Journal of Integrative Agriculture 2016, 15(10): 2343–2352

RESEARCH ARTICLE

Available online at www.sciencedirect.com

ScienceDirect

Initial detection of the quorum sensing autoinducer activity in the rumen of goats in vivo and in vitro

RAN Tao1, 2, ZHOU Chuan-she1, 3, XU Li-wei1, GENG Mei-mei1, TAN Zhi-liang1, 3, TANG Shao-xun1, 3, WANG Min1, 3, HAN Xue-feng1, 4, KANG Jin-he1

1 Key Laboratory for Agro-Ecological Processes in Subtropical Region/Hunan Research Center of Livestock & Poultry Sciences/South-Central Experimental Station of Animal Nutrition and Feed Science, Ministry of Agriculture/Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, P.R.China

2 University of the Chinese Academy of Sciences, Beijing 100049, P.R.China3 Hunan Co-Innovation Center of Animal Production Safety, CICAPS, Changsha 410128, P.R.China4 Hunan Co-Innovation Center for Utilization of Botanical Functional Ingredients, Changsha 410128, P.R.China

AbstractQuorum sensing (QS) is a type of microbe-microbe communication system that is widespread among the microbial world, particularly among microorganisms that are symbiotic with plants and animals. Thereby, the cell-cell signalling is likely to occur in an anaerobic rumen environment, which is a complex microbial ecosystem. In this study, using six ruminally fis-tulated Liuyang black goats as experimental animals, we aimed to detect the activity of quorum sensing autoinducers (AI) both in vivo and in vitro and to clone the luxS gene that encoded autoinducer-2 (AI-2) synthase of microbial samples that were collected from the rumen of goats. Neutral detergent fiber (NDF) and soluble starch were the two types of substrates that were used for in vitro fermentation. The fermented fluid samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The acyl-homoserine lactones (AHLs) activity was determined using gas chromatography-mass spectrometer (GC-MS) analysis. However, none of the rumen fluid extracts that were collected from the goat rumen showed the same or similar fragmentation pattern to AHLs standards. Meanwhile, the AI-2 activity, assayed using a Vibrio harveyi BB170 bioassay, was negative in all samples that were collected from the goat rumen and from in vitro fermentation fluids. Our results indicated that the activities of AHLs and AI-2 were not detected in the ruminal contents from six goats and in ruminal fluids obtained from in vitro fermentation at different sampling time-points. However, the homologues of luxS in Prevotella ruminicola were cloned from in vivo and in vitro ruminal fluids. We concluded that AHLs and AI-2 could not be detected in in vivo and in vitro ruminal fluids of goats using the current detection techniques under current dietary conditions. However, the microbes that inhabited the goat rumen had the potential ability to secrete AI-2 signaling molecules and to communicate with each other via AI-2-mediated QS because of the presence of luxS.

Keywords: quorum sensing, AHLs, AI-2, luxS, rumen bacteria, goat

1. Introduction

Quorum sensing (QS) is one of microbe-microbe inter-actions. The interactions occur via signal transduction mechanisms by secretion, release, and sensing of signal

Received 29 October, 2015 Accepted 24 May, 2016RAN Tao, Mobile: +86-13755047025, E-mail: xinweiyuejun@126.com; Correspondence TAN Zhi-liang, Tel: +86-731-84619702, Fax: +86-731-84612685, E-mail: zltan@isa.ac.cn

© 2016, CAAS. Published by Elsevier Ltd. This is an open access art ic le under the CC BY-NC-ND l icense (http:/ /creativecommons.org/licenses/by-nc-nd/4.0/)doi: 10.1016/S2095-3119(16)61417-X

2344 RAN Tao et al. Journal of Integrative Agriculture 2016, 15(10): 2343–2352

molecules, called autoinducers (AI), to coordinate gene expression and to facilitate the collective behavior at the microbial population level (Bassler and Losick 2006; Ban-dara et al. 2012). In general, the coordination of microbial population behavior enhances access to nutrients or to specific environmental niches and facilitates the collective defense against other competitive organisms or communi-ties (Williams 2007). It was determined that many physi-ological processes of various bacteria (e.g., competence (Lee and Morrison 1999), bioluminescence (Bassler et al. 1993), virulence factor secretion (Wagner et al. 2004; Zhao et al. 2010), and biofilm formation (Li et al. 2002; Merritt et al. 2003)) were regulated by QS. Therefore, QS enables bacteria to optimize their adaptability to the environment and increases the chance to survive in bad surroundings, especially via symbiotic or pathogenic relationships with plants or animals.

