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MB4002 Research Project

Title: Search for inhibitors of bacterial


quorum sensing among the microbiota of
marine sponges

Student’s name: Marcas O’ Muineachain

Student number: 106003290

Year: 2009/2010

Supervisor’s name: Dr. Teresa Barbosa

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Table of contents

1. Cover page

2. Table of contents

3. Abstract and Introduction

4 – 10. Introduction

10. Aims and objectives of project

11 – 17. Materials and Methods

18 – 26. Results

26 – 32. Discussion

32. Conclusion and Acknowledgements

33 – 37. References

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Abstract

N-acyl-homoserine lactone (AHL) mediated quorum sensing plays an important role in the
control of the expression of multiple virulence factors in some pathogens such as
Pseudomonas aeruginosa and consequently is a potential target for the development of
novel therapeutic agents. Therefore, it is imperative to screen complex microbial
communities, such as the marine environment for producers of quorum sensing inhibitory
compounds. In this study, we used a Chromobacterium violaceum based assay to screen for
the production of quorum sensing inhibitors by 30 sporeformers isolated from the marine
sponge Haliclona simulans. A similar assay was also applied to screen 27,648 clones from a
H. simulans metagenomic library. Of the 30 culturable sporeformers, we identified nine
potential quorum sensing inhibitor (QSI) producers, including two that carried the lactonase
gene aiiA, which encodes for a lactonase enzyme, a potent degrader of AHL signal
molecules. Initial screening of the metagenomic library identified 13 clones, which appear to
carry sequences mediating quorum sensing inhibition, however only 2 proved to be
reproducible. We also cloned the aiiA gene from the sponge B. cereus group isolate #11
into P. aeruginosa and tested for impacts on AHL quorum sensing regulated swarming
motility and pyocyanin production, but we could draw no conclusions from the results as
sequence analysis of the cloned insert revealed the absence of the first 12 nucleotides
encoding the first four amino acids of the protein. This study is the first to apply a function
based metagenomic approach to screen for QSIs in the marine environment and establishes
a template for future assays using a biosensor such as C. violaceum for QSI detection.

Introduction

Quorum sensing (QS) is a phenomenon of cell-density gene expression where bacterial cells
co-ordinate the expression of certain genes in a population dependent manner by releasing,
detecting, and responding to a signalling molecule (Waters & Bassler, 2005). QS has evolved
independently in Gram positive and Gram negative bacteria and the mechanism differs by
the nature of the diffusible signalling molecule. In Pseudomonas aeruginosa and over 70
other Gram negative bacteria which are known to utilise QS, intercellular signalling is
achieved through hormone-like N-acylated homoserine lactone molecules (AHLs), which are
termed autoinducers (Fuqua et al., 2001; Taga & Bassler, 2003). The gram positive QS

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mechanism is based on peptide autoinducers and two component pathways (Fuqua et al.,
2006). Because QS systems are key regulators for the expression of virulence factors in
certain pathogenic bacteria, interfering with QS is an attractive antimicrobial target in the
context of widespread antibiotic resistance. Such therapies would interfere with cell
signalling, not affecting viability (Rasmussen et al., 2006), thereby reducing the selective
pressure on bacteria to develop resistance (Dong et al., 2007).

Biofouling refers to a process whereby unprotected artificial and natural substrata are
quickly colonised by biota in an aquatic environment (Railkin, 2004). Biofilm formation,
which can be mediated by QS, is the initial stage of biofouling (Dobretsov et al., 2009). It is
therefore reasonable to expect to find compounds produced by marine organisms, such as
algae and sponges, which inhibits QS as a means of defence against colonisation (Sauer et
al., 2002). In addition, it is believed that sponges form a symbiosis with microbes, such as
bacteria and fungi (Kubanek et al., 2003) which results in the production of many bioactive
natural compounds, some of which were shown to be QS inhibitors (Mohamed et al., 2008).
Increasing research is confirming that new marine microbes discovered among microbial
communities in sponges are producers of natural bioactive compounds (Wang., 2006).
Therefore, screening for potential QS inhibitors among the microbiota of marine sponges is
a good strategy to identify QS inhibitors which may be of potential therapeutic use in the
future.

AHL mediated Quorum sensing

As referred to above, QS is based on the secretion and detection of small signal molecules
termed autoinducers. When the QS signals reach the minimal threshold stimulatory
concentration, they bind to specific receptor proteins which initiate transcription of the QS-
controlled genes, enabling most of the bacterial population to simultaneously express a
specific phenotype and thereby adapt particular behaviours on a population-wide scale
(Waters & Bassler, 2005). The general structure of the Gram negative bacteria signalling
molecules is dhown in figure 1a. These are AHLs that are synthesised by homologues of the
AHL synthase LuxI from S-adenosyl methionine and an intracellular pool of carrier proteins,
with each AHL distinguished by the length, degree of saturation, and substitution of the acyl

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side chains (Parsek et al., 1999; Dobrestor et al., 2009). LuxI functions as the auotoinducer
synthase which synthesises an AHL and the LuxR protein is an AHL-responsive DNA binding
transcriptional activator. After synthesis the AHL freely diffuses in and out of the cell and the
concentration increases as the cell density increases. Upon reaching the threshold, AHL is
bound by LuxR which initiates transcription of the operon. A positive feedback loop is
created, because the LuxR-AHL complex also induces the expression of LuxI as it is encoded
on the operon. This floods the environment with the AHL signal causing the entire
population to go into “quorum sensing mode” (Kaplan & Greenberg, 1985; Stevens et al.,
1994; Waters & Bassler, 2005) (fig 1b).

Figure 1. The core AHL molecule and the paradigm Vibrio fischeri LuxI/LuxR QS system.
(a) Each AHL is distinguished by characteristics of its side chain. (b) The model QS system in
V. fischeri.The red triangles indicate the autoinducer that is produced by LuxI. See text for a
detailed explanation. (Modified from Waters & Bassler, 2005).

