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Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Edward Llwyd Building, Penglais Campus, Aberystwyth, Ceredigion, Wales UK, SY23 3DA. Details of the fellowship Contact in UK: Dr Sharon Huws The Institute of Biological, Environmental and Rural Sciences (IBERS) Edward Llwyd Building, Aberystwyth University, Aberystwyth, SY23 3DA U.K. Tel: +44 1970 823202 E mail: [email protected] Research and Fellowship Dates: 1 January 30 April 2014 Background and purpose of fellowship The main Purpose of the Fellowship was to undertake a research project contributing to an existent BBSRC funded grant centred upon increasing understanding of the function of rumen microorganisms in terms of their effects on meat and milk production as a cause of their interactions with forage.
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Page 1: Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin · 2019. 9. 16. · Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin Institute of Biological, Environmental

Report to the Stapledon Memorial Trust

Dr Lucy A. Akinmosin

Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,

Edward Llwyd Building, Penglais Campus, Aberystwyth, Ceredigion, Wales UK, SY23 3DA.

Details of the fellowship

Contact in UK:

Dr Sharon Huws

The Institute of Biological, Environmental and Rural Sciences (IBERS)

Edward Llwyd Building,

Aberystwyth University,

Aberystwyth, SY23 3DA U.K.

Tel: +44 1970 823202

E mail: [email protected]

Research and Fellowship Dates: 1 January –30 April 2014

Background and purpose of fellowship

The main Purpose of the Fellowship was to undertake a research project contributing to an

existent BBSRC funded grant centred upon increasing understanding of the function of

rumen microorganisms in terms of their effects on meat and milk production as a cause of

their interactions with forage.

Page 2: Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin · 2019. 9. 16. · Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin Institute of Biological, Environmental

Research report 1

Title

Perturbation of the forage attached rumen microbiome through addition of rumen

protozoa increases plant fermentation

L.A. Onime, T. Robinson, M.B. Scott, A.H. Kingston-Smith, S.A. Huws

Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University,

Penglais Campus, SY23 3DA, UK.

Running Title: Protozoa affect plant degradation within the rumen

Keywords: Rumen, protozoa, bacteria, plant degradation, fermentation gas production

*Correspondence: Lucy A.Onime, Animal and Microbial Sciences, Institute of Biological,

Environmental and Rural Sciences (IBERS), Aberystwyth University, Edward Llwyd

Building, Penglais Campus, Aberystwyth, Ceredigion, UK, SY23 3DA.. E-mail:

[email protected]. Tel: +44 1970 823631

Significance and Impact of the Study: The role of protozoa within the rumen is widely

disputed with some data suggesting that absence of protozoa increases ruminant nutrient use

efficiency. In this study we demonstrate that presence of the rumen protozoa increases plant

degradation and fermentation, suggesting that their presence increases the nutrients available

to the ruminant following feeding on fresh perennial ryegrass.

Abstract

The effects of presence or absence of rumen protozoa on the density and diversity of fresh

perennial ryegrass (PRG) attached microbiota, dry matter degradation and gas production was

monitored, in vitro, over time. QPCR data showed significant (P<0.05) increases in PRG

attached total bacterial 16S rDNA (3.32 log10g-1

vs. 2.1 log10g-1

)and Prevotella 16S rDNA

(3.21 log10g-1

vs. 2.23 log10g-1

) concentration following 2 h of incubation in the presence of

protozoa compared with absence of protozoa. However no differences in 16S rDNA

concentration were seen at all other incubation times. Plant degradation results revealed that

PRG degraded to a greater extent (P= 0.051) after 4 h of incubation in the presence of

protozoa compared with their absence. Cumulative gas production was also significantly

higher (P<0.001) in the presence of protozoa than in their absence. Terminal restriction

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fragment length polymorphism (T-RFLP) generated dendrograms demonstrated that the

attached microbiota showed clustering dependent on the absence/presence of protozoa at 2 h

post incubation only. This study suggests that presence of protozoa increase the degradation

and fermentation of perennial ryegrass within the rumen and therefore probably the nutrients

available to the ruminant.

Introduction

The rumen is highly complex but stable environment that contains a diverse microbiota and

microflora, many of which are as yet unculturable (Edwards et al. 2008; Kingston-Smith et

al. 2010). The microbiota and microflora have complex interactions and relationships which

makes it challenging to understand their functionality. Studies on the microbial ruminal

microbiota have shown that there is substantial functional redundancy which increases their

resilience but increases the challenge of understanding their function (Edwards et al. 2008).

Rumen bacteria are generally credited for playing a crucial role in the breakdown of

plant material in the rumen due to their prevalence and ability to metabolise a large variety of

organic matter. However rumen protozoa, which make up a large biomass in the rumen, also

possess cellulolytic activity and consequently have been suggested to play a role in digestion

of cellulose post ingestion of forages by ruminants (Coleman 1986; Ricard et al. 2006).

Nevertheless it has been established that ruminants can survive without ciliated protozoa

thereby making their role controversial (Williams and Coleman 1992). It has also been

observed that defaunation of ruminants can increase ruminant productivity and decreases

methanogenesis and total-tract digestion (Franzolin and Dehority 1996; Koenig et al. 2000;

Belanche et al. 2011) without any critical consequences to the animal and to the persisting

microbial population. Rumen protozoa are known to function in the rumen by recycling

ruminal microbial N (Ushida et al. 1990), producing steady ruminal fermentation, relatively

lower numbers of bacteria by ingesting them, increase in percentage dry matter, liquid

volume and turnover rate of ruminal contents (Veira 1986).

Our previous work showed that PRG colonising bacteria in the rumen were enveloped

in extracellular polymeric substances which implies biofilm production(Huws et al. 2013).

This process involved 2 main phases which encompasses primary colonising bacteria which

later detach to an extent from plant to be partially replaced with a population of secondary

colonising bacteria between 2 – 4 h after entry of PRG into the rumen. Some protozoa

species have been reported to have an impact on development of biofilm communities;

however protozoan predation of biofilms is hypothesized to be sustainable and doesn’t aim to

drastically reduce the community (Huws et al. 2005; Alexander et al. 2008).

In this study we determined the effects of rumen protozoa on the attached biofilm

diversity and function. Specifically, we examined the effect of protozoa on the density and

diversity of fresh perennial ryegrass (PRG) attached microbiota, dry matter degradation and

gas production, in vitro.