Several chemically distinct families of AI with different signal transduction modes were identified. These families of AI include acyl-homoserine lactones (AHLs, commonly signified as AI-1), post-translationally modified autoinducer peptides (AIPs), and autoinducer-2 (AI-2). AI-1 is secret-ed by Gram-negative bacteria, and AIPs are secreted by Gram-positive bacteria (Whitehead et al. 2001; Sturme et al. 2002). AI-2 is secreted by both Gram-positive and Gram-negative bacteria. Because it is widely distributed, it is thought to be a universal signal that enables interspecies communication (Schauder et al. 2001; Xavier and Bassler 2003; Sun et al. 2004). Meanwhile, LuxS, which is encod-ed by the luxS gene, was proven to be a critical enzyme for activating the bacterial methyl cycle and for producing AI-2 from S-adenosylmethionine (SAM) (Sigrid et al. 2006). Therefore, LuxS is commonly recognized as AI-2 synthase, and the existence of luxS gene is used to judge whether a certain bacteria has the potential to secret AI-2 or not. Currently, several qualitative and quantitative methods of AI detection are available and include a reporter strains bioassay (Bassler et al. 1997; Zhu et al. 2003) as well as liquid chromatography-mass spectrometric (LC-MS) (Morin et al. 2003; Campagna et al. 2009) and gas chromatogra-phy-mass spectrometric (GC-MS) assays (Cataldi et al. 2004; Thiel et al. 2009).

In the rumen microbial ecosystem, many different micro-bial groups (e.g., bacteria, archaea, fungi, and protozoa) live in symbiotic relationship with the host (Kamra 2005; Makkar 2005). Rumen microbes inhabit anaerobic environment, and bacterial cells can increase in numbers up to 1011–12 mL–1 in rumen fluid. Therefore, it is speculated that there are various inter- and intra-species communication that may be important for regulating the community structure and for assuring the supply of nutrients for the host in the form of volatile fatty acids and microbial proteins. However, the mi-

crobe-microbe interactions among different organisms have not been studied well in the rumen of ruminants. Recently, several research groups have explored QS in the rumen ecosystem of bovine and have proven that rumen microbes use QS to communicate with each other (Erickson et al. 2002; Edrington et al. 2009; Swearingen et al. 2013). This was reviewed in our previous study (Ran and Tan 2012). Biofilm formation is one of the mechanisms that rumen mi-crobes use to attach to solid feed materials to initiate feed digestion (Miron et al. 2001). Because biofilm formation is generally regulated by QS (Li et al. 2002; Merritt et al. 2003), it is of particular interest to know whether biofilm formation by rumen microbes is also regulated by QS. In addition, it is important to determine whether rumen bacteria can regulate their behaviors using the QS signaling systems to survive under competitive circumstances or whether they maintain the mutualism relationship. However, the detection of AI-activity from a complex ruminal microbial ecosystem remains challenging. Therefore, in vitro fermentation, which is commonly used in ruminant research to artificially imitate rumen environment under more simplified and controllable conditions, is also taken into consideration. In this study, we attempted to detect AHLs and AI-2 activities in the ruminal contents of goats in vitro and in vivo to pave the way for further understanding of physiological functions of AI and QS in ruminants.

2. Materials and methods

2.1. Experimental animals

The animal study was approved by the Animal Care Com-mittee, the Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, China.

In total, six ruminally fistulated Liuyang black goats (a local breed in south China) were raised individually in an animal house at a controlled temperature of 21°C. All goats had free access to fresh water. A maize stover-con-centrate diet was offered to each goat in equal amounts at 08:00 and 18:00 every day. Before the rumen fluid sample collection, a pre-feeding of two weeks was performed to allow experimental animals to get used to the diet and the environment.