Quorum sensing in Pseudomonas aeruginosa

The best studied QS system in a pathogen is that of P. aeruginosa, a bacterium associated


with nosocomial and life-threatening infections of the immunocompromised (van Delden &
Iglewski, 1998). The QS system in P. aeruginosa consists of two hierarchically arranged QS
circuits that have an interrelated effect (Pearson et al., 1997) (Fig. 2)

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Figure 2. The QS system in P. aeruginosa. See text for a detailed explanation (Modified from
Raina et al., 2009).

P. aeruginosa has two luxR homologues: LasR and RhlR. LasI and RhlI are two Lux-I type
synthases for autoinducer synthesis (Ni et al., 2008). The primary circuit is the Las system,
which encodes the proteins LasI and LasR (Gambella & Iglwski., 1991). LasI catalyses the
synthesis of the AHL ccompound N-3-oxodecanoyl-L-homoserine lactone (3-oxo-C12-HSL)
(Pearson et al., 1994), which activates the transcription regulator LasR, allowing LasR to bind
to the promoters of genes regulated by QS to enable virulence factor production, as shown
in figure 3 (Wilcox et al., 2008) (fig. 2). This also leads to formation of the PQS 2-heptyl-3-
hydroxy-4-quinolone causing RhII to be induced (Raina et al., 2009).

In the Rhl circuit, RhlI synthesises N-butyryl-homoserine lactone (C4 – HSL) which binds to
the receptor RhlR upon accumulation of a sufficient concentration of C4 – HSL (Pearson et
al., 1997). The RhlR-AHL complex then activates other virulence genes (fig. 2).

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Interfering with Quorum Sensing

Interference with QS circuits can be achieved in a number of ways and the most commonly
researched approaches target AHL synthesis, the signalling molecule and the signal receptor
(Ni et al., 2008).

A. Targeting AHL synthesis

The first step in AHL-mediated QS is the synthesis of AHL compounds by LuxI homologues.
Because S-adenosylmethionine (SAM) is the AHL precursor, inhibitors of enzymes which
require SAM to function can inhibit AHL-mediated QS (Ni et al., 2008). L/D-S-
adenosylhomocysteine (SAH), sinefungin, butyryl-SAM and holo-ACP are analogues of SAM
which are known to be strong inhibitors of RhII (Rasmussen et al., 2006). In vitro tests with
SAH showed it to decrease the activity of RhII by 97% (Parsek et al., 1999). The main
stumbling block regarding the use of SAM analogues as therapeutic agents is that SAM is
ubiquitous in biological systems, so its use could have undesirable side-effects (Ni et al.,
2008).

B. Targeting the signalling molecule

AHL lactonases and AHL-acylases (fig. 3) are two groups of enzymes which inactivate
bacterial AHLs. To date, 19 bacterial species are known to produce these enzymes, with
Bacillus species the best known producer of AHL lactonases. Streptomyces species,
Acinetobacter species, and P. aeruginosa are examples of AHL acylase producers (Czajkowski
& Jafra., 2009). AHL lactonases hydrolyse the lactone ring of AHLs, producing acyl
homoserines which leads to a 1000-fold reduction of signal activity (Dong & Zhang., 2005).
The best characterised of the AHL lactonases is AiiA (encoded by aiiA gene) first isolated
from Bacillus sp. 24B1 (Dong et al., 2000) and known to be a metalloprotein (Liu et al.,
2005). Homologues of AiiA lactonase have been discovered in a significant number of
bacteria from the Bacillus genus (Kim et al., 2005). A number of studies have shown AiiA to
attenuate the virulence of different pathogenic bacteria by enzymatic degradation of AHLs.
For example, the introduction of the aiiA gene in P. aeruginosa was found to reduce the

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production of cytotoxic compounds (Dong et al., 2000), prevent swarming motility and most
significantly to adversely impact on biofilm formation and dispersion (Wang et al., 2007).

Figure 3. AHL lactonases and AHL acylases enzymatically degrade AHLs. (From Zhang et al.,
2007).

AHL acylases hydrolyse the amide bond of AHL compounds releasing fatty acids and a
homoserine lactone (Zhang & Dong, 2004). Most AHL acylases among the nine characterised
to date are N-terminal hydrolases, consisting of two or more subunits with high substrate
specificity that degarades only AHLs, especially those with long chains (Czajkowski & Jafra.,
2009). Produced by both gram positive and gram negative bacteria, the role of AHL acylases
is believed to be mainly competitive (Dobretosov et al., 2009).

C. Targeting the signal receptor

The most researched approach of inhibiting QS is the search for, and use of receptor
antagonists which bind to the AHL receptor, blocking the activation of LuxR homologues.
The first effective QS inhibitors discovered were halogenated furanones produced by the
alga Delisea pulchra (Nys et al., 1993), which inhibit AHL-mediated gene expression by
binding to and blocking the AHL receptor (Manfield et al., 1999). Subsequent research has
focused on enhancing the potency of furanones by creating synthetic analogues.

Another approach in targeting the signal receptor is modification of the acyl side chain of
the AHL. Studies on P. aeruginosa (Kline et al., 1999), Agrobacterium tumefaciens (Zhu et al.,
1998), and V. fischeri (Reverchon et al., 2002) have shown that the modified AHL analogues
can antagonise the effect of natural ligands by binding the receptor.

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Rasmussen and colleagues recently identified a number of potent compounds that could
block LuxR and LasR – based QS. A DNA array – based analysis of the most potent
compound, 4-nitro-pyridine-N-oxide (4-NPO), showed that it down – regulated 37% of QS
regulated genes by targeting signal receptors (Rasmussen et al., 2005a).

Screening for QS inhibitors among the microbiota of marine sponges

Marine sponges are one of the oldest animal phylum (Miller & Muller., 2003) and harbour a
diverse range of microbes, estimated in some sponges to make up to 40% of the sponge
tissue volume (Usher et al., 2004). Bacteria and fungi have been isolated from a wide range
of marine sponges (Wang., 2006). The result of the sponge – microbe symbiosis is the
production of bioactive secondary metabolites (mostly by the bacteria) which are of
biotechnological interest for their potential therapeutic or cytotoxic effects (Faulkner et al.,
2000). The sponges provide a protected nutrient – rich niche and the symbionts are believed
to aid the sponge by producing antimicrobial compounds and providing the sponge with
limiting nutrients (Mohamed et al., 2008). It is hypothesised that AHLs produced by sponge
– associated bacteria could be pivotal in the regulation of the sponge – bacteria symbioses,
such as in the regulation of colonisation and the production of bioactive compounds (Taylor
et al., 2004). Skindersoe and colleagues (Skindersoe et al., 2008) screened extracts from the
great barrier reef for Quorum sensing inhibitory (QSI) activity and found QSIs in all of the
major groups of organisms screened including the marine sponge Luffariella variabilis.