Materials and methods

Plant growth conditions

Perennial ryegrass, (Lolium perenne cv. Aberdart; PRG) was grown from seed in plastic seed

trays filled with general purpose compost. Trays were housed in a greenhouse under natural

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light with supplementary illumination provided during the winter months (minimum 8h

photoperiod) or in a growth cabinet (Sanyo, Osaka) with 16 h of light (irradiance ~ 300 µmol

m-2s-1) per day. Temperature was maintained at 22/19°C for day/night and plants were

watered twice a week. After 6 weeks plants were harvested and cut 3 cm above soil level,

washed in cold distilled water and cut into 1 cm in length. 0h samples were also freeze-dried

and stored at -20oC for bacterial profiling.

In vitro incubation, in vitro dry matter degradability and gas production

A total of 2L of rumen fluid was collected from three rumen fistulated Holstein x Freisian

non-lactating cows and then passed through 3 layers of cheese cloth. The trials were

performed in triplicate in flasks (150 mL) containing 5g fresh perennial ryegrass cut into ~ 1

cm length particles used as substrate and a 10% (v/v) rumen fluid inoculum prepared in Van

Soest buffer.

In terms of protozoal preparation, protozoa from rumen fluid inoculum was prepared

in Van Soest Buffer as described by Huws et al. (2009). Microcopy was used to enumerate

the resultant protozoal numbers such that the added volume would result in approx. 5 x 104

protozoa/mL, in incubations with protozoa. For incubations without protozoa the supernatant

obtained after protozoa where pelleted was sonicated for 3 x 1 min with 1 min intervals on

ice to further ensure no protozoa were present. An equivalent volume of this preparation was

added to all incubations to ensure that the flask content were as similar as possible with the

only difference being presence or absence of protozoa. All flasks were put in shaking

incubator maintained at 100 RPM at 39°C.

At 1, 2, 4, 6 and 8 h time intervals gas production was monitored using a digital gas

syringe (Bailey & Mackey Ltd., Birmingham, UK,). The contents of the flasks were

subsequently vacuum filtered through filter paper to trap the plant material (11 µm2 pore

size; ®QL100, Fisher Scientific, Leicestershire, UK). Then the plant material was washed

with 200 mL PBS, to get rid of loosely attached microbes, and then placed in 4%

gluteraldehyde overnight at 4oC to remove attached microbes (efficient removal of bacteria

has previously been checked using scanning electron microscopy using the technique by

Huws et al 2013. The plant material was then vacuum filtered and washed (50 mL sterile

distilled water) with the resultant filtrate centrifuged at 13,000 rpm, for 20 mins. The

resulting pellets were stored at -20oC for later DNA extraction and T-RFLP analysis. Washed

plant material was placed in drying oven for 24-48 h (60°C) as described by (Van Soest et al.

1991), and the dry matter recorded.

DNA extraction

DNA was extracted from the microbial pellet residues using the FastDNA SPIN Kit for Soil

(QBiogene, Cambridge, U.K.) following manufactures instructions except that the samples

were homogenised and lysed in the FastPrep instrument (QBiogene, Cambridge, UK) for 3

intervals of 30 s with incubation of 30 s on ice between bead-beating The extracted DNA was

run on 0.8% agarose gel to check integrity and ultraviolet (UV) absorbance at 280, 260 and

230 nm were measured using EPOCH (BioTek, Bedfordshire, UK) .

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Terminal-Restriction fragment length polymorphisms (T-RFLP)

Amplification of 16S rDNA was accomplished in triplicate using primers 799F2 (FAM

labelled on 5′end) 5′-AACAGGATTAGATACCCTG -3’ and R1401 5’-

CGGTGTGTACAAGACCC -3’ as described by (Huws et al. 2009). All PCR amplifications

were performed using an Applied Biosystems 2720 thermal cycler (Applied Biosystems,

Warrington, UK). Equal amount DNA (50 ng) was added to a 25ul reaction mix containing 1

mM each primer, 0.8 mM dNTPs, 1.5 mM MgCl2, 50 mM KCl and 25 U Taq DNA

polymerase in 10 mM Tris/HCl (pH9.0). Thermal cycling conditions consisted of an initial 5

min denaturation then 30 cycles with 0.45 sec at 940C for denaturation, 45 sec at 55

0C for

annealing and 1.30 min at 720C for extension, and 7 min extension in the last cycle.

Amplicons were then pooled and purified using the QIAquick PCR purification kit (Qiagene,

West Sussex, U.K.). The concentration of the purified PCR product was determined by using

the Epoch spectrophotometer (BioTek, Bad Friedrichshall, Germany) and the samples were

then diluted to 20ng/ul. Restriction enzyme digestion was performed using HaeIII and Msp1

(Promega, Madison, USA) according to manufacturer’s instructions and incubated for 5 h at

37°C in order to produce terminal restriction fragments. The digested fragments were size

separated on an ABI3130xl DNA Applied Biosystems capillary sequencer (Life Technologies

Corporation, California, and USA), T-RFs were exported to excel to remove peaks below 50

formatted and imported into Gene mapper software (Applied Biosystems) to view the

profiles. Clustering analysis was undertaken with a band tolerance limit of ±0.5 bp and the

Pearson coefficient after data were imported into Bio-Rad fingerprinting (Bio-Rad,

Hertfordshire, UK).

16S rDNA Quantitative PCR

QPCR was performed on the total bacterial population and Prevotella spp as previously

described by (Huws et al. 2010). The following primers were utilised 520F 5’-

AGCAGCCGCGGTAAT – 3’ AND 799R2 5’ – CAGATCTAATCCTGTT – 3’, (Maeda et

al. 2003) and for Prevotella spp 5’-CRCGGTAAACG TGGAT-3 and 5’GGT CGG GTT

GCA GAC C -3’ (Huws et al., 2013 ref). QPCR analyses were performed using a 7500 real-

time PCR system (Applied Biosystems, Warrington, UK. The total bacterial standard was

generated using equal amounts of genomic DNA from 8 different pure cultures of bacteria:

Butyrivibrio fibrisolvens (JW11), Clostridium aminophilum (49906), Fibrobacter

succinogenes (S85), Peptostreptococcus anaerobius (27337), Prevotella bryantii (B14),

Ruminococcus albus (SY3), Selenomonas ruminantium (Z108) and Streptococcus bovis

(ES1). The Prevotella standard was generated with equal concentrations of genomic DNA

from P. albensis, P. brevis, P. bryantii and P. ruminicola. The Absolute quantities were

analysed using 7500 SYSTEM SDS software (Applied Biosystems, Warrington, UK). All