2.2. In vitro fermentation

In vitro fermentation was performed according to the mod-ified procedures of Sun et al. (2009). In the current study, neutral detergent fiber (NDF) and soluble starch, which represented fiber and water-soluble carbohydrates, were used as fermentation substrates. Briefly, rumen fluids were collected from all experimental goats before morning feed-

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ing, mixed and filtered through four layers of cheesecloth, and were transferred to a pre-warmed thermos. Then, an anaerobic buffer (1:9, v/v) and a mineral solution were added to the fluids under continuous flow of CO2. Subsequently, 50 mL of the mixture was transferred to fermentation bottles, which contained previously weighed 500 mg substrates of either NDF or soluble starch. Finally, the fermentation bottles were incubated at 39°C with shaking at 50 r min–1. The samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The 0 h bottles were used as blank bottles to adjust in vitro fermentation. At each sampling time, three bottles with the same substrate were taken out and immediately sampled, as described below.

2.3. Sample collection and preparation

To detect the AHLs activity in the in vivo study, the sampling process was performed, as described by Erickson et al. (2002). Briefly, approximately 1 L of rumen contents was collected from each of six goats via the rumen fistulae 2 h before and after the morning feeding. After filtering through four layers of cheesecloth, the collected rumen fluids were centrifuged at 15 000×g for 15 min at 4°C. For each sam-ple, the centrifuged supernatants (approximately 500 mL) were mixed with an equal volume of acetic ether to extract AHLs. The extraction procedures were run thrice, and the organic phases were combined together. Subsequently, the organic extracts were dried using a rotary-evaporator, then were dissolved in 1 mL of chromatographic-grade methanol, and were stored at –20°C.

Regarding the in vivo AI-2 activity assay, 5 mL of filtered rumen fluids were centrifuged at 15 000×g for 15 min at 4°C, and the centrifuged supernatants of rumen fluids were carefully removed. The pH was adjusted to 7.0 using 1 mol L–1 NaOH, and rumen fluids were filtered through the syringe filter with 0.2 μm pore sizes. Finally, the supernatant was stored at –20°C for further AI-2 bioassay. The precipitated cell pellets were collected and stored at –20°C for DNA extraction.

For in vitro analysis, the sampled fermentation fluids were pretreated using the same approach as for the in vivo assay.

2.4. AHLs activity detection using GC-MS analysis

The AHLs detection using GC-MS was conducted according to Cataldi et al. (2004). The standard AHLs (purchased from Sigma-Aldrich, USA; Table 1) were dissolved in a chro-matographic-grade methanol to achieve a concentration of 1 mg mL–1 as stock solutions, which were stored at –20°C. For the GC-MS analysis, the stock solutions of standard AHLs were mixed and further diluted with a chromatograph-ic-grade methanol to a final concentration of 100 μg mL–1.

The obtained samples were subjected to GC-MS analysis using an Agilent 7890A/5975C GC-MS system (Agilent Technologies, USA) under conditions described by Cataldi et al. (2004). The structures of N-acyl chains of AHLs were identified from the obtained retention times and fragmen-tation patterns. Common fragmentation patterns, with the most abundant ion at m/z 143 and other minor peaks at m/z 71, 57, and 43, were detected under the abovementioned GC-MS conditions.

2.5. AI-2 activity detection using the V. harveyi bio-assay

AI-2 detection was performed using the V. harveyi bioas-say, as reported by Bassler et al. (1997) and by Vilchez et al. (2007). The autoinducer bioassay liquid medium (AB medium) and AI-2 bioassay working solution of BB170 was prepared according to Vilchez et al. (2007). The cell free supernatant (CFS) of V. harveyi BB120 was prepared using the same procedure as for the preparation of AI-2 bioassay samples. Then, 180 μL of the working solution and 20 μL of CFS samples were added to the wells of a 96-well plate. The CFS of V. harveyi BB120 was used as a positive control, while autoinducer bioassay (AB) medium was used as a negative control. All samples and controls had three replicates and were assayed at least four times. The plates were incubated at 30°C with shaking at 100 r min–1, and light production was measured every 15 min using an Lminoskan Ascent (Thermo Fisher Scientific, USA) until the light production reached the maximal value approximately within the initial 3–4 h of incubation (Bassler et al. 1997).