Screening strategies

A number of screening methods have been developed to screen for Quorum sensing
inhibitors (QSIs). Violacein is a distinctive purple pigment produced by Chromobacterium
violaceum and its biosynthesis is controlled by AHL mediated QS. Inhibition of the QS system
by AHL analogues and other antagonists prevents violacein biosynthesis (Periara et al.,
2004). McClean and colleagues exploited this phenotype to screen for QSIs where a positive
result is indicated by the lack of pigmentation surrounding the subject being screened
(McClean et al., 2004).

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An alternative screening method developed by Rasmussen and colleagues uses genetically
modified bacteria which are termed QSI selectors (QSIS) which are killed if AHLs are present
but survive if AHLs are present with a potential QSI (Rasmussen et al., 2005a).

Screening for QSI producers among the microbiota of marine sponges or any other complex
environment must also utilise modern and developing molecular technologies given the
major limitations of traditional culturable methods (99% of microorganisms estimated to be
unculturable). In this regard, metagenomics will greatly assist in identifying QSIs. Isolating
total DNA from an environment, cloning into a suitable vector and screening for genes and
metabolic pathways which may be involved in the production of novel biosynthetic
compounds offers a major advantage over traditional approaches.

Aims of project

We had 30 sporeformers which had been characterised in detail, and PCR screening
identified the lactonase gene (aiiA) in two isolates; CH8a and #11. We also had a
metgenomic library from H. simulans. There were three objectives of the study:

1. Devolop an assay to screen for QSIs using Chromobacterium violaceum as an


indicator.
2. Apply the assay to the sporeformers and the metagenomic library.
3. Evaluate the effect of lactonases from sponge associated sporeformers CH8a and
#11 on QS regulated virulence determinants of P. aeruginosa.

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Materials and Methods

Bacterial strains, plasmids and growth conditions

Sporeformers from the marine sponge Haliclona simulans (see table 1) previously isolated in
the laboratory were maintained in Marine agar/broth (MA/MB) (Difco), at 30 °C with
aeration.
The QSI indictor organism, Chromobacterium violaceum Bergonzini 1880 (DSMZ; DSM No.
30191) was maintained in nutrient medium (1L: peptone 3 g, meat extract 3 g and 15 g
(1.5%) agar for solidification), aerobically, with agitation at 30 °C. P. aeruginosa PA01 and E.
coli strains used in cloning were cultivated aerobically, with agitation, in Luria - Bertani (LB)
(1L: 10 g tryptone, 5 g yeast extract, 10 g NaCl) medium at 37 °C.
The antibiotic concentrations used (unless otherwise stated) were gentamicin 10 µg ml-1,
kanamycin 40 µg ml-1, and chloramphenicol 12.5 µg ml-1. See tables 1 and 2 for a
comprehensive list of the strains and plasmids used.

Table 1. Identification of H. Simulans associated sporeformers by 16S rRNA gene sequencing


analysis

Isolate # Closest known species % ID


1 Bacillus aquimaris 99
Bacillus vietnamensis 99
2 Paenibacillus amylolyticus 99
3 Bacillus pumilus 100
Bacillus altitudinis 100
Bacillus aerophilus 100
4 Bacillus baekryungensis 99
Bacillus hwajinpoensis 99
8 Paenibacillus amylolyticus 99
10 Bacillus hwajinpoensis 99
Bacillus baekryungensis 99
11 Bacillus cereus group: 100

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B. cereus; B. Thuringiensis; B. Anthracis
12 Bacillus mycoides 100
Bacillus weihenstephanensis 100
Bacillus cereus group 99
14 Bacillus pumilus 100
Bacillus altitudinis 100
Bacillus aerophilus 100
Bacillus stratosphericus 100
15 Bacillus baekryungensis 99
Bacillus hwajinpoensis 99
22 Paenibacillus amylolyticus 100
Paenibacillus pabuli 99
23 Bacillus aquimaris 99
Bacillus vietnamensis 99
24 Bacillus lehensis 96
27 Bacillus baekryungensis 99
Bacillus hwajinpoensis 99
42 Bacillus algicola 99
43 Bacillus lehensis 96
49 Bacillus licheniformis 100
51 B. cereus group: 100
B. cereus; B. Thuringiensis; B. Anthracis
52 B.cereus group: 100
B. cereus; B. Thuringiensis; B. Anthracis
56 Bacillus pumilus 100
Bacillus altitudinis 100
Bacillus aerophilus 100
73 Bacillus barbaricus 99
Bacillus arsenicus 99
74 Bacillus hwajinpoensis 100
Bacillus baekryungensis 99
78 Bacillus baekryungensis 99
Bacillus hwajinpoensis 99
83 Bacillus baekryungensis 99

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Bacillus hwajinpoensis 99
84 Bacillus pumilus strain 100
91 Bacillus licheniformis 100
146 Bacillus hwajinpoensis 100
Bacillus baekryungensis 99
147 Bacillus licheniformis 100
MMA7 Bacillus subtilis 100
CH8a B.cereus group: 100
B. cereus; B. Thuringiensis; B. Anthracis

Table 2. Other strains and plasmids used in this study.


Strain/Plasmid Relevant properties/origin
Chromobacterium violaceum DSM30191 Biosensor; Inhibition of violacein production
indicates QSI activity. DSMZ.
P. aeruginosa PA01 Positive control for QSI assay, host for
pMM11aiiA construct. HDCC, UCC.
E. coli DH5α Host for pBBR1MCS5. HDCC, UCC.
E. coli HB101 Host for pRK2013. HDCC, UCC.
pBBR1MCS5 Broad – host – range vector, gentamicin
resistant. Kovach et al. 1995.
pRK2013 Helper plasmid for mobilization of pBBR1MCS5
derivatives, kanamycin resistant. HDCC, UCC.
pMM11aiiA Construct containing coding region of lactonase
Gene aiiA from sponge isolate #11.