QPCR were performed in duplicate and PCR assay efficiency was calculated as follows

Efficiency = 10(-1/slope) × 100. QPCR efficiency was over 90%

18S rDNA Quantitative PCR

Total protozoa 18S rDNA was enumerated using the primers 316 forward: 5’ GCT

TTCGWTGGTAGTGTATT 3’ and 539 reverse 5’ CTTGCCCTCYAATCGTWCT 3’

(Sylvester et al. 2004). Standard curves were generated by serial diluting rumen protozoal

DNA standard obtained from previous published study (Huws et al. 2012), which was

previously determined to be low in plant and bacterial contamination. Amplification was

Page 6: Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin · 2019. 9. 16. · Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin Institute of Biological, Environmental

carried out in a final volume of 25 μL containing 12·5 μL Sybr green, 250 nm each the

primers and 2 μL of a 1:10 dilution of extracted genomic DNA. The thermal cycling

programme was forty cycles of 94°C for 15 s and 54°C for 36s and 72°C for 30 s, with an

initial cycle of 94°C for 10 min. After PCR, a dissociation curve (melting curve) was

constructed in the range of 55–95°C. All samples for the three experimental runs were run in

duplicate for the validation of results.

Statistical analysis

QPCR values were Log10-transformed (to attain normality) and subjected to analyses of

variance. Statistical analysis of all data was performed using the GenStat program (Tenth

Edition, Hemstead,UK) (Payne 2004)

Results and discussion

The rumen microbiota rapidly attach to plant materiel entering the rumen (Koike et al. 2003;

Huws et al. 2013). From previous studies Prevotella spp seems to be the predominant

microbe attached to PRG (Huws et al. 2013). The diversity of fresh perennial ryegrass

attached microbiota change between 2-4 h of incubation in situ indicative of primary

bacterial colonisation to secondary bacterial colonisation events occurring (Huws et al. 2013).

In this study we investigated the role that protozoa play in terms of altering primary and

secondary PRG attached microbiota diversity and abundance. Defaunation has been generally

used to study the effect of protozoa on rumen fermentation and microbial population in

vivo/in Situ (Ushida et al. 1990; Koenig et al. 2000; Ranilla et al. 2007). In a previous study

we attempted to assess the effects of protozoa on the PRG colonising microbiota in vivo/in

Situ using PRG suspended within the rumen in dacron bags of varying pore sizes. To restrict

protozoal but allow influx of the microbiota to the bags (negative control) we used 5µm pore

size dacron bags and 100µm pore size dacron bags to provide unrestricted flow (positive

control). The bags were sealed tightly using heat also. Nonetheless, we discovered that the

rumen protozoa would find a way of penetrating the 5µm pore size bags to gain access to the

plant material and microbiota (data not shown). Therefore, in this study, we employed an in-

vitro defaunation technique to address the role protozoa play in plant colonization and hence

in the fermentation and degradation of plant material.

There were significant differences between In vitro dry matter digestibility IVDMD in

the incubations with protozoa present and incubations without protozoa, with more

degradation seen in the presence of protozoa (P= 0.051, S.E.D =1.706; Figure 1).

Specifically, dry matter degradation was stable until the 4 h time point and then subsequent

readings (from 6 h) revealed that in the presence of protozoa, PRG was degraded to a greater

extent compared with control conditions (Figure 3). The increase in fiber degradation in the

presence of protozoa potentially confirms protozoal contribution to fibrolytic activity

(Williams and Withers 1991; Dijkstra and Tamminga 1995). However, it is uncertain if this

increase in fiber degradation is attributed specifically to protozoal activity or to some form of

interactions between them and the attached microbiota (Delfosse-Debusscher et al. 1979).

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Protozoa have been shown to perform selective predation on cellulolytic bacteria (Dehority

and Odenyo 2003), while species such as Epidinium caudatum and

Polyplastronmultivesuculatum, which are the main fibrolytic ciliates in the rumen may adhere

to plant and have been found to ingest small plant particles (Williams and Coleman 1992,

Huws et al. 2009, Huws et al. 2012) thereby contributing to degradation of plant material.

Previous studies have shown that protozoa ingest cellulose (Morgavi et al. 1994; Huws et al.

2009); therefore, it is plausible that they contribute to the degradation in synergy with

bacteria (Ricard et al. 2006). There was a rapid initial gas production, without a lag, after

which gas production stayed at a high level. The evolution of gas during fermentation was

altered to a significant degree by the presence of protozoa (Figure 2). The volume of gas

produced was persistently greater (P<0.001, S.E.D = 3.56) in the presence of protozoa than in

absence for all time intervals sampled after 0 h of incubation.

Changes in microbial population were assessed using QPCR to enumerate the

population of total colonising rumen bacteria and the prevalence of Prevotella spp. We also

evaluated protozoal 18S rDNA concentration to ensure that there was a protozoal differential

between control and test incubations. QPCR efficiency for all QPCR reactions was between

90% and 105%. Correlations of genomic DNA standards for all QPCRs were >0.95. The

QPCR for protozoa showed significant difference (P<0.05) between the control and the test

with the test showing higher number of protozoa compared to the control (Data not shown).

At 2 h the total bacterial 16S rDNA concentration was higher (P<0.05) in incubations without

protozoa as opposed to the incubations with protozoa (Table 2). Similarly Prevotella 16S

rDNA concentration was correspondingly higher (P<0.05) at 2 h of incubation without

protozoa compared with incubations with protozoa (Table 2). The removal of protozoa at all

other incubation times resulted in no significant overall change in 16S rDNA concentrations

for either total bacterial or Prevotella spp (Table 2). Initial (0h) bacterial 16S rDNA

concentrations for 0 h are not presented as these were very low (about 2ng/g for total

bacterial 16S rDNA) and correspond to the low levels of epiphytic bacteria present pre-

incubation. Prevotella spp are also known to be a fibrolytic and their presence in high

numbers suggests that Prevotella is a core bacterial species. This result is consistent with data

published by (Stevenson and Weimer 2007) and (Huws et al. 2010) in which DNA

concentration of Prevotella spp. approached total bacterial DNA concentration, confirming

that Prevotella spp. are dominant bacteria in the rumen.

