Characterization of N-Acylhomoserine Lactones Produced by Bacteria Isolated from Industrial Cooling Water Systems

The cooling water systems are used to remove heat generated in the various industries. Biofouling of the cooling water systems causes blocking of condenser pipes and the heat exchanger tubes. In many Gram-negative bacteria, N-acylhomoserine lactone (AHL) are used as quorum-sensing signal molecule and associated with biofilm formation. To investigate the relationship between quorum sensing and biofouling in the cooling water system, we isolated a total of 192 bacterial strains from the five cooling water systems, and screened for AHL production. Seven isolates stimulated AHL-mediated purple pigment production in AHL reporter strain Chromobacterium violaceum CV026 or VIR07. Based on their 16S rRNA gene sequences, AHL-producing isolates were assigned to Aeromonas hydrophila, Lysobacter sp., Methylobacterium oryzae, and Bosea massiliensis. To the best of our knowledge, B. massiliensis and Lysobacter sp. have not been reported as AHL-producing species in the previous researches. AHLs extracted from the culture supernatants of B. massiliensis and Lysobacter sp. were identified by liquid chromatography-mass spectrometry. AHLs produced by B. massiliensis were assigned as N-hexanoyl-l-homoserine lactone (C6-HSL), N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL), and N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL). AHLs produced by Lysobacter sp. were assigned as N-decanoyl-l-homoserine lactone (C10-HSL) and N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL). This is the first report of identification of AHLs produced by B. massiliensis and Lysobacter sp. isolated from the cooling water system.


Introduction
Quorum sensing is one of the cell-cell communication systems depending on cell population density [1]. Quorum-sensing signaling is mediated by an autoinducer, which is a self-produced signal molecule [1]. In many Gram-negative bacteria, several kinds of N-acylhomoserine lactone (AHL) have been identified as autoinducer signal compounds involved in this mechanism [2]. The substrates in AHL synthesis are S-adenosyl methionine (SAM) and acyl-acyl carrier proteins. AHLs are synthesized bacteria by LuxI family protein, and diffused outside and inside the cell. Some AHLs requires active transport through an efflux pump [3]. When intracellular concentration of AHLs increases, LuxR family protein binds AHL and regulates the expression of specific genes [2]. AHL-mediated quorum sensing regulates the various phenotypes such as bioluminescence, motility, pigment production, and biofilm formation [1,2]. In general, AHL-deficient mutants show defects in quorum-sensing regulated gene expression. Therefore, it is expected that disrupting or manipulating the AHL signals could inhibit the expression of quorum-sensing regulated phenotypes such as virulence and infection of host cells [4].
The cooling water systems are used to remove heat generated in the various industries such as chemical and petrochemical, electric power generating stations, and paper mills [5]. Biofouling, which is accumulation of microorganisms on the wet surfaces, is one of the serious problems in the cooling water systems. Biofouling of the cooling water systems causes blocking of condenser pipes and the heat exchanger tubes [5]. Chlorine is one of the most used chemicals for controlling biofouling in the industrial cooling water systems. However, to inhibit biofouling with effect, it is necessary for continuous or intermittent chlorination in the industrial cooling water systems. In the case of the membrane bioreactor (MBR) wastewater treatment systems, quorum sensing is associated with the formation of a biofouling layer on the membrane surface [6]. Quorum-quenching technique has been investigated as an approach to controlling biofouling in MBR wastewater treatment systems [6,7]. However, there is no research example that describes the relationship between biofouling of the cooling water systems and quorum sensing. In a previous study, the AHL synthase gene homolog were found within 26% of the 265 completed Proteobacterial genomes [8]. It was revealed that the majority of the bacterial community in an industrial cooling water system was Proteobacteria [5]. From these facts, there is a high possibility that AHL-mediated quorum sensing affects the development of biofouling in the cooling water system. To our knowledge, the AHL-producing bacterial community in the industrial cooling water system has not been examined. In this study, we screened for AHL-producing bacteria from the industrial cooling water systems, and identified AHL structures produced by them.

Isolation of Bacteria from Industrial Cooling Water Systems
The cooling water samples were collected from the industrial cooling water systems of factory A, B, C, D, and E. The water samples were serially diluted in sterilized distilled water and spread on 1/5-diluted trypticase soy broth (TSB) agar plate (Becton, Dickinson and Company). The plates were incubated in the dark at 30˝C for 72 h. Thereafter, bacterial colonies were randomly selected from among those that grew on the 1/5-diluted TSB plate. Colonies from each sample were randomly selected and transferred onto a new 1/5-diluted TSB agar medium.