^DSMZ – DSMZ cell culture collection, Braunschweig, Germany. HBCC, UCC – Microbiology
department culture collection.

Screening for quorum – sensing inhibitory activity of sponge associated sporeformers

Since sponge associated sporeformers are quite peculiar in their growth properties, it was
necessary to select a suitable medium for growth. Initially, attempts to develop an assay
were carried out in marine agar/broth (MA/MB) and subsequent attempts included LB agar,
nutrient agar/broth (NA/NB) and Mueller Hinton agar (MH). Overnight cultures of the 30

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sporeformers (in MB and/or NB) were streaked or spotted (5 µl) onto a 20 ml plate. In some
cases, multiple test sporeformers were streaked/spotted on different sectors of the same
plate. After 24 – 48 h incubation at 30 °C, plates were overlayed with 10 ml nutrient soft
agar (NSA) inoculated with 150 µl overnight C. violaceum culture (overnight cultivated in
5 ml NB and/or MB at 30 °C with agitation) . Plates were then incubated at 30 °C and
monitored for zones of lack of pigmentation (positive result) at 24, 48 and 72 h. PA01 was
used as a positive control for all assays carried out.

The final optimised assay involved overnight cultivation of the sporeformers in MB and NB
which were streaked on a 20 ml NA plate, incubated for 48 h. Presence of growth was
recorded and plates were overlayed with the biosensor and monitored for a zone of lack of
pigmentation at 24, 48 and 72h.

Screening of the metagenomic library for potential QSIs

Figure 4. Outline of the procedure for screening the metagenomic library. See text for a
detailed explanation.

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A metagenomic library constructed from total DNA from the sponge Haliclona simulans
using the CopyControl Fosmid Library Production Kit (Epicentre) was used (prepared by Dr
David Lejon). This library consists of ~60,000 fosmids stored in 384 well plates, with each
fosmid containing 35 - 40 kb of metagenomic DNA present as a single copy until induced by
arabinose. Clones contain a chloramphenicol resistance cassette. 27,648 clones from the
library were screened for QSI activity. Before every experiment the metagenomic library
was replicated overnight in a storage medium (1 L: 10 g tryptone, 5 g yeast extract, 5 g NaCl,
6.3 g K2HP04, 1.8 g KH2PO4, 0.45 g Na3 – citrate, 0.9g (NH4)2S04, 0.09g MgS04.7H2O, and 44
ml glycerol; autoclaved 121 °C for 15 mins) with chloramphenicol in 384 well microtiter
plates. 384 clones were provided per 384 well microtiter plate and the first screen involved
application of six 384 well plates to one 20 cm2 Q plate (Genetix). 260 ml LB containing
arabinose (0.01% solution) and chloramphenicol was poured in a 20 cm2 Q plate (Genetix).
The metagenomic library was then applied to the Q plates by an automated robot. After 48
h, the Q plates were overlayed with 100 ml nutrient soft agar containing 0.75 ml
C. violaceum overnight culture. The plates were dried and incubated at 37 °C and the results
were recorded at 24 and 48 or 72 h.
The second screen (and a repeat of the second screen to establish reproducibilty) was
repeated under the same conditions as above but with one 384 well plate per Q plate to
expand the size of the clones and refine the search from areas of interest in the first screen
(where the clones were tightly packed together).

Cloning of the aiiA gene from B. Cereus group isolates #11 and CH8a into PA01

The coding sequence of the lactonase gene (aiiA) from isolates #11 and CH8a was amplified
by PCR using the primers described by Lu and colleagues (Lu et al., 2006) using different
cloning sites than those described by the authors. The sequences of the degenerated
primers were aiiA-F, 5’ CCAAGCTTATGACAGTAAARAARAARCT 3’ with HindIII (underlined)
site added before the 5’ start codon and aiiA-R, 5’ TTTGGATCCCTATATATAYTCHGGG 3’ with
BamHI site (underlined) added behind the 3’ stop codon. (R: G or A; Y: T or C; H: A, T or C).
The reagents used for the PCR (50 µl reaction) were as follows: 5 µl 10 x Pfu buffer, 1.5 µl
MgCl2 (50 mM), 1 µl dNTP (10 mM), 1 µl aiiA-F (25 pmol ml-1), 1 µl aiiA-R (25 pmol ml-1), 2 µl
Pfu DNA polymerase (Promega)( 2-3U/µl) , 37.5 µl dH20 and 1 µl genomic DNA.

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Optimisation of the MgCl2 concentration proved to be essential for amplification.
The PCR conditions involved denaturation at 94 °C for 5 minutes, followed by 30 cycles of
denaturation at 94 °C for 30 seconds, annealing at 52 °C for 30 seconds, and extension at
72 °C for 2 minutes and a final extension at 72 °C for 10 minutes. PCR products were
purified using the QIA quick PCR purification kit (Qiagen, GmBH Germany) before and after
restriction digestion.
The cloning vector pBBR1MCS5 (fig. 2) was purified from an overnight culture of E. coli
DH5α using the QIAprep Spin Miniprep Kit (Qiagen), restricted with HindIII and BamHI
(double digest) and purified from a 0.8% agarose gel using the QIAquick Gel Extraction Kit
(Qiagen).

Figure 5. Broad – Host - Range cloning vector pBBR1MCS5. Arrows point to our selected
restriction sites HindIII and BamHI (figure from LCG-UNAM-MEXICIO).

The double digested inserts were then ligated to the vector using T4 DNA ligase (Promega)
in a standard ligation reaction. Chemically competent DH5α cells were used for
transformation of ligated DNA and transformants were selected on gentamicin. Clones were
confirmed by by restriction analysis. Fidelity of amplified sequences in the resulting
construct pMM11aiiA was confirmed by sequence analysis with the M13 forwar primer in
the vector (GATC, Biotech AG, Konstang, Germany).