Bacterial diversity and community structure in the samples were analysed by T-RFLP

which generates a DNA “fingerprint” for each bacterial community based on restriction

fragments of DNA from each sample. Two different restriction enzymes (HaeIII and Msp1)

were used to generate TRFs. Hae III generated more peaks, therefore only results for the

HaeIII enzyme are presented (Figures 3A and 3B). T-RFLP generated dendrograms were

very similar between the population structure of the samples incubated in the absence of

protozoa and those in the presence of protozoa except for the 2 h time point. This effect

which was evident on bacterial profiles for the 2 h time points observed for all the replicated

experiments (Data not shown).This observation was also consistent with QPCR results at 2 h

post incubation, where a significant increase was observed in the presence of protozoa for

both total bacteria and Prevotella spp. (Table 1.) This supports the notion of a 2-4 h shift and

validates our previous assertion that there is a shift from primary colonisers to secondary

colonisers at this time point (Huws et al. 2013).

This study demonstrates that the rumen protozoa increase plant degradation and

change the attached PRG biofilm diversity after 2 h of incubation in vitro which may also

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partially explain increased plant degradation. This is important because it increases our

understanding of plant–microbe interaction in the rumen and the role of protozoa in this

interactome. This knowledge is of immense importance to strategically manipulate rumen

microbes to increase fiber degradation and therefore improve address and the future increased

demand for meat and milk

Conclusions

The role that rumen microbes play in terms of colonisation is essential to developing novel

strategies to enhance efficiency of feed utilisation .In this study, we have demonstrated that

protozoa play a role also in the colonisation of plant material specifically in primary

colonisation events. We have also confirmed previous data on the role of Prevotella spp. in

the early colonisation of plant. This study illustrates that the presence of rumen protozoa can

widely increase the rumen fermentation pattern and colonisation of fresh PRG thus

potentially releasing more nutrients for the ruminant .This knowledge is important in

understanding the plant-microbe interactome at the level of functionality which is key to

improving ruminant nutrient use efficiently.

Acknowledgements

We acknowledge funding from Stapledon fellowship and the Biotechnology and Biological

Sciences Research Council (BBSRC, UK). The authors have no conflict of interest.

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3583-3597.

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Veira, D.M. (1986) The role of ciliate protozoa in nutrition of the ruminant. J Anim Sci 63,

1547-1560.

Williams and Coleman (1992) The rumen protozoa. In: P. N. Hobson (Ed.) The Rumen

Microbial Ecosystem pp. 77-128. Elsevier Science Publishing, New York.

Williams, A.G. and Withers, S.E. (1991) Effect of ciliate protozoa on the activity of

polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem. Journal of

Applied Bacteriology 70, 144-155.

Table 1: 16S rDNA concentration (Log10 μg DNA concentration g−1

plant Dry Matter) of total

bacteria and Prevotella spp attached to perennial ryegrass over time in the presence and absence

of protozoa

Total bacteria Prevotella spp

Time

(h) Treatments Treatments

Absence Presence SED P-

Value

Absence Presence SED P-

Value

1 2.24 2.4 0.51 0.77 2.07 2.66 0.35 0.11

2 2.1 3.26 0.32 0.00 2.23 3.21 0.37 0.01

4 3.53 3.26 0.39 0.49 3.22 3.53 0.34 0.38

6 4.18 4.35 0.45 0.70 4.08 3.71 0.24 0.14

8 4.72 3.95 0.59 0.21 4.01 4.17 0.19 0.41

SED = Standard error of mean.

Figure Legends

Figure 1: In -vitro Dry matter degradability (%) of PRG in the absence and presence of

protozoa, where ■ =presence of protozoa; ▴ = absence of protozoa.

Figure 2: Cumulative gas production (ml gas/g DM) from the in vitro fermentation of PRG

in the absence and presence of protozoa, where ■=presence of protozoa; ▴ = absence of

protozoa.

Figure 3: Unweighted pair group method with arithmetic mean dendrogram showing the

impact of protozoa on bacterial diversity for all time points (A) and for the 2 h incubation

only (B) following Hae111 based T-RFLP. Scale relates to percentage similarity with results

from experiment 1 and samples taken at different time point (0, 1, 2, 4, 6, and 8 h).

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Figure 1

Figure 2

0

5

10

15

20

25

30

35

40

45

0 2 4 6 8 10

%In

Vit

ro d

ry m

atte

r d

egra

dab

ility

Incubation time (h)

0

20

40

60

80

100

120

140

0 2 4 6 8 10

Gas

pro

du

ctio

n(m

l gas

/g D

M)

Incubation time (h)

S.E.D 1.706

P-Value 0.051

S.E.D 3.56

P-Value >0.001

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Figure 3A.

Figure 3B

Page 13: Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin · 2019. 9. 16. · Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin Institute of Biological, Environmental

Research report 1

Title

Homoserine lactone based bacterial cell-cell communication within the rumen

SA Huwsa, LA Onime, LB Oyama

a, CJ Newbold

a, P Golyshin

b, O Golyshina

b, A Winters, F

Privéa, B Hauke, AH Kingston-Smith

a.

aInstitute of Biological, Environmental and Rural

Sciences (IBERS), Aberystwyth University, Penglais Campus, Aberystwyth, Ceredigion, UK,

SY23 3FG, bSchool of Biological Sciences, Bangor University, Deiniol Road, Bangor,

Gwynedd, UK, LL57 2UW.

Introduction

Bacteria are able to regulate community levels of gene expression by intercellular

communication through diffusible signal molecules through quorum sensing. Quorum

sensing was first observed in Vibrio fischeri to activate bio-luminescence in large cell

colonies but it has been observed in several other species including Agrobacterium spp.,

Rhizobium spp., Pseudomonas spp., Brucella spp., and Vibrio spp. These communication

signals assess cell-density and then present a coordinated response in the regulation of gene

expression in the community. At high population density the concentration of the signal

molecules produced increases; the outcome is an activated transcriptional regulator which

promotes the transcription of the signal molecule ligases resulting in a magnification of the

signal. Several gram-negative bacteria produce acylated homoserine lactones (AHLs) as

quorum-sensing signal molecules. Most bacteria AHLs have the same core chemical structure

with variations in the length of the acyl-chain functional group and modifications with an oxo

or hydroxyl group at the C3 positions. Another group of quorum sensing signal are the

Autoinducer 2 (AI-2), these have been demonstrated to be involved in inter species

communication in both gram positive and gram-negative bacteria. Kirisits, and Parsek. (2006)

have shown that AI-2 signals play a role in the formation of biofilms. Morover Erickson et al.