Screening of AHL-Producing Bacteria
AHLs were detected by two AHL biosensors, Chromobacterium violaceum CV026 and VIR07. Strain CV026 and VIR07 respond to short-chain and long-chain AHLs respectively, by producing the purple pigment violacein [9,10]. The bacterial isolates were inoculated onto a 1/5-diluted TSB agar medium prepared in the 96-well plates. Strain CV026 and VIR07 were inoculated onto the lower right or left positions of the same well. The bacterial colonies were incubated at 30˝C for 24 h. AHL-producing colonies, which induced the production of purple pigments of the AHL reporter strains, were selected.

Identification of AHL-Producing Bacteria
Chromosomal DNA of AHL-producing bacteria was extracted using DNeasy Blood and Tissue Kit (QIAGEN). To identify the bacterial species of AHL-producing bacteria, the 16S rRNA gene-encoding regions were amplified by PCR with Blend Taq plus DNA polymerase (Toyobo, Osaka, Japan) and the previously described universal primers, 27f (5 1 -AGA GTT TGA TCM TGG CTC AG-3 1 ) and 1525r (5 1 -AGG AGG TGW TCC ARC C-3 1 ) [11]. After electrophoretic separation, 16S rRNA fragments were purified from agarose by using MagExtractor PCR and Gel Clean up (Toyobo). Sequencing reactions were performed with the BigDye Terminator, ver. 3.1 and Applied Biosystems 3500 Series Genetic Analyzer (Applied Biosystems). Closest type-strain 16S rRNA gene relatives to each clone sequence were determined using the RDP II sequence match tool [12]. A phylogenetic tree by the neighbor-joining method was constructed by Molecular Evolutionary Genetic Analysis (MEGA) [13].

Extraction of AHLs
AHL-producing bacterial strains were inoculated into 4 mL of 1/5-diluted TSB medium and incubated at 30˝C for 18 h with shaking. Two milliliter of full-grown cultures of bacterial strains were transferred into 200 mL of fresh 1/5-diluted TSB medium and incubated at 30˝C for 24 h with shaking. After cultivation, cells were removed by centrifugation at 10,000ˆg for 5 min. The culture supernatant was concentrated by evaporation using a rotary evaporator. The concentrated supernatant was extracted with three times its volume of ethyl acetate in a separatory funnel. The extract was evaporated to dryness using a rotary evaporator and was then dissolved in 500 µL of dimethylsulfoxide. To check the presence of AHL, full-grown culture of CV026 or VIR07 was mixed with LB agar medium. Eight-millimeter paper disks, which were applied AHL samples, were placed on LB agar medium containing CV026 or VIR07. After overnight incubation at 30˝C, the appearance of purple pigment was determined as the presence of AHLs.

AHL Identification by Liquid Chromatography-Mass Spectrometry (LC-MS)
Identification of AHL were performed using a triple quadrupole/linear ion trap instrument (LIT) (QTRAP5500; AB Sciex) with an electrospray ionization (ESI) source and coupled to an UHPLC system (Nexera X2; Shimadzu). Chromatographic separation was achieved on a C18 column (Kinetex F5, ϕ 2.1ˆ150 mm, 2.6 µm; Phenomenex). For the analysis of AHL, the elution of the samples was carried out using acetonitrile (solvent A) and water (solvent B), both of which contained 0.1% (vol/vol) acetic acid the following gradient (vol/vol): initially 10% A, rising to 50% A at 1.5 min, followed by a 7 min gradient to 99% A. Finally, the column was equilibrated for 3 min, using this solvent composition. The column was operated at 30˝C with a flow rate of 0.2 mL/min. MS/MS spectra were recorded in product ion scan mode using LIT. Ion source was maintained at 400˝C with curtain gas at 20, collisional activated dissociation (CAD) gas at 7 psi (12 psi for LIT), ion source gas at 80 psi, and ion source gas2 at 70 psi. Ionspray voltage was set at 5500 V in positive ion mode. Declustering, entrance, and collision cell exit potentials were maintained at 15 V. Injection volume (2 µL) and the settings used was as described previously. Precursor ion scanning experiments were performed in positive ion mode for analysis, where Q1 was set to scan a mass range of m/z 80 to 500 Da and Q3 was monitor for the product ion that indicates presence of lactone ring at m/z 102. AHLs were analyzed by LC-MS/MS with MS/MS and precursor ion scanning experiments by identification from synthetic AHLs standards. AHL standards, N-hexanoyl-L-homoserine lactone (C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL), and N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL), were synthesized using a previously described method [14].

Nucleotide Sequence Accession Numbers
The nucleotide sequences of 16S rRNA were deposited in the DDBJ/ENA/GenBank database with the following accession numbers, LC102389-LC102396.