Data analysis was conducted using the SPSS Statistics 17.0 software, and the relative light unit (RLU) of AI-2 activity was calculated as described by Kirthiram et al. (2012):

RLU=Average value of the sample/Average value of the negative control

2.6. Cloning and sequencing of the luxS gene ho-mologues

DNA of the obtained cell pellets was extracted using a QIAamp® DNA Stool Mini Kit (Qiagen GmbH, Hilden, Ger-many) according to the supplier recommendations. The primers that were used in this study are shown in Table 2. The degenerate primers (LuxS-F1/luxS-R1 and luxS-F2/luxS-R2) were used in a Nested PCR with a reference to the procedures of Mitsumori et al. (2003). The other primers were designed based on the reported parameters, either luxS or a putative region of luxS from genome sequences of several rumen bacteria (GenBank accession numbers: CP002006.1, CP002403.1 and ACOK00000000.1). PCR amplification was performed in a 50-μL reaction system

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Table 2 Primers used in this study

Item SequenceDegenerate primers reported by Mitsumori et al. (2003)

luxS-F1 5´-AGYTTYRMHDTVGAYCAYAC-3´luxS-R1 5´-ARHTCSTYATTBYTRTTYAC-3´luxS-F2 5´-ACBGTDTTYGAYYTRCGTTT-3´luxS-R2 5´-TGRTADSYRTTYARYTCMGG-3´

Primers designed based on the reported luxS gene sequence of Prevotella ruminicola

luxS PR-S1 5´-ACCGAACCCAATAAGGAGC-3´luxS PR-A1 5´-TTCAGACGGTCAAGATAGCG-3´luxS PR-S2 5´-GATTTACGCATTACCGAACCC-3´luxS PR-A2 5´-CATCGGCAAACACCTTACCAT-3´

Primers designed based on the reported gene sequence of Rumincoccus flavefaciens FD-1

luxS RF-S 5´-CAAGGGCGTTCTTTGCGACTAC-3´lux S RF-A 5´-ACGGCGGCATAACAAGGGAC-3´

Primers designed based on the reported gene sequence of Ruminococcus albus R7

luxS RA-S1 5´-TCCCTGGACTTTATGTTTCACG-3´luxS RA-A1 5´-AATAGTTTCCGCATTCCTCC-3´luxS RA-S2 5´-ACAGCAAGGGTGAGGCAGTAAC-3´luxS RA-A2 5´-AACGAATCTCTTGTTCTCGGTGA-3´

consisting of 2 μL of bacterial genomic DNA and of 48 μL of reaction mixtures that contained 0.5 μL of each primer, 2 μL of dNTP-Mix (TaKaRa, China), 5 μL of a dream-Taq buf-fer, and 2.5 U of dream-Taq DNA polymerase (Fermentas, Canada). PCR was performed, as follows: heating at 95°C for 4 min followed by a touchdown program for 10 cycles with successive annealing temperature decrements of 1°C in every cycle of denaturing at 95°C for 30 s, annealing at 60–50°C for 30 s, and extension at 72°C for 30 s. Then, an-other 25 cycles were performed at an annealing temperature of 50°C for 30 s and a final extension at 72°C for 10 min, followed by storage at 4°C. Then, the PCR products were recovered and cloned using pMD®18-T vector (TaKaRa, Dalian, China). Sequencing was performed by Sangon Biotech Co. (Shanghai, China).

3. Results and discussion

3.1. Detection of the AHLs activity

None of the fluid extracts obtained from the goat rumen and

Table 1 Information about the acyl-homoserine lactones (AHLs) standards used and retention times during the gas chromatography-mass spectrometer (GC-MS) analysis1)

Name Abbreviations Molecular formula Structure Molecular

ion [M]+

Retention time (min)This study

Cataldi et al. (2004)

N-Hexanoyl HL C6-HSL C10H17NO3 O

NH

O

O

199 4.94 7.17

N-(3-Oxohexanoyl) HL 3-oxo-C6-HSL C10H15NO4O

NH

O

O

O213 UD ND

N-Octanoyl HL C8-HSL C12H21NO3O

NH

O

O

227 6.26 8.81

N-(3-Oxooctanoyl) HL 3-oxo-C8-HSL C12H19NO4O

NH

O

O

O241 UD ND

N-Decanoyl HL C10-HSL C14H25NO3O

NH

O

O

255 7.55 10.23

N-(3-Oxodecanoyl) HL 3-oxo-C10-HSL C14H23NO4O

NH

O

O

O

269 UD ND

1) UD, undetectable; ND, not detected; other information about the AHLs standards was offered by Sigma-Aldrich (USA).