(2002) also described the presence of AHLs and also AI-2-mediated quorum sensing, in

rumen fluid by means of bioassay. These demonstrate that AHL and AI-2-mediated quorum-

sensing signal systems are ways by which rumen bacteria may possibly perform important

and often fundamental functions, such as biofilm formation, symbiosis, and extracellular

enzyme production regulation. In the rumen, biofilms consists of complex matrix making up

a suitable stable microenvironment where bacteria are protected to perform cellulolytic

breakdown of plant fiber Huws et al. (2013). These biofilms play an important role in feed

digestion in the rumen. As a first step in understanding the microb-microb interaction taking

place in the rumen, specifically in identifying the mechanism involved in biofilm formation,

we employed a bioinformatics strategy to identify genes whose expression might be regulate

by quorum sensing mechanism. In this present study we attempt to provide substantial

confirmation of the presence of AHL mediated Quorum sensing system in the rumen by using

an open source public metagenome analysis tool and databases such as MG-Rast, DNA-

Master, and BLAST X to detect genes that are involved in quorum sensing from the rumen.

Materials and strains

All chemicals used for enzymatic tests and library screening were of the purest grade

available and were purchased from Sigma-Aldrich Company Ltd. (Dorset, UK), Fisher

Scientific (Leicestershire, UK) and TCI Europe (Zwijndrecht, Belgium). Restriction

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enzymes were from Promega UK Ltd. (Southampton, UK). Escherichia coli strains EPI300-

T1R (Epicentre, Cambio Ltd., Cambridge, UK) were used for library construction and

screening, and TOP10 (Invitrogen, Carlsbad, CA, USA), for protein expression. All bacterial

hosts were maintained and cultivated according to the supplier’s recommendations.

Rumen sampling and DNA extraction

Rumen contents were collected from four rumen-fistulated, non-lactating Holstein cows

(average weight of 731 kg) housed at Trawsgoed experimental farm (Aberystwyth,

Ceredigion, Wales). The animals were fed a diet composed of a mixture of grass silage and

straw (75:25) ad libitum and ~1 kg of sugar beet nuts at 0700 with constant access to fresh

water. Sampling was completed 2 h after concentrate feeding. Strained ruminal fluid (SRF),

solid-attached bacteria (SAB) and liquid associated bacteria (LAB) were harvested as

described by Huws et al. (2010).

Construction of metagenomic libraries

Metagenomic DNA was extracted from 200 μl of SRF, SAB and LAB using the BIO101

FastDNA® Spin Kit for Soil (Qbiogene, Cambridge, UK) following the supplier’s protocol

except that after the first step the samples were shaken three times for 30 s, in a FastPrep

bead beater (Qbiogene, Cambridge, UK) at 6 m/s with cooling on ice for 30 s between each

shake. The libraries were constructed using the CopyControlTM

pCC1FOSTM

vector and the

reagents supplied in the CopyControlTM

Fosmid Library Production Kit (Epicentre, Cambio

Ltd., Cambridge, UK), following the supplier’s recommendations. All clones were picked

using a colony picker Genetix QPix2 XT (Genetix Ltd., New Milton, England), and sub-

cultured for 20 h in 384-well plates (Genetix Ltd., New Milton, England) containing LB

broth with 12.5 µg/ml chloramphenicol and 20% glycerol. They were then stored at -80°C in

30% glycerol.

Screening for AHL activity

Selective screening of the clones for AHL activity was accomplished using a rapid screening

assay as described by Kouker and Jaeger (1987).

First the clones were grown to early exponential phase and then harvested by centrifugation

at 12,000 × g for 10 min. The resulting pellets were collected and resuspended in 5 ml of

KH2PO4 buffer and before sonication for 30 s three times and centrifuged (12,000 × g) at

4°C for 30 min to obtain cell extract. In each well of a 96 well plate, 50 μl of cell extract from

the clones was added to 100 μl of 20 mM KH2PO4, this mixture was incubated at 30°C.

After 2h, 1 μl of X-Gal (20 mg ml-1) was added to the mixture and further incubated at 30°C

for 1hr. Absorbance in each well was measured at 635 nm by spectrophotometry. After

incubation the positive blue clones with were selected and their fosmids extracted using the

QIAprep® Spin Miniprep kit (Qiagen, Crawley, UK). The fosmids were digested with

digested using the BamHI restriction enzyme and the digested fragments were separated by

running on 1% agarose gels to determine fosmid size.

454Pyrosequencing and sequence analysis

The fosmid sequences were determined using a high throughput pyrosequencing GS FLX

(454 Life Sciences) at Aberystwyth University, UK. The purified AHL-positive fosmids were

fragmented to 600-900 bp fragments by nebulisation. The sheared fosmids were ligated to

molecular barcodes (Multiplex Identifiers, MID, Roche Life Sciences) containing short

oligonucleotide adaptors “A” and “B”. This was in order to specifically tag each sample in

the sequencing run. The MID-adaptor ligated DNA libraries were mixed in an equimolar

amount and clonally amplified by emulsion PCR using 13.6x106 Sepharose beads per

emulsion reaction. Emulsions were then broken with isopropanol and emulsion PCR beads

were enriched for template-positive beads and bead-attached DNAs were denatured with

NaOH and sequencing primers were annealed. Approximately 790,000 beads with clonally

Page 15: Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin · 2019. 9. 16. · Report to the Stapledon Memorial Trust Dr Lucy A. Akinmosin Institute of Biological, Environmental

amplified DNA were then deposited on two of four 70x75mm regions of the PicoTiterPlate.

The PicoTiterPlate device was then loaded onto the 454 instrument along with the sequencing

reagents, and sequences were obtained according to the manufacturer’s protocol. An SFF

(Standard Flowgram Format) file was obtained for each sample. The reads from each of the

pooled libraries were identified by their MID tags by the data analysis software gsAssembler

v2.5.3 (Roche Life Sciences) after the sequencing run. The assembly was done using the

default parameters on gsAssembler. BLASTN on NCBI was used to trim the vector sequence

from the contigs and then 3 different approaches were used to analyze the sequences.