Isolation and Identification of AHL-Producing Bacteria
A total of 192 bacterial strains were isolated from 5 different factories and examined for the further study. As the results of screening of AHL-producing bacteria using C. violaceum CV026 and VIR07, 7 bacterial strains showed AHL-producing activity. 16S rRNA gene were amplified by PCR and sequenced for identification of bacterial species (Table 1). AHL-producing strain D10, which was isolated from factory B, was assigned to the Aeromonas hydrophila. Two AHL-producing strains F6 and F13, which were isolated from factory C, were assigned to the A. hydrophila and Lysobacter brunescens, respectively. Four AHL-producing isolates, which were isolated from factory D, were assigned to the Methylobacterium oryzae (strain D7 and F9) and Bosea massiliensis (strain E9 and F12). Table 1. Identification and characterization of N-acylhomoserine lactone (AHL)-producing strains on 96-well plates. Induction of AHL-responsible violacein production by CV026 or VIR07 is represented by the number of plus signs; +, low induction; ++, high induction; -, no induction.

Phylogenetic Analysis of AHL-Producing Isolates
Phylogenetic analysis was carried out to examine the relationships of AHL-producing isolates ( Figure 1). A. hydrophila, which is close to strain D10 and F6, is found in freshwater environments and well-known AHL-producing bacteria [15]. It has been reported that AHL-producing Aeromonas strains were isolated from activated sludge [11,15]. It has been reported that Aeromonas strains mainly produced N-butyryl-L-homoserine lactone (C4-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) [11]. From the results of AHL-production assay, it was assumed that the A. hydrophila D10 and F6 can produce the short chain AHLs such as C4-HSL and C6-HSL as well as reported AHL-producing Aeromonas species. Methylobacterium species were often isolated from the tap water and well-known AHL-producing bacteria [16,17]. Methylobacterium have been reported to exhibit higher stress-tolerance than the other species found in biofilms, particularly under conditions of extreme nutrient limitation [18]. On the other hand, Lysobacter sp. and Bosea massiliensis have not been reported as AHL-producing bacteria. This is the first report of an AHL-producing bacterial strain belonging to the genus Lysobacter and Bosea. F6 and F13, which were isolated from factory C, were assigned to the A. hydrophila and Lysobacter brunescens, respectively. Four AHL-producing isolates, which were isolated from factory D, were assigned to the Methylobacterium oryzae (strain D7 and F9) and Bosea massiliensis (strain E9 and F12).

Phylogenetic Analysis of AHL-Producing Isolates
Phylogenetic analysis was carried out to examine the relationships of AHL-producing isolates ( Figure 1). A. hydrophila, which is close to strain D10 and F6, is found in freshwater environments and well-known AHL-producing bacteria [15]. It has been reported that AHL-producing Aeromonas strains were isolated from activated sludge [11,15]. It has been reported that Aeromonas strains mainly produced N-butyryl-L-homoserine lactone (C4-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) [11]. From the results of AHL-production assay, it was assumed that the A. hydrophila D10 and F6 can produce the short chain AHLs such as C4-HSL and C6-HSL as well as reported AHL-producing Aeromonas species. Methylobacterium species were often isolated from the tap water and well-known AHL-producing bacteria [16,17]. Methylobacterium have been reported to exhibit higher stress-tolerance than the other species found in biofilms, particularly under conditions of extreme nutrient limitation [18]. On the other hand, Lysobacter sp. and Bosea massiliensis have not been reported as AHL-producing bacteria. This is the first report of an AHL-producing bacterial strain belonging to the genus Lysobacter and Bosea.

Analytical Identification of AHL Molecules
In this study, we identified the AHL profiles of B. massiliensis E9 and F12, and Lysobacter sp. F13, which have not been reported as AHL-producing species. To identify the structure of AHL produced by these strains, AHL samples were extracted from the culture supernatants of these strains. The presence of AHLs was checked by LB agar plates containing C. violaceum CV026 and VIR07. As the results, AHL extracts from B. massiliensis and Lysobacter sp. strongly stimulated C. violaceum CV026 and VIR07, respectively (Figure 2). These results corresponded to those of 96-well plate assay in Table 1. HPLC fractionation followed by MS analysis of the AHL extracts of these strains were performed to identify the types of AHLs. The presence of C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C8-HSL was identified in the AHL extracts of B. massiliensis E9 (Figures 3-5). The profile of the AHL extracts of strain F12 completely corresponded to that of strain E9. The presence of C10-HSL and 3-oxo-C10-HSL was identified in the AHL extracts of Lysobacter sp. F13 (Figures 6 and 7). Two short-chain AHLs, C6-HSL and 3-oxo-C6-HSL, can stimulate the production of violacein by C. violaceum CV026 [9]. Three long chain AHLs, 3-oxo-C8-HSL, C10-HSL, and 3-oxo-C10-HSL, can stimulate the production of violacein by C. violaceum VIR07 [10]. AHL profiles of these three strains support the results of the AHL-production assay shown in Table 1 and Figure 2.