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Plasmid pMM11aiiA containing the aiiA gene and the vector pBBR1MCS5 were transformed
into PA01 by triparental mating with the helper strain E. coli HB101 bearing plasmid
pRK2013 as follows:
Overnight cultures of the following were prepared: 5 ml Donor, 5 ml Helper and 5 ml
recipient which were centrifuged at 4,000 rpm for 7 minutes, the supernatant discarded and
the pellet resuspended in 1 ml Ringers solution 1/4 strength (kindly provided by Julianna O’
Callagan), centrifuged at 4,000 rpm for 5 minutes, the supernatant discarded and the pellet
resuspended in 100 µL ¼ Ringers.
20 µL donor, 20 µL helper and 40 µL of recipient were combined and 100 µL spotted onto
the middle of LB/Gentamicin 30 µg ml-1 plates which were incubated overnight at 37 °C.
The following day, transformants were selected on M9 medium by resuspending growth in
¼ Ringers and plating serial dilutions (10-1 to 10-3) on M9 medium (1.5% agar) containing
citrate (30%), MgSO4 (1 M), FeCl3 (50 mM) and gentamicin (30 µg ml-1). The plates were
then incubated at 37 °C overnight.

Pyocyanin production assay


Strains PA01-pMM11aiiA and PA01-pBBR1MCS5 were grown at 37 °C in LB broth
supplemented with gentamicin (30 µg ml-1) and IPTG (10 µg ml-1) for induction of the aiiA
gene. Differences in coloration were noted.

Swarming motility assay


The swarming motility assay was performed as described by Rashid & Kornberg (Rashid &
Kornberg., 2000). 5 µL of overnight culture supplemented with Gentamicin and IPTG as
described above (PA01-pMM11aiiA and PA01-pBBR1MCS5) was spotted in the centre of an
Eiken agar plate supplemented with gentamicin and IPTG as described above. Plates were
incubated at 37 °C for 24 h and the swarming patterns noted.

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Results

Screening for quorum – sensing inhibitory activity of sponge associated sporeformers


The media tested for growth of the sporeformers and violacein production by C. violaceum
was Marine agar (MA), Nutrient agar(NA), Mueller – Hinton agar (MH) and LB agar (LB). All
sporeformers grow on MA but growth and violacein production by C. violaceum is very poor,
unlike on NA (fig 6a and 6b). The area of non – pigmentation surrounding a QSI producer is
also very distinctive in NA (fig. 6a and b). Some sporeformers have very specific nutritional
requirements, not satisfied by NA, LB or MH. Violacein production is strong and
homogenous in LB (fig. 6c) but is less so in MH (fig. 6d). In overlayed sporeformers which
showed a zone of clearing, the halo was non – transparent and opaque indicating that the C.
violaceum cells were viable (i.e the halo is not due to non – growth of C. violaceum). It was
determined that Nutrient medium was the most reliable for the assay and all 30
sporeformers were assayed on this medium.

Figure 6. Testing violacein production by C. violaceum in different media with PA01 and
some test sporeformers to evaluate the distinctiveness of any zone of non – pigmentation

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surrounding growth. (a) A 5 µL spot of PA01 (overnight culture in NB) on an MA base (left)
and an NA base (right) shown 48 h after overlaying as described. (b) A CH8a streak on an MA
and NA base 48 h after overlaying. (c) A 5 µL spot of test sporeformers on the NSA overlay
with an LB base. (d) A 5 µL spot (MB) of test sporeformers on the NSA overlay with a
Mueller – Hinton base.

In screening for sporeformer QS inhibition, four separate assays were performed, all in the
same manner except for assays 3 and 4 which had a streak from a marine broth overnight
culture (to give those sporeformers which grew poorly in NB a better opportunity to
become established and grow on an NA plate) in addition to the nutrient broth culture (fig
4. and 3a). Table 4 shows results of the four assays. Sporeformers #1, #3, #11, #12, #23, #51,
#52, #56 and CH8a consistently showed QSI activity highlighted in red (fig. 7 and fig. 8). Five
of the sporeformers (highlighted in green) did not grow in NA and therefore it could not be
determined if they produce any QSIs. Nine grew in NA but did not produce QSIs detectable
by this assay (blue) and the results of six sporeformers (purple) are inconclusive.

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Table 4. Results of the four assays screening H. simulans associated sporeformers^.

Sporeformer # Growth in Nutrient agar after 48 hr QSI producer


A1 A2 A3 A4 A1 A2 A3 A4
#1 + + + + Yes F Yes F Yes F Yes F
#2 + ND + +/- No ND No No
#3 + + + + Yes Yes Yes Yes
#4 - - IC +/- ND ND No Yes F
#8 + + + ND No No No ND
#10 +/- +/- ND +/- No No ND No
#11 + + + + Yes Yes Yes Yes
#12 + + + + Yes Yes Yes Yes
#14 + + + + No No No No
#15 - +/- +/- +/- ND No No No
#22 + +/- +/- +/- No No No Yes F
#23 +/- +/- ND +/- Yes F Yes F ND Yes F
#24 - - ND +/- ND ND ND No
#27 - - ND +/- ND ND ND No
#42 - - ND +/- ND ND ND No
#43 - - +/- +/- ND ND No No
#49 + + + + No No No No
#51 + + + + Yes Yes Yes Yes
#52 + + + + Yes Yes Yes Yes
#56 + + + + Yes Yes Yes F Yes
#73 ND + ND + ND No No Yes
#74 - - - +/- No ND No Yes
#78 IC - ND IC No ND ND Yes
#83 - - IC IC ND ND No Yes
#84 + + + + No No No No
#91 + + + + No No No No
#146 - - ND - ND ND ND No
#147 + + + + No No No No
CH8a + + + + Yes Yes Yes Yes
MMA7 + + + + No No No No
^ Legend: IC = Isolated colonies, Yes F = Yes but faint zone of non pigmentation, ND = Not
determined, - = no growth, + = growth, +/- = weak growth,.

20
Figure 7. Sporeformer QSI assay. Left: Sporeformers #11, #12, #51, #52, #23, #56 and CH8a after 48
h growing at 30 °C .MB streak and NB streak per plate. Right: 24 h after overlaying with C. violaceum.