In the first approach we utilized BlastX to compare the six-frame conceptual translation

products of our nucleotide query sequence against protein sequences for AHL in a local

database for AHL that we developed. A second method was DNA Master, a genome editor

was used as a tool to predict the open reading frames in the nucleotide sequence. It runs a

collection of analysis programs: Glimmer (version 3.02) and GeneMark (version 2.0), for

auto-annotation and analysis sequences. Both predict the coding potential of open reading

frames and then a BLAST search was done saving hits with an E value 10E-3.

A third approach involved uploading sequence data into Mg-RAST server which used several

bioinformatics tools for analysis, protein prediction, clustering and annotation of our

sequence datasets.

Result

We prospected AHL production within the rumen microbiome using fosmid-based

metagenomic DNA libraries created from forage-attached bacteria. The library consisted of

8,448 clones with an insert size of 30 to 35 kbp. Transformants were grown overnight at 37oC

in 384 wells containing LB broth and selectively screened for AHL production using the cell-

free supernatant of Agrobacterium tumefaciens NTL4 in the presence of x-gal (1µg/ml)3.

Fifty-six of the clones exhibited β-galactosidase activity. Twenty two of these putatively

positive clones have also undergone 454 pyrosequencing and sequences were subsequently

prospected for AHL synthase genes using DNA Master, BLAST, and MG-RAST.

Bioinformatic data revealed that up to 50% of the fosmids possibly containing AHL synthase

genes (based on similarities of 30-100% and e-values of 0.1 to 1.0 x 10-6

).

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Clone/fossmids Blast X DNA master MG-RAST SAB 27 P2 sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.22

ribosomal protein L11 methyltransferase Succinatimonas sp.

CAG 777

N-3-oxooctanoyl-L-homoserine lactone quorum-

sensing transcriptional activator beta-lactamase Helicobacter pylori N-3-oxooctanoyl-L-homoserine lactone quorum-

sensing transcriptional activator recA protein Dialister invisus CAG 218 N-3-oxooctanoyl-L-homoserine lactone quorum-

sensing transcriptional activator dEAD/DEAH box helicase Succinatimonas sp. CAG 777 N-3-oxooctanoyl-L-homoserine lactone quorum-

sensing transcriptional activator DEAD/DEAH box helicase Succinatimonas hippei BarA-associated response regulator UvrY (=

GacA = SirA) s-ribosylhomocysteine lyase Succinatimonas sp. CAG 777 BarA-associated response regulator UvrY (=

GacA = SirA) S-ribosylhomocysteinase Succinatimonas hippei S-ribosylhomocysteine lyase (EC 4.4.1.21) /

Autoinducer-2 production protein LuxS quorum-sensing autoinducer 2 AI-2 LuxS Dickeya dadantii

Ech586

S-ribosylhomocysteine lyase (EC 4.4.1.21) /

Autoinducer-2 production protein LuxS SAB 27 O2 sp|P54298|LUXM_VIBHA Acyl-homoserine-

lactone synthase LuxM OS=V... 30.8 0.10

Thiol disulfide interchange protein DsbG Escherichia coli P12b sp|A7MRY3|LUXM_VIBCB Acyl-homoserine-

lactone synthase LuxM OS=V... 30.8 0.10

LysR family transcripitonal regulator Bacteria sp|Q87NA8|OPAM_VIBPA Acyl-homoserine-

lactone synthase OpaM OS=V... 26.9 1.3

carbon starvation protein involved in peptide utilization

Escherichia coli str. K-12 substr. MG1655

sp|P12747|LUXI_ALIFS Acyl-homoserine-lactone

synthase OS=Aliivi... 25.8 2.1

sp|Q4KCM5|PVDQ_PSEF5 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 25.4 4.7

sp|Q9FDA3|VANM_VIBAN Acyl-homoserine-

lactone synthase VanM OS=V... 24.6 6.6

sp|P33882|EXPI_PECSS Acyl-homoserine-lactone

synthase OS=Pectob... 23.9 9.4

SAB 27 F7 sp|Q87NA8|OPAM_VIBPA Acyl-homoserine-

lactone synthase OpaM OS=V... 25.0 5.3

Ser/Thr protein phosphatase family protein Prevotella

ruminicola 23

S-adenosylmethionine synthetase (EC 2.5.1.6) sp|B7VQM9|LUXM_VIBSL Acyl-homoserine-

lactone synthase LuxM OS=V... 24.3 8.6

Ser/Thr protein phosphatase family protein Prevotella

ruminicola 23

S-ribosylhomocysteine lyase (EC 4.4.1.21) /

Autoinducer-2 production protein LuxS ABC transporter ATP-binding protein Prevotella ruminicola 23 SAB 27 E6 sp|A7MRY3|LUXM_VIBCB Acyl-homoserine-

lactone synthase LuxM OS=V... 30.8 0.11

sp|P54298|LUXM_VIBHA Acyl-homoserine-

lactone synthase LuxM OS=V... 30.8 0.11

ABC transporter family protein Escherichia coli sp|P12747|LUXI_ALIFS Acyl-homoserine-lactone

synthase OS=Aliivi... 25.8 2.3

LysR family transcripitonal regulator Escherichia coli sp|Q9I194|PVDQ_PSEAE Acyl-homoserine lactone

acylase PvdQ OS=Ps... 25.8 4.2

Thiol disulfide interchange protein DsbG Escherichia coli P12b sp|Q4KCM5|PVDQ_PSEF5 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 25.4 4.9

sp|Q89VI2|BJAI_BRADU Isovaleryl-homoserine

lactone synthase OS=... 24.3 8.6

sp|P33882|EXPI_PECSS Acyl-homoserine-lactone

synthase OS=Pectob... 23.9 9.9

SAB 27 F9 sp|P54298|LUXM_VIBHA Acyl-homoserine-

lactone synthase LuxM OS=V... 30.8 0.14

LysR family transcriptional regulator Escherichia coli sp|A7MRY3|LUXM_VIBCB Acyl-homoserine-

lactone synthase LuxM OS=V... 30.8 0.14

50S rRNA methyltransferase Shigella flexneri sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 27.3 1.0

ABC transporter family protein Escherichia coli sp|Q87NA8|OPAM_VIBPA Acyl-homoserine-

lactone synthase OpaM OS=V... 26.9 1.8

sp|Q9FDA3|VANM_VIBAN Acyl-homoserine-

lactone synthase VanM OS=V... 26.9 2.0

sp|Q88NU6|QUIP_PSEPK Acyl-homoserine

lactone acylase QuiP OS=Ps... 25.4 7.4

SAB 27 N10 sp|P54291|RHLI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 25.8 2.4

