, 6h) were tested, using BB170 as a control.

The comparison of V. harveyi wild type and BB170 would help in identifying the spot

unique to the wild type strain, which would suggest the presence of AI-2. 1,2-

phenylenediamine was also tested to account for the excess 1,2-phenylenediamine in the

supernatant samples.

Gas Chromatography

GC analysis was done at Capilano University using the PerkinElmer Clarus 500

with the Oven program (initial temp. 95 °C, hold 2.8 min, increase by 4.5 °C/min to 195

°C, hold 0.5 min), and the Flow program (initial flow 8 ml/min, hold 2.8 min, increase by

10 ml/min/min to 10 ml/min). Helium was used as the carrier gas and 1 mL samples at a

split ratio of 1:5. The injector temperature was 195 °C, the detector temperature was175

°C, and the total run time was 22 minutes. Samples included V. harveyi wildtype, V.

harveyi BB170, BL21- pSB1C3- BBa_K1202110 24h and 9h, 1,2-phenylenediamine in

dichloromethane, and chlorotrimethylsilane in dichloromethane.

RESULTS & DISCUSSION

Biological Production Results

The transformations of pSB1C3- BBa_K1202110 into BL21 were successful, as

the SDS-PAGE protein assay showed accentuated protein production at LuxS (17.3 kDa)

and Pfs (26.5 kDa) (Sewald et al., 2007; uniprot.org). LuxS and Pfs production increased

over time in both BL21 and DH10β. However, BL21 produced the most protein at 24

hours of culture time (Figure 14.). Optical density was recorded for each aliquot taken at

the various time intervals (Table 1.). Lumninescence of the wild type V. harveyi was

recorded at 3 hours (.08 OD600).

Figure 14. SDS-PAGE protein assay. Pfs and LuxS are found at 26 kDa and 17 kDa respectively. E. coli BL21 at 24 hours showed the most protein production for LuxS and Pfs. In both BL21 and DH10β there was an increase in protein production proportionate to time. Table 1. Absorbance (OD600) of Vibrio harveyi wild type, BL21- pSB1C3- BBa_K1202110, DH10β- pSB1C3- BBa_K1202110, and Vibro harveyi BB170 at various time intervals. Highlighted data corresponds to the SDS-PAGE gel.

Time  (hours)  

Absorbance  (OD600)  VHWT   DH10β   BL21   VH  BB170  

1   0.03   0.065   0.132   0.217  2   0.048   0.031   0.163   0.211  3   .080  

(LUM)  .050   .133   .282  

4   .134   .062   .184   .193  5   .114   .050   .135   .161  6   .172   .104   .166   .246  7   .244     .196   .358  11   .390   .204   0.266   .486  24   0.621   0.277   0.478   0.56  

The BL21 bacteria were effective in producing excess amounts of LuxS and Pfs.

The bands on the SDS-PAGE gel corresponding to both proteins grew thicker as time

progressed. However, it is unknown whether a higher yield of LuxS and Pfs would equate

to a greater production of AI-2. Additionally, it is still uncertain that engineered bacteria

would produce more protein, or AI-2, than the wild type V. harveyi. Further

quantification assays are necessary to determine which bacteria produce the most AI-2,

and if there is a correlation between higher levels of protein production and AI-2

production. Alternatively, the gene could be placed into a plasmid with an inducible

promoter to see if that results in a higher protein yield (Schauder et al., 2001).

Isolation & Quantification Results

Figure 15. The positive control, luminescing V. harveyi wild type, can be seen in the two rows of a 96 well plate. The negative control, V. harveyi BB170 was not detected. Image taken using the alpha imager.

Figure 16. Vibrio harveyi wild type (.205 OD600) at increasing volumes left to right: 20 µL --240 µL (left), and 180 µL – 400 µL (right). The Alpha Imager was successful in picking up the V. harveyi wild type

luminescence, although adjustments in the focus could be made to reduce the noise. The

V. harveyi BB170 bioassay was unsuccessful, and no luminescence appeared even 2

hours post induction (mixing of AI-2 containing supernatant with BB170). This could

have been a contamination error, and there could have been other bacteria in the culture

instead of V. harveyi BB170. The cells will be re-cultured from the original plate and

agar stab cultures and frozen stocks will be made for more attempts of the bioassay in the

fall. The detection method has yet to be optimized to capture more consistent images.

Chemical Quantification

1. TLC

There were slight differences between the various bacterial supernatants. 1,2-

phenylenediamine accounted for the larger spot at the top of the TLC plate. Although

every supernatant sample had streaks leading up to the first spots, the streaks were all

slightly different in appearance, and prevalence.

Figure 17. TLC on samples (from left to right) BL21-9h, BL21-24h, V. harveyi wild type-3h, V. harveyi wild type-6h, V. harveyi BB170, 1,2-phenylenediamine.

    Although the TLC results were largely inconclusive, BL21-24h showed an

encouraging spot at the bottom of the plate. BL21-24h produced the most LuxS/Pfs

protein and therefore should produce the most AI-2, leading us to believe that the spot

could be more than just a blotting inconsistency. However, other AI-2 producing bacteria

did not have a spot in the same location and instead only showed a streak. To determine

the identity of the BL21-24h spot, it could be extracted and tested using gas

chromatography. The DPD derivative is a small molecule and TLC may not be sensitive

enough for its detection. It could be one of the particles found in the streaks between the

start line and the first dot in each supernatant sample. In the future, larger samples could

be cultured and filter sterilized, and further concentrated by evaporating off some of

solution using a rotary evaporator. Dichloromethane has a relatively low boiling point

(39.6°C) and therefore will evaporate more effectively than water. Lastly, an increase in

TLC sample application to 20 spots per sample may increase the probability of

identifying the molecule.