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from in vitro fermentation showed the same or similar frag-mentation pattern with AHLs standards (Figs. 1 and 2). This indicated that neither in vivo nor in vitro ruminal fluid sam-ples contained AHLs-like compounds in the current study. Multiple AHLs were detected in the rumen fluids of cattle (Erickson et al. 2002; Edrington et al. 2009). However, all samples from the sheep inoculated with enterohemorrhagic Escherichia coli (EHEC) were AHL negative (Edrington et al. 2009). Erickson et al. (2002) detected AHLs from 6 out of 8 cattle that were fed with a different barley/alfalfa ratio. The researchers determined that the cattle that was fed high concentrate diets tended to have longer chain AHLs than the cattle that was fed high forage diets. This indicated that AHLs secretion by the rumen microbes was related to diet. Edrington et al. (2009) reported that AHLs were detectable in the rumen contents of feedlot cattle in spring, summer, and in fall, but not in winter. In this study, no AHLs activity was detected in vivo or in vitro, which could be explained in two ways. First, no AHLs were secreted by the rumen microbes under the current maize stover-concentrate diet, and when NDF and soluble starch were used as in vitro fermentation substrates. Second, AHLs-mediated QS was not evolved by the microbes that inhabited the rumen of goat and sheep. The observed variation between cattle and goat/sheep was due to the difference in ruminal microbial communities. Indeed, EHEC is largely prevalent in cattle herds, and its colonization in cattle is accomplished by AHLs-mediated QS via SdiA (Hughes et al. 2010; Sperandio 2010). Interestingly, because EHEC is responsible for the outbreaks of bloody diarrhea worldwide, the interference with SdiA-mediated EHEC colonization in cattle is an exciting approach for reducing the cattle vulnerability to this human pathogen and for decreasing the contamination of meat

products (Hughes et al. 2010; Sperandio 2010). Therefore, our results implied that no pathogenic microorganisms, such as EHEC, inhabited in the rumen of experimental goats that were used in this study.

It was noticeable that the retention time of AHLs stan-dards in the current study was relatively short (Table 1) com-pared with the results reported by Cataldi et al. (2004). This could have been caused by the difference in sensitivities of different GC-MS systems. More work needs to be performed to determine the exact nature and function of substances that were detected using GC-MS analysis in this study.

3.2. Detection of the AI-2 activity

All samples that were collected from the goat rumen and from in vitro fermentation fluids were AI-2 negative. When the AI-2 activity was evaluated using in vivo samples, the light production of V. harveyi BB170 was below that of the negative control. This suggested that no AI-2 was detected in in vivo samples. Thus, we speculated that the ruminal contents contained chemical compounds, which inhibited the V. harveyi BB170 light production. No regular changes in the relatively light unit (RLU) were founded for all detected ruminal contents at different sampling times from the same and different goats (Fig. 3-A). This could have been caused by the individual differences of the experimental animals.

To reduce the influence of animals and of the complex contents of ruminal fluids, the in vitro fermentation fluids were assayed for the AI-2 activity. The RLU values of all samples were lower than those of the negative control. The RLU of samples at the same time-point, which were collected from in vitro fermentation fluids using soluble starch as the substrate, was lower compared with the RLU when NDF

Fig. 1 Retention times of C6-HSL, C8-HSL, C10-HSL (A), and of the representative ruminal fluid samples (B).

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Fig. 2 Fragmentation patterns of C6-HSL (A), C8-HSL (B), C10-HSL (C), and of the representative ruminal fluid samples (D, E, F, G, H). RT, retention time. The AHLs standards without the side chain substituent showed a common fragmentation pattern with the most abundant ion at m/z 143.

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was used as the substrate (Fig. 3-B). During the in vitro fermentation, soluble starch was fermented faster than NDF and led to higher production of chemical compounds, which inhibited the light production by V. harveyi BB170.

Mitsumori et al. (2003) and Lukas et al. (2008) detected the AI-2 activity in the rumen contents of three cows as well as in pure culture fluids of several rumen bacteria. Howev-er, it was clear that the AI-2 detection ability of V. harveyi BB170 as a reporter strain could be interfered with by the components of culture medium and by time, which led to either false negative or positive results (DeKeersmaecker et al. 2003; Turovskiy and Chikindas 2006; Vilchez et al. 2007). It was noticeable that the AI-2 detection procedures of Mitsumori et al. (2003) could have given a false positive detection because Mitsumori et al. (2003) measured the light production only at 7 h after incubation, which was long enough for V. harveyi BB170 itself to produce high level of light without the addition of exogenous AI-2. Bassler et al. (1997) indicated that the maximal light production occurred between 3 and 4 h during the detection process, and that light production should be measured every 15 min after 3 h of incubation. On the other hand, Lukas et al. (2008) ignored the effect of glucose, which was one of the culture medium components on V. harveyi BB170. However, Turovskiy and Chikindas (2006) reported that exogenous glucose, which was added to the culture medium, inhibited bioluminescence of V. harveyi. The results in this study suggested that V. harveyi BB170 was unsuitable for detecting the AI-2 activity of rumen fluids in vivo and in vitro as a reporter strain, which was due to the complexity of the rumen contents and in vitro fermentation fluids. Future studies using other AI-2 detection methods, such as FRET-based AI-2 assay (Zhu and Pei 2008; Raut et al. 2015) and liquid chromatography-tandem mass spectrometry analysis (Campagna et al. 2009), are