Ser/Thr protein phosphatase family protein Prevotella

ruminicola 23

sp|Q4ZPM1|QUIP_PSEU2 Acyl-homoserine

lactone acylase QuiP OS=Ps... 25.8 3.4

sp|Q48LS4|QUIP_PSE14 Acyl-homoserine lactone

acylase QuiP OS=Ps... 25.0 5.5

sp|Q87NA8|OPAM_VIBPA Acyl-homoserine-

lactone synthase OpaM OS=V... 23.9 9.3

sp|Q87XP3|QUIP_PSESM Acyl-homoserine

lactone acylase QuiP OS=Ps... 24.3 9.8

SAB 27 E18 sp|Q48KB0|PVDQ_PSE14 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 26.9 1.3

LysM domain-containing protein Prevotella ruminicola 23 sp|Q88NU6|QUIP_PSEPK Acyl-homoserine

lactone acylase QuiP OS=Ps... 24.6 5.3

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SAB 5 G7 sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.36

ABC transporter ATP-binding protein Prevotella ruminicola 23 sp|P52988|YENI_YEREN Acyl-homoserine-

lactone synthase OS=Yersin... 26.6 2.0

acyltransferase family protein Prevotella ruminicola 23 sp|P33880|CARI_PECCC Acyl-homoserine-lactone

synthase OS=Pectob... 25.4 4.3

acyltransferase family protein Prevotella ruminicola 23 sp|P52990|PSYI_PSESZ Acyl-homoserine-lactone

synthase OS=Pseudo... 24.3 8.9

SAB 15 P22 sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.37

signal peptide protein Prevotella sp|Q9I194|PVDQ_PSEAE Acyl-homoserine lactone

acylase PvdQ OS=Ps... 29.3 0.45

BexD/CtrA/VexA family polysaccharide export protein

Bacteroides gallinarum

sp|Q88IU8|PVDQ_PSEPK Acyl-homoserine

lactone acylase PvdQ OS=Ps... 26.9 2.1

putative serine/threonine-protein kinase Bacteroides sp. CAG

545

sp|Q4KCM5|PVDQ_PSEF5 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 25.4 6.7

sp|Q3KD51|PVDQ_PSEPF Acyl-homoserine

lactone acylase PvdQ OS=Ps... 24.6 9.1

SAB 12(1) B11 sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.36

sp|P52988|YENI_YEREN Acyl-homoserine-

lactone synthase OS=Yersin... 26.6 2.0

sp|P33880|CARI_PECCC Acyl-homoserine-lactone

synthase OS=Pectob... 25.4 4.3

sp|P52990|PSYI_PSESZ Acyl-homoserine-lactone

synthase OS=Pseudo... 24.3 8.9

SAB 12(1) C11 sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.36

LysR family transcripitonal regulator Escherichia coli sp|P52988|YENI_YEREN Acyl-homoserine-

lactone synthase OS=Yersin... 26.6 2.0

Thiol disulfide interchange protein DsbG Escherichia coli P12b sp|P33880|CARI_PECCC Acyl-homoserine-lactone

synthase OS=Pectob... 25.4 4.3

alkyl hydroperoxide reductase subunit C Escherichia coli sp|P52990|PSYI_PSESZ Acyl-homoserine-lactone

synthase OS=Pseudo... 24.3 8.9

alkyl hydroperoxide reductase subunit F Escherichia coli SAB 12(1) D11 sp|Q9FDA3|VANM_VIBAN Acyl-homoserine-

lactone synthase VanM OS=V... 23.5 3.2

sp|P52988|YENI_YEREN Acyl-homoserine-

lactone synthase OS=Yersin... 22.3 5.4

sp|P33904|TRAI_RHIRD Acyl-homoserine-lactone

synthase OS=Rhizob... 21.6 9.2

sp|Q87XP3|QUIP_PSESM Acyl-homoserine

lactone acylase QuiP OS=Ps... 21.9 9.4

sp|Q4ZPM1|QUIP_PSEU2 Acyl-homoserine

lactone acylase QuiP OS=Ps... 21.9 9.5

sp|Q3KH00|QUIP_PSEPF Acyl-homoserine lactone

acylase QuiP OS=Ps... 21.9 9.6

sp|Q48LS4|QUIP_PSE14 Acyl-homoserine lactone

acylase QuiP OS=Ps... 21.9 9.7

SAB 12(1) E11 sp|Q48KB0|PVDQ_PSE14 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 26.9 1.3

LysM domain-containing protein Prevotella ruminicola 23

sp|Q47187|EXPI_DICD3 Acyl-homoserine-lactone

synthase OS=Dickey... 24.3 5.8

SAB 11 O9 sp|B7VQM9|LUXM_VIBSL Acyl-homoserine-

lactone synthase LuxM OS=V... 21.6 9.5

TetR family transcriptional regulator Staphylococcus pasteuri

SP1

SAB 11 P1 sp|Q3KD51|PVDQ_PSEPF Acyl-homoserine

lactone acylase PvdQ OS=Ps... 23.5 4.3

uncharacterized protein YhcH/YjgK/YiaL family Prevotella

dentalis DSM 3688

sp|Q9I4U2|QUIP_PSEAE Acyl-homoserine lactone

acylase QuiP OS=Ps... 23.1 5.0

sp|Q48KB0|PVDQ_PSE14 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 22.7 5.7

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sp|P33880|CARI_PECCC Acyl-homoserine-lactone

synthase OS=Pectob... 25.4 2.7

sp|Q88NU6|QUIP_PSEPK Acyl-homoserine

lactone acylase QuiP OS=Ps... 24.6 5.3

sp|Q47187|EXPI_DICD3 Acyl-homoserine-lactone

synthase OS=Dickey... 24.3 5.8

SAB 12(1) H11 sp|Q9I194|PVDQ_PSEAE Acyl-homoserine lactone

acylase PvdQ OS=Ps... 26.2 1.9

O-acetylhomoserine aminocarboxypropyltransferase/cysteine

synthase family protein

sp|Q884D2|PVDQ_PSESM Acyl-homoserine

lactone acylase PvdQ OS=Ps... 25.8 2.5

SAB 12(1) I11 sp|P12747|LUXI_ALIFS Acyl-homoserine-lactone

synthase OS=Aliivi... 28.9 0.35

metallo-beta-lactamase Prevotella bryantii sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.42