Negative control is an NA base


with no growth overlayed

Figure 8. Sporeformer assay. Explained in figure. MMA7, which showed no area of non –
pigmentation surrounding growth is included as a comparison to those that produce QSIs.

21
Screening of the metagenomic library for QSI

Screen 1 was carried out in two rounds, with six 384 well microtiter plates per Q plate.
There were 2,304 clones per Q plate and the total number of clones screened in the initial
screen was 27,648 clones. A halo (zone of non pigmentation) surrounding an individual
clone was considered a potential hit and its approximate position on the plate noted. This
occurred for 18 microtiter plates (red), and these were rescreened with one microtiter plate
per Q plate, where it would be possible to get the exact coordinates of a clone of interest.
As is apparent in table 5, there were false positives (e.g, no halo recurred on plate 33).
However there were also very distinctive haloes (for example, see fig. 12). A repeat of the
second screen was carried out and of the 13 clones identified (Figs 9, 10 and 11 shows a
representative sample), it was only possible to establish the reproducibility of two clones as
QSIs (O3 from plate 1001 and M23 from plate 1002) (Fig 9 and 10).

Table 5. Summary of the results of the metagenomic library screen for QSI.

Screen 1: Six microtiter plates Screen 2: One microtiter plate Repeat of Screen 2: To
per Q plate – plate numbers. per Q plate. Rescreened plate establish the reproducibility of
Red plate numbers show number and coordinate of screen 2
clone(s) of interest (possible clone shown
QSI producers)
1001, 1002, 1003, 33, 34, 35 1001: O3. 1002: M23. 1001: O3. 1002: M23.
33: Negative. 34: F16. 33: Negative. 34: Inconclusive.
1004, 1005, 1006, 138, 139, 1004: Negative. 138: Negative. 1004: Negative. 138: Negative.
140 139: Negative. 139: Negative.
1007, 1008, 1009, 141, 142, 1007: Negative. 142: E8, P23, 1007: Negative. 142: E8
143 G7. 143: I18, K18, L20. negative, P23 negative, G7
Positive. 143: I18 Negative, K18
Negative, L20 inconclusive.
1010, 25, 26, 144, 145, 146 None repeated None repeated
27, 28, 29, 147, 83, 84 None repeated None repeated
30, 31, 32, 85, 86, 87 None repeated None repeated
88, 89, 90, 156, 157, 158 None repeated None repeated

22
91, 92, 93, 159, 1011, 1012 92: Negative. 1012: Negative. 92: Negative. 1012: D11.
94, 95, 136, 1013, 1014, 1015 136: A12 C11 and P3. 136: A12 Inconclusive, C11
Negative, P3 Negative.
137, 148, 149, 1016, 1017, 137: Negative. 137: Negative.
1018
150, 151, 152, 1019, 1020, None repeated None repeated
1021
153, 154, 155, 1022, 1023, 153: Negative. 154: F14. 1022: 153: Negative. 154: Negative.
1024 Negative. 1024: Negative. 1022: Negative. 1024: Negative

Figure 9. Clone M23 produced a halo in both screens. A small zone of non - pigmentation in
the 1st screen (not shown) on plate 1012 prompted a rescreen (“2nd screen”) which
identified clone M23 as a potential QSI producer. A repeat of the second screen showed a
less prominent but still noticeable clearing surrounding the clone.

23
Figure 10. Clone 03 gave reproducible results. Half of plate 1001 (Coordinates A to H not
shown) showing (a) the distinctive zone of clearing surrounding clone O3 in the second
screen and (b) in a repeat of the second screen.

Figure 11. Second screen and its repeat for plate 154 and 136. (a) Plate 154, second screen
pictured after 72h (Top image) showing the clearing surrounding clone F14. A repeat of the
screen (bottom image) did not highlight F14. (b) Plate 136, emphasising clones A12, C11 and
P3 – not replicated in rescreen (bottom image).

24
Figure 12. Prominent potential putative positives. (a) Clones I18, K18 and L20. (b) Clone D11.

Cloning of aiiA gene into PA01


PCR amplification of aiiA from both #11 and CH8a was successful, however transformation
of aiiA from CH8a was unsuccessful and we proceeded successfully with aiiA from #11 (fig.
13). Triparental mating was successful (fig. 14a), however sequencing of the construct and
BLAST analysis showed that 4 codons were missing from the start of the aiiA gene (i.e, 4
amino acids not coded for making the aiiA insert inactive) (fig. 14b)

Figure 13. 0.8% agarose gels showing successful amplification of aiiA and transformation.
(a) Successful PCR amplification of aiiA from #11 and CH8a with Pfu polymerase (two empty
lanes after the marker was a PCR using different conditions). (b) aiiA #11, aiiA CH8a and
pBBR1MCS5 shown after digestion. (c) Confirming the presence of the insert in

25
transformants. 1 = pMM11aiiA cut with HindIII and BamHI (aiiA insert released). 2 = pBBR
cut with HindIII and BamHI. 3 = pMM11aiiA cut with SalI (Linearises construct – 710 bp aiiA
+ 4768 bp vector = 5478 bp band). 4 – PBBR cut with SalI. 5 = pMM11aiiA cut with SpeI and
ClaI (releases aiiA insert). 6 = pBBR cut with SpeI and ClaI.

Figure 14. Successful cloning of aiiA:#11 into PA01. (a) Confirming the presence of
pMM11aiiA in PAO1. The first lane shows the presence of the construct in several forms
(Linear, coiled and suprcoiled in descending order). The second lane (beside the marker)
shows the vector in the same forms. Note that the bands showing the construct are larger
and thicker than those of the vector. (b) A BLAST search of the aiiA sequence from the
construct showed that 12 nucleotides (4 codons) were missing from the aiiA insert (based
on a comparison with the top match B. thuriniegensis).

PA01 virulence factor assays


The swarming motility assay and the pyocyanin production assays were carried out before
the results of the sequencing were known. No differences in swarming or pyocyanin
production (PA01-pMM11aiiA compared to PA01-pBBR1MCS5) were observed, which was
not surprising given that the insert was found to be inert (4 amino acids not coded for) after
BLAST analysis of the sequence.