Analytical Identification of AHL Molecules
In this study, we identified the AHL profiles of B. massiliensis E9 and F12, and Lysobacter sp. F13, which have not been reported as AHL-producing species. To identify the structure of AHL produced by these strains, AHL samples were extracted from the culture supernatants of these strains. The presence of AHLs was checked by LB agar plates containing C. violaceum CV026 and VIR07. As the results, AHL extracts from B. massiliensis and Lysobacter sp. strongly stimulated C. violaceum CV026 and VIR07, respectively (Figure 2). These results corresponded to those of 96-well plate assay in Table 1. HPLC fractionation followed by MS analysis of the AHL extracts of these strains were performed to identify the types of AHLs. The presence of C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C8-HSL was identified in the AHL extracts of B. massiliensis E9 (Figures 3-5). The profile of the AHL extracts of strain F12 completely corresponded to that of strain E9. The presence of C10-HSL and 3-oxo-C10-HSL was identified in the AHL extracts of Lysobacter sp. F13 (Figures 6 and 7). Two short-chain AHLs, C6-HSL and 3-oxo-C6-HSL, can stimulate the production of violacein by C. violaceum CV026 [9]. Three long chain AHLs, 3-oxo-C8-HSL, C10-HSL, and 3-oxo-C10-HSL, can stimulate the production of violacein by C. violaceum VIR07 [10]. AHL profiles of these three strains support the results of the AHL-production assay shown in Table 1 and Figure 2.

Analytical Identification of AHL Molecules
In this study, we identified the AHL profiles of B. massiliensis E9 and F12, and Lysobacter sp. F13, which have not been reported as AHL-producing species. To identify the structure of AHL produced by these strains, AHL samples were extracted from the culture supernatants of these strains. The presence of AHLs was checked by LB agar plates containing C. violaceum CV026 and VIR07. As the results, AHL extracts from B. massiliensis and Lysobacter sp. strongly stimulated C. violaceum CV026 and VIR07, respectively (Figure 2). These results corresponded to those of 96-well plate assay in Table 1. HPLC fractionation followed by MS analysis of the AHL extracts of these strains were performed to identify the types of AHLs. The presence of C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C8-HSL was identified in the AHL extracts of B. massiliensis E9 (Figures 3-5). The profile of the AHL extracts of strain F12 completely corresponded to that of strain E9. The presence of C10-HSL and 3-oxo-C10-HSL was identified in the AHL extracts of Lysobacter sp. F13 (Figures 6 and 7). Two short-chain AHLs, C6-HSL and 3-oxo-C6-HSL, can stimulate the production of violacein by C. violaceum CV026 [9]. Three long chain AHLs, 3-oxo-C8-HSL, C10-HSL, and 3-oxo-C10-HSL, can stimulate the production of violacein by C. violaceum VIR07 [10]. AHL profiles of these three strains support the results of the AHL-production assay shown in Table 1 and Figure 2.

Conclusions/Outlook
In this study, it was found that there were various AHL-producing bacteria in the industrial cooling water system. Bosea have been reported as opine degraders in rhizosphere and an amoeba-resisting bacteria [19]. Interestingly, Bosea thiooxidans isolated from the tobacco rhizosphere exhibited the ability to inactivate AHL [20]. Lysobacter have been reported as ubiquitous beneficial

Conclusions/Outlook
In this study, it was found that there were various AHL-producing bacteria in the industrial cooling water system. Bosea have been reported as opine degraders in rhizosphere and an amoeba-resisting bacteria [19]. Interestingly, Bosea thiooxidans isolated from the tobacco rhizosphere exhibited the ability to inactivate AHL [20]. Lysobacter have been reported as ubiquitous beneficial

Conclusions/Outlook
In this study, it was found that there were various AHL-producing bacteria in the industrial cooling water system. Bosea have been reported as opine degraders in rhizosphere and an amoeba-resisting bacteria [19]. Interestingly, Bosea thiooxidans isolated from the tobacco rhizosphere exhibited the ability to inactivate AHL [20]. Lysobacter have been reported as ubiquitous beneficial bacteria emerging as novel biocontrol agents and a new potential source of anti-infectives [21]. Another quorum-sensing system, which was mediated by the diffusible signal factor (DSF), has been identified from Lysobacter enzymogenes [21]. However, there is no example of AHL-producing bacteria belonging to genus Bosea and Lysobacter. Many gram-negative bacteria use AHL-mediated quorum sensing to regulate the secretion of sticky extracellular polysaccharide (EPS), which is cause for biofouling [22]. AHL-degrading techniques have been investigated as an approach to controlling biofouling in membrane bioreactor (MBR) wastewater treatment systems [7,23]. Future studies of the relationship between EPS formation and AHL-mediated quorum sensing in cooling water isolates may provide useful information for the control of biofouling.