GNAT family acetyltransferase Prevotella ruminicola 23 sp|Q9I194|PVDQ_PSEAE Acyl-homoserine lactone

acylase PvdQ OS=Ps... 26.2 3.6

acetyltransferase Prevotella bryantii sp|Q46968|ECHI_ERWCH Acyl-homoserine-

lactone synthase OS=Erwini... 24.6 8.4

thiol disulfide interchange protein Parabacteroides merdae 23S rRNA uracil-5- -methyltransferase RumA Prevotella

ruminicola 23

SAB 12(2) E15 sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.38

AraC family transcriptional regulator Pedobacter heparinus

DSM 2366

N-3-oxohexanoyl-L-homoserine lactone quorum-

sensing transcriptional activator sp|P74945|VANI_VIBAN Acyl-homoserine-lactone

synthase OS=Vibrio... 26.6 1.9

tRNA 5-methylaminomethyl-2-thiouridylate -methyltransferase

Prevotella ruminicola 23

N-3-oxooctanoyl-L-homoserine lactone quorum-

sensing transcriptional activator sp|Q4ZV08|PVDQ_PSEU2 Acyl-homoserine

lactone acylase PvdQ OS=Ps... 25.8 5.2

S-adenosylmethionine synthetase (EC 2.5.1.6) sp|Q9FDA3|VANM_VIBAN Acyl-homoserine-

lactone synthase VanM OS=V... 25.4 5.4

SAB 9 H11 sp|P12747|LUXI_ALIFS Acyl-homoserine-lactone

synthase OS=Aliivi... 28.9 0.35

thioredoxin domain-containing protein Prevotella ruminicola 23 S-adenosylmethionine synthetase (EC 2.5.1.6) sp|P33883|LASI_PSEAE Acyl-homoserine-lactone

synthase OS=Pseudo... 28.9 0.42

glycosyltransferase group 2 family Prevotella sp. CAG 5226 sp|Q9I194|PVDQ_PSEAE Acyl-homoserine lactone

acylase PvdQ OS=Ps... 26.2 3.6

GNAT family acetyltransferase Prevotella ruminicola 23 sp|Q46968|ECHI_ERWCH Acyl-homoserine-

lactone synthase OS=Erwini... 24.6 8.4

metallo-beta-lactamase Prevotella bryantii metallo-beta-lactamase family protein Prevotella ruminicola 23 23S rRNA uracil-5- -methyltransferase RumA Prevotella

ruminicola 23

SAB 27 J22 sp|Q88NU6|QUIP_PSEPK Acyl-homoserine

lactone acylase QuiP OS=Ps... 26.9 2.0

hypothetical protein Lachnospiraceae bacterium NK4A136 S-ribosylhomocysteine lyase (EC 4.4.1.21) /

Autoinducer-2 production protein LuxS XRE family transcriptional regulator Streptococcus

pneumoniae

Table 1 : Blastx, DNA –master and MG-RAST results showing homologous acyl homoserine lactone (AHL) synthase within the sequenced

fosmids

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LUCY. A. AKINMOSIN

STAPLEDON MEMORIAL TRUST FELLOWSHIP 2014

Conclusion

This data, show that quorum sensing using homoserine lactone based signalling is likely to be

present within the rumen. Confirmation of AHL production is also underway using thin layer

chromatography (TLC) and HPLC. Understanding bacterial cell communication and its

ecological importance is key to obtaining a clearer understanding of the rumen system.

Understanding how bacteria communicate in the rumen and the consequences of cell-cell

signaling are key to obtaining a clearer understanding of the rumen system and its impact

upon nutrient availability to the ruminant.

Acknowledgments

We are grateful for support from the Biotechnology and Biological Sciences Research

Council, Coleg Cymraeg Cenedlaethol, and the Stapeldon Trust.

References

1. Erickson et al. (2002). Can. J. Microbiol., 48, 374-378.

2. Huws et al. (2010). FEMS Microbiol. Ecol., 73: 396-407.

3. Huws et al. (2013). Lett. Appl. Enviro., 56: 186-196.

4. Kawaguchi et al. (2008). Appl. Envoro. Microbiol., 74, 3667-3671.

5. Kirisits, M. J., and M. R. Parsek. (2006.) Cell. Microbiol. 8:1841-1849.

6. Kouker, G. and Jaeger, K.E. (1987) Appl. and Enviro Microbiol 53, 211–213.

Outcomes of the Fellowship and experience gained.

This fellowship has given me the opportunity to practice in the Institute of Biological,

Environmental and Rural Sciences (IBERS) which an internationally recognised

research centre world class institution in the U.K

The research results from this fellowship have been prepared for submission for

publication in a peer- reviewed journal

The research results have also been presented at 2 conferences : The 2014 Annual

Society of experimental biology meeting took place from the 1st - 4th July at the

University of Manchester, United Kingdom and at the 9th Joint Rowett/INRA

Symposium, Gut Microbiology: from Sequence to Function which took place in

Aberdeen from 16th

-18 June.2014.

This fellowship has significantly increased my expertise and knowledge of different

areas in ruminant microbiology research which are in response to global challenges

such as food security, bioenergy and sustainability.

It has allowed me to gained new insight on research methodologies in bioinformatics

and also in metagenomics which are vital to my future scientific development.

This fellowship has also afforded me the opportunity to work and to integrate with the

Herbivore Gut Ecosystem group at IBERS which represents one of the largest group

of scientists in Europe dedicated to work on rumen microbiology and rumen function.

Strengthening of research ties with possibility of future collaboration between

University of Udine in Italy and the Aberystwyth University U.K. have also been

achieved.

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LUCY. A. AKINMOSIN

STAPLEDON MEMORIAL TRUST FELLOWSHIP 2014

Acknowledgements

I would like to thank all the staff of the Institute of Biological, Environmental and Rural

Sciences (IBERS) for all their support during my stay, particularly for providing me with

many opportunities to learn new techniques and become a better scientist .Particularly I

would like to express my thanks to Dr Sharon Huws, my host for the hospitality at University

of Aberystwyth (IBERS) in the U.K, and this work that resulted in a paper that is been

prepared for publication. I am grateful for her mentoring during my time at IBERs. I would

also like to acknowledge lab mates at IBERS for always being helpful and contributing to

creating a stimulating research environment. Finally, I would like to thank the Stapledon

Memorial Trust for giving me opportunity to come to Aberystwyth and providing me with the

funding required to make this work feasible. Overall, it was a great pleasure and honour to be

a part of this fellowship.


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