26
Discussion

Increasing levels of antibiotic resistance coupled with few recent discoveries of effective
bactericidal drugs makes it a priority to screen for and develop novel therapeutic agents.
One such approach may be through the use of bacteriostatic agents such as quorum sensing
inhibitors, the focus of this study. The screening strategy of this project is a good example of
the modern approach in characterising novel microbes and elucidating their metabolic
potential, especially in regard to the production of novel bioactive compounds which could
have biotechnological or pharmaceutical applications. Firstly, culturable H. Simulans isolates
were previously identified down to the genus and species level by 16S rRNA sequencing to
establish bacterial molecular phylogeny (Mignard & Flandrois., 2006). After the isolates
were identified by BLAST analysis (table 1) (The results of the 16S rRNA sequencing and
BLAST analysis was made available to us prior to commencement of the study), the next
approach was to develop a reliable phenotypic assay to screen for quorum sensing
inhibitors (QSIs).
In addition, we also followed a culture – independent approach by screening a metagenomic
library of total DNA from H. simulans using the assay developed for screening the culturable
isolates (i.e, an activity based screen).
First used as a biosensor in 1997 by McClean and colleagues (McClean et al., 1997),
Chromobacterium violaceum has since been extensively used to detect QSIs in assays with
various different approaches. The common feature of all assays involving the wild type C.
violaceum is its strong purple pigmentation, which is the compound violacein, production of
which is regulated by AHL mediated quorum sensing. QSIs (such as lactonases) inhibit this
QS mediated violacein, causing inhibition of violacein production, leading to a distinctive
zone of non pigmentation surrounding the QSI producer. This biosensor was selected for its
distictive qualitative aspect (i.e., Is there a zone of non – pigmentation surrounding the test
marine isolate? If so, we have identified a QSI producer) and the relative ease of screening.
To develop a reliable assay for our particular H. Simulans isolates, it was necessary to test
the growth of these isolates (all sporeformers) in different media, test the growth
consisitency of violacein production by C. violaceum in the media and to strike a balance
between these considerations to select the medium in which to perform the assay. C.
violaceum exhibits exceptional adaptability to both environmental and nutritional

27
variability, observed at a phenotypic level and confirmed by elucidation of its complete
genome sequence (Brazilian National Genome Project Consortium, 2003). Therefore, it was
expected to grow on most media tested.
Tryptophan is believed to be the only precursor required for violacein biosynthesis and the
carbon source and its concentration in the medium also have an impact on violacein
production (Antonio & Creczynski-Pasa, 2004).
In our test assays growth and violacein production are almost completely absent on the
marine agar overlay. The reason for this is not known however some component of marine
agar must be inhibitory. Tryptone (a rich source of tryptophan) is present in all media
tested. Tryptone is present in LB (10 g L-1) and in Nutrient agar and Mueller – Hinton agar as
part of the meat extract composition (3 g L-1 and 2 g L-1 respectively). This is consistent with
the assay findings, as violacein was consistently produced in LB, nutrient medium, and
Mueller – Hinton, although the latter tended to have more faint pigmentation (fig 3d).
A selection of the 30 sporeformers were tested for growth on the media described above
and it was noted that some of them were particularly fastidious and were unable to grow in
LB, NA or MH. Due to the strong and homogenous pigmentation of C. violaceum on the NSA
overlay with an NA base, and the fact that C. violaceum did not grow/produce pigment on
MA, NA was ultimately selected.
In testing the 30 sporeformers, we were already aware that CH8a and #11 had the aiiA gene
for lactonases as all of the sporeformers were PCR screened by others in the laboratory
using primers designed by Lu and colleagues (Lu et al., 2007). Additionally, Bioinformatics
analysis identified the presence of the catalytic residue Tyr-194, which is highly conserved in
AHL-lactonases (Lu et al., 2006). A negative result in the PCR screen however could not rule
out the presence of a lactonase encoding gene in the other isolates that could not be
detected with these specific primers. On the other hand, identification of the presence of
the aiiA gene does not tell us if this gene is expressed in this host or elsewhere.
Results of the assay confirm that the gene is expressed in CH8a and #11. In addition, marine
isolates #1, #3, #12,#23, #51, #52 and #56 (table 4) consistently showed inhibition of
violacein production surrounding their streaks in multiple assays leading to the finding that
these produce QSIs. No finding can be made on the 6 sporeformers which gave inconsistent
results (see table 4). The 9 sporeformers which grew but did not inhibit violacein cannot be
ruled out as QSI producers. All we can conclude is that they did not produce a QSI

28
detectable by a C .violaceum based assay. Of the nine sponge – associated sporeformers
which demonstrated some level of inhibition of violaceum pigmentation 5 belonged to the
B. cereus group (#11, #12, #51, #52, CH8a), two to B. pumilis/B. altitudinis/B. aerophilus
group (#3 and #56) and the final two being part of the B. aquimaris/B. vietnamensis group
(#1 and #23). Previous studies have demonstrated the widespread presence of the
lactonase gene, aiiA among strains belonging to the B. cereus group (Dong, 2000). Despite
this a previous PCR screening in this laboratory failed to identify these sequences, with the
exception of isolates CH8a and #11.
The finding that 9 of the 30 (or 30%) culturable sporeformers produce QSIs is in general
agreement with research by Kanagasabhapathy and colleagues (2009) who concluded that
12% of bacteria associated with the marine brown alga Colpomenia sinuosa produce QSIs.
The higher percentage for sponges would be expected as they tend to have a larger
symbiont population with more complex activities (Mohamed et al., 2006). The major
limitation of our assay was that 8 of the sporeformers could not be assayed due to non –
growth or very poor growth on nutrient medium. This problem was overcome by
Kanagasabhapathy and colleagues (Kanagasabhapathy et al., 2009) who used Serratia
rubidaea JCM 14263 as a QSI biosensor. This biosensor was similar in mechanism to C.
violaceum but it was salt tolerant, allowing a marine agar based assay. This strain will be
considered for future QSI assays of marine isolates.
The next stage of screening the marine isolates would involve carrying out a Southern blot
using specific sequences as probes to identify the presence of known QSI encoding genes
(e.g aiiA gene in lactonases).