2. Gas Chromatography

See appendix A for results.

In looking for an additional peak in the AI-2 containing samples, results were

inconclusive. Differences in the chromatographs of V. harveyi wild type versus BB170

were insignificant. The GC likely did not have the sensitivity necessary to pick up the

presence of the DPD derivative. In addition, the methods used for preparation were a

simplified version of those used in Thiel et al. Thiel et al. carried out a more extensive

isolation process using more advanced technology, and used a varying program for GC

analysis. The AI-2 derivative was only detected as a small peak on the Thiel et al. GC-

MS results, and therefore the simplification of methods may have diminished the

probability of detection. Therefore, more closely following the methods of Thiel et al.

may produce more conclusive results.

FUTURE RESEARCH

In the next few years at Quest I plan to continue the project beyond what I have

accomplished this summer. Initially, changes must be made to the extraction methods in

order to successfully isolate AI-2 (discussed in prior discussion sections). However, once

I have isolated a relatively concentrated AI-2 solution, the project can progress to

studying the genetic engineering applications and potential bacterial manipulation

methods AI-2 may provide. Two of the possible methods include using AI-2 in a highly

specified inducer system, and studying the effect of AI-2 on biofilm formation.

1. AI-2 as a Tool in Genetic Engineering

With genetic libraries (i.e. iGem) easily accessible, there is much opportunity to

explore the role AI-2 could play in a genetically engineered system. Similarly to V.

harveyi BB170, which is induced by AI-2 to luminesce, synthetic cloned systems can be

designed to respond in a particular way to quorum sensing molecules. Furthermore, since

AI-2 is considered the universal signaling molecule, AI-2 inducible systems may further

the specificity in which our genetic engineering functions. For example, the pLsrA-YFP-

K117008 plasmid (iGem) can be induced by AI-2 to produce yellow fluorescence

(figure18.). This summer, the plasmid was transformed into DH5α E. coli, which have a

frame shift mutation on LuxS and therefore they do not produce AI-2 (Winzer et al.,

2002). Various filter-sterilized supernatants were added to the DH5α-pLsrA-YFP-

K117008 bacteria and yellow fluorescence was visible with UV light. However, it was

minimally florescent and could not be picked up by the alpha imager. In the future, I will

explore how DH5α-pLsrA-YFP-K117008 E. coli responds to various levels of AI-2, as a

genetic engineering tool.

Figure 18. The K1170008 system is inducible by AI-2 and produces yellow fluorescence protein. Image retrieved from igem.org. 2. AI-2 as a Method to Modulate Bacterial Behaviour

AI-2 has been deemed partially responsible for the formation, and deformation of

biofilm (Kong et al., 2006; Garcia-Aljaro et al., 2012). For example, in Staphyloccoci AI-

2 has been noted to reduce biofilms, whereas on other bacteria, such as E. coli, AI-2 has

been shown to promote the biofilm state (Kong  et  al.,  2006,  Thompson  et  al.,  2015).  

65%  of  all  bacterial  infections  in  humans  are  attributed  to  biofilms,  and  therefore  

looking  at  AI-­‐2  as  a  possible  method  for  the  manipulation  of  biofilms  is  highly  

relevant  to  human  health  (Arranga  et  al.,  2013).  

  To  begin  studying  the  effect  of  AI-­‐2  on  biofilm  formation,  a  96  wells  plate  in  

conjunction  with  a  Calgary  Biofilm  Device  (Figure  19.)  will  be  used  to  grow  DH5α

E.coli with chloramphenicol resistance. Once biofilm has formed, a concentrated AI-2

solution can be added in varying amounts to each well. The Calgary Biofilm Device can

then be rinsed and the biofilm can be quantified using a crystal violet staining technique

(O’Toole, 2011). This procedure would be a good starting point for showing the effect of

AI-2 on E. coli biofilms.

 

Figure  19.  The  Calgary  Biofilm  Device  provides  a  surface  for  the  bacteria  in  96  well  plates  (bottom)  to  adhere  to  for  biofilm  formation.  Image  retrieved  from  cdeworld.com.        

CONCLUSION    

  Autoinducer-­‐2  is  revealing  itself  to  be  a  potentially  valuable  molecule  in  the  

medical  world.  In  March  2015,  Thompson  et  al.  released  a  paper  showing  a  strong  

correlation  between  AI-­‐2  and  the  rehabilitation  of  the  gut  microbiome  after  

antibiotic  induced  dysbiosis  (2015).  Although  the  biochemical  mechanisms  for  such  

correlations  are  not  yet  known,  it  is  hypothesized  that  AI-­‐2  could  have  an  effect  on  

the  gene  that  leads  to  the  production  of  polysaccharide  intercellular  adhesion  (PIA),  

which  holds  biofilms  together  (Kong  et  al.,  2006).  In  addition,  since  AI-­‐2  is  a  

byproduct  of  the  AMC,  it  could  influence  the  methylation  cycle  leading  to  changes  in  

metabolism,  and  therefore  effecting  gene  regulation  and  biofilm  formation  

(metabolism  slows  down  in  biofilm  state)  (Vendeville  et  al.,  2005).  Although  we  

have  already  shown  that  the  DH10β-pSB1C3- BBa_K1202110 production method

generates a larger quantity of LuxS/Pfs proteins, the AI-2 has yet to be isolated.

Extracting a relatively high concentrated solution of AI-2 would allow for numerous

possibilities in studying its effect on bacteria. Therefore, I hope this research will

eventually contribute to the widespread scientific effort being made towards the

classification of AI-2’s role in living systems.

     

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Appendix A

1. Vibrio harveyi wild type

2. Vibrio Harveyi BB170

3. BL21- pSB1C3- BBa_K1202110

4. 1,2-phenelynediamine

5. Chlorotrimethylsilane