necessary to verify the presence or absence of AI-2 activities in the rumen of goats.

3.3. Cloning and sequencing homologues of the luxS gene

The homologues of luxS gene (GenBank accession num-bers: KC493139–KC493150) were successfully amplified using the primer pair luxS PR-S1/luxS PR-A1. Amplification of possible luxS homologues using primers designed by putative luxS sequences of Ruminococcus flavefaciens FD-1 and Ruminococcus albus R7 was unsuccessful. This revealed that the luxS sequences of R. flavefaciens FD-1 and R. albus R7 were merely draft genomes and needed further correction. The sequences obtained in this study were aligned with reference sequences (obtained from the GenBank using MEGA 5.1 software) and with a phylogenetic tree, which was evaluated using the bootstrap test based on 1 000 resamplings and that was constructed using the neighbor-joining method (Fig. 4). The amino acid sequenc-es, which were deduced from the luxS homologues that were detected in this study and from the amino acid sequence of LuxS of Prevotella ruminicola 23, showed that they were closely related. However, the amino acid sequences were not closely related to the other reference sequences.

Mitsumori et al. (2003) cloned the luxS gene homologues from the rumen of cows using the degenerate primers of luxS-F1/luxS-R1 and luxS-F2/luxS-R2. However, using the same primers, we failed to amplify the same target gene from the ruminal bacteria of goats, whilst the luxS gene homologues were successfully amplified using the primer pair luxS PR-S1/luxS PR-A1. In addition, the phylogenetic placement of amino acid sequences supported the fact that the luxS homologues, which were detected in this

Fig. 3 AI-2 detection of in vivo ruminal fluid samples from six goats (A), where 0 means the morning feeding time, –2 means 2 h before the morning feeding; and in vitro ruminal fluids at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h (B). RLU, relative light unit; NDF, neutral detergent fiber.

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study, were not close to the luxS homologues that were obtained from the rumen of cows by Mitsumori et al. (2003) (see Fig. 4). This further confirms the speculation that the ruminal bacteria of secreting AI-2 may be different between cows and goats. In this study, the observation of luxS gene homologues without the AI-2 activities demonstrated that the AI-2 detection methods should be further improved for mixed ruminal fluids.

4. Conclusion

The AHLs and AI-2 were not detected either in vivo or in vitro ruminal fluids of goats under current feeding condi-tions. The successfully cloned and sequenced homologues of the luxS gene indicated that the ruminal microbes in goats may potentially have the ability of secreting AI-2 signaling molecules. Therefore, the AI-2-mediated QS may be in-volved in rumen environment, and new methods need to be developed for detecting AI-2 activities in mixed ruminal fluids. In the future, more work needs to be performed to determine the possible role that luxS and QS played in rumen microbe-microbe communication during the feed digestion or microbial pathogenesis. This will finally allow us to adjust the QS procedure to improve the ruminant

animal feed efficiency and to reduce methane emission. In addition, it will be possible to disturb the QS of pathogenic microorganisms to improve animal health to gain better economic benefits.

Acknowledgements

We want to express our thanks to Sun Baolin, School of Life Sciences, University of Science & Technology of China for kindly providing the reporter strains (V. harveyi BB120, 170), and Dr. Guan Leluo of University of Alberta, Edmonton, Canada for valuable suggestions. The author gratefully ac-knowledges the financially support of the Chinese Academy of Sciences (KZCX2-YW-455), the CAS/SAFEA Internation-al Partnership Program for Creative Research Teams, China (KZCX2-YW-T07) and K C Wong Education, Hong Kong.

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(Managing editor ZHANG Juan)