As the vast majority of marine isolates are not culturable, a culture – based screening
approach has significant limitations. The metagenomic approach is a culture independent
method that involved extraction of genomic DNA from H. simulans, cloning of the DNA into
pCC1fos and construction of a library which was transformed into a host (E. coli in this case)
which was then screened for QSI activity. To our knowledge, this is the first study in which a
metagenomic library from the marine sponges was screened for QSIs using a biosensor such
as C. violaceum. Using the C. violaceum based assay we screened 27,648 clones initially. Any
clone which produced a zone of non – pigmentation was noted, however due to the the
tightly packed nature of the clones (six 384 well plates per Q plate) on the plate, it was not

29
possible to determine the exact coordinates of the clones. The purpose of our initial screen
was to refine our search to 384 well plates which contain clones which could be QSIs. Of the
seventy - two 384 well plates initially screened, 18 of them had at least one potential clone
with QSI activity. These 18 were rescreened twice and 13 putative positives were identified,
of which we could only establish the reproducibility of two: Clone M23 and clone O3.
Regarding the other 11, eight clones produced a zone of non pigmentation in the second
screen, but not when repeated. It is possible that a number of these could be false positives,
however they would still be considered as potential QSIs until ruled out but further screens
will need to be performed in order to establish if these are false positives or lack of
reproducibility was associated with mistakes introduced in specific assays. Importantly some
of these clones produced very distinctive zones of lack of pigmentation those that produced
very distinctive zones of non pigmentation (as in figures 11 and 12). We observed an
unusual pattern of violacein production in our assays. The centre of the plate containing no
clones was often almost free of violacein other than isolated spots whereas the sectors
containing the clones mostly showed strong violacein production. We speculate that this
may occur for one of two reasons. Firstly, that C. violaceum produces violacein as a
starvation response when growing over the sectors containing the clones as this is an area
of reduced nutrient availability. Alternatively, some signal produced by the E. coli clones is
inducing violacein production.

Due to time constraints it was not possible to carry out further assays to confirm whether or
not a clone was definitively a QSI producer. Future work should focus on this and inserts of
reproducible putative positive clones should be sequenced and analysed to try to identify
the source of QSI activity. As per all activity based assays of metagenomic libraries, the most
significant limitation is the requirement of the surrogate host to be able to express the
cloned genes and synthesise the resulting gene products (Lorenz & Schleper, 2002).

Should any of the bioactive compounds produced by the culturable isolates or the
metagenomic library be deemed to have potential as therapeutic QSIs, the producer could
be cultivated on a large scale or if this were not feasible, the producers biosynthetic genes
could be isolated and the biosynthetic pathway expressed in a culturable bacteria (Schmidt,
2008). However, the latter approach would require knowledge of the isolates taxonomy

30
which may be lacking due to the huge prokaryotic diversity associated with sponges.
Consequently, selection of an appropriate expression host would be a major limitation
(Hochmuth & Piel, 2009).

Research has shown that the QS system in P. aeruginosa is a significant factor in its
pathogenicity, mediating biofilm formation, production of toxins such as pyocyanin and
affecting motility (Smith et al., 2002; Wang et al., 2007).
We attemped to clone the lactonase gene (aiiA) from the sponge B. Cereus group isolates
marine isolates CH8a and #11 into P. aeruginosa PA01 to examine if the expression of aiiA
would affect the expression of different virulence factors such as pyocyanin production and
swarming motility. We used the primers described by Lu (Lu et al., 2006) and colleagues but
selected different restriction sites to suit our cloning vector. After selecting the HindIII and
BamHI restriction sites, we analysed the sequences obtained from the aiiA PCR screening of
isolate #11 and CH8a for the presence of these restriction sites and none were found.
Amplification of the gene from isolates CH8a and #11 was successful as was the ligation with
the vector, however transformation of the CH8a construct was not successful. We
proceeded with the construct containing the aiiA gene from #11 was successfully cloned
into P. aeruginosa PA01. No differences were noted in phenotype between PAO1-
pMM11aiiA and PA01-pBBR1MCS5 in regard to pyocyanin production and swarming
motility. This was not in agreement with a study by Wang and colleagues (Wang et al., 2007)
who found that a PAO1 mutant containing the coding region of the aiiA gene from Bacillus
thuringiensis degraded PA01 AHLs leading to decreased biosynthesis of pyocyanin and
negatively impacted swarming motility, among other virulence factors. Because a BLAST
search of aiiA from isolate #11 against the database showed the closest similarity was with
an aiiA gene from a strain of B. thuringiensis, we were expecting similar findings.
Upon receipt of the aiiA sequence in pMM11aiiA a BLAST analysis revealed that 12
nucleotides were missing from the start of the coding region of the aiiA gene, effectively
making the gene inert. This was very surprising as we had checked the sequence for HindIII
sites before selecting restriction sites and none were discovered. Upon consideration of the
matter, we determined that there was a HindIII restriction site within the first twenty
nucleotides of the aiiA gene that were missed from the sequencing of the PCR product

31
obtained in the initial screen. Therefore, the repeat of this experiment will need to make
use of a different restriction site.

Conclusion
Although the ecological and biotechnological significance of the sponge-microbe symbiosis
is now better understood, there are major limitations due to the inability to culture most
microbes. Even a culturable isolate which produces a certain bioactive compound in its
natural environment may not do so on artificial laboratory growth media. This study
attempted to overcome the non - culturable limitation by screening a metagenomic library
and the results were promising although further work would need to be carried out before
we draw any definitive conclusions. Quorum sensing is undoubtedly implicated in the
virulence of many pathogens and attempts to screen for QSIs and develop them as
therapeutic agents continues to be the focus of intensive research. Our contribution
provides an example of the methodology in screening for QSIs, where some potential
positives were identified which future researchers may expand on and optimise.

Acknowledgements

I would like to thank my supervisor Dr. Teresa Barbosa for her sound advice and patience
during this project. Thanks also to Robert Phelan, a postgraduate student in the lab who
kindly offered advice and to Mark McCarthy for operating the robot which applied the
metagenomic library.

32
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