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Keywords:

  • bioassay;
  • LC-MS;
  • AHLs;
  • morphological observation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Quorum sensing (QS) is a system of cell-to-cell communication by means of intercellular signaling molecules to coordinate a set of targeted gene expression or repression in many Gram-negative bacteria; it plays important roles for bacteria in adaptation to adverse environmental conditions. In this study, we first demonstrated that Microcystis aeruginosa PCC-7820 could produce QS-related signal acylated homoserine lactones (AHLs) among the metabolite of axenic M. aeruginosa, based on bioassay and liquid chromatography–mass spectrometry (LC-MS) analysis. The concentration of the AHLs in the culture medium was cell density dependent and reached a maximum of 18 nM at 1.03 × 107 cells mL−1, 30 days after inoculation. The regulation mechanism of QS in M. aeruginosa and its possible role in bloom formation are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Quorum sensing (QS) is a system of stimulus and response that is correlated to population density by means of inter- or intracellular signaling molecules (autoinducers) (Kaplan & Greenberg, 1985). Many species of bacteria use QS to coordinate sets of targeted gene expression or repression that relies on the density of their local population, which experiences a concentration threshold of signal molecules such as N-acyl-homoserine lactone (AHL), cyclic thiolactone, furanosylborate, methyl dodecenoic acid, hydroxypalmitic acid methyl ester, and farnesoic acid (Dong & Zhang, 2005; Williams, 2007; von Bodman et al., 2008).

QS and its mediated signals have been described in more than 70 different Gram-negative species of bacteria. However, to date, there have been a few investigations of its occurrence in cyanobacteria, which are photosynthetic Gram-negative prokaryotes. Sharif et al. (2008) indicated that the epilithic colonial cyanobacterial species Gloeothece PCC6909 had a QS system that was mediated by a signal molecule of C8-AHL. It was suspected that QS was able to improve Gloeothece's adaptation to environmental stress and acquire species competition advantages in the natural ecosystems.

Cyanobacteria, a group of photosynthetic prokaryotes, are the dominant bloom-forming species because of its strong adaptation to environmental stress by utilization of various sensing mechanisms and intracellular signaling systems. The involvement of signal transduction and cell-to-cell cooperation indicates that a role for autoinducer-like compounds may exist in such responses (Sharif et al., 2008). In fact, such QS molecules have been reported in cyanobacterial assemblages (Bachofen & Schenk, 1998; Braun & Bachofen, 2004). However, there are no reports of AHL production that is attributed specifically to axenic cyanobacteria, except for Gloeothece (Sharif et al., 2008). Microcystis aeruginosa is a species of freshwater cyanobacteria that can form harmful algal blooms that are of economic and ecological importance. Cells of this species usually are organized into colonies as a biofilm-like sheath. As QS has proven to be an important factor in the control of biofilm architecture (Davies et al., 1998; Huber et al., 2001; Lynch et al., 2002), is it possible that M. aeruginosa has a QS regulation system?

This study aims at detecting whether M. aeruginosa has a QS phenomenon by bioreporter assay and liquid chromatography–mass spectrometry (LC-MS) analysis. The ecological role of QS in M. aeruginosa has been discussed. The understanding of the role of QS in the regulation of M. aeruginosa growth and environmental adaptation may be useful in developing new strategies to control bloom formation and outbreak in freshwater ecosystems.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Growth and culture conditions of Maeruginosa

An axenic strain of M. aeruginosa PCC-7820, which was kindly supplied by Dr Pengfu Li at Nanjing University, China, was grown and maintained in a growth chamber at 28 °C day−1 and 22 °C night−1, with a 14 : 10 h light–dark regime under an illumination of 3000 lx (Mu et al., 2007). For determination of growth rate and AHLs, a 200-mL culture of M. aeruginosa at the exponential phase was harvested under sterile conditions and centrifuged at 8000 g for 10 min. The pellet was resuspended in 500 mL of fresh sterile BG11 medium to a final cell concentration of 1 × 10mL−1 in 1-L flasks. The flasks were incubated in a growth chamber as described above. The cultures were sampled at 10, 20, 30, and 40 days after inoculation for growth measurement at 680 nm with an ultraviolet/visible spectrophotometer (PGENERAL, China), bioreporter assay, and AHLs detection with LC-MS analysis. Each sample was replicated for three times.

Extraction of AHLs

AHLs were extracted from the culture in accordance with reported literature (Yates et al., 2002) with some modifications. Three hundred milliliter of algal culture was centrifuged at 8000 g for 10 min to remove cells, and then the supernatant was adjusted to pH 2 and stored at 4 °C for 16 h. After that, the sample was extracted three times with 150 mL of dichloromethane. The combined dichloromethane extracts were dried by anhydrous MgSO4 and evaporated to dry. The resulting residue was dissolved in 1 mL of HPLC-grade methanol, sealed, and stored at −20 °C until they were required.

Bioreporter assay of AHLs

Three bioreporters were used to test whether M. aeruginosa can produce a QS signal. Agrobacterium tumefaciens (AT) bioassay strain KYC55 (pJZ410) (pJZ372) (pJZ384), which was kindly supplied by Dr Jun Zhu at Nanjing Agricultural University and was cultivated in AT medium supplemented with appropriate antibiotics (Zhu et al., 2003). The dichloromethane extracts were added to AT medium containing the AHL monitor strain A. tumefaciens KYC55 and tested for β-galactosidase activity (Miller, 1972). Chromobacterium violaceum bioassay strain CV026 mutated in the AHL synthase gene cviI was kindly supplied by Dr Yonghua Yang at Nanjing University and was grown in liquid LB medium at 28 °C to test whether the strain is able to produce violacein when the dichloromethane extract of M. aeruginosa was supplied to the growth medium (McClean et al., 1997). Vibrio harveyi bioassay strain BB170 was purchased from Guangdong Institute of Microbiology, China, and was grown in autoinducer bioassay medium to determine the presence of AI-2-like molecules in the dichloromethane extracts of M. aeruginosa by examination of the bioluminescence (Bassler et al., 1997).

Mass spectrometric identification

AHLs were studied by LC-MS on a C18 stationary phase column [150 × 2.1 mm, Patricle Sz. (u) Dim.]. The mobile phases consisted of water (A) and acetonitrile (B) at a flow rate of 0.2 mL min−1. The organic content in the gradient was increased from 15% B to 65% B over 40 min and then to 15% B over 5 min with an additional 5 min at 15% B. Injection volume was 10 μL, and UV detection was set at 210 nm.

The eluent from the HPLC was linked directly to a liquid chromatography (LCQ) Advantage MAX mass spectrometer (Finnigan). For identification of AHLs, the mass spectrometer was operated in full-scan mode from 50–800 Da to determine whether any signals indicative of the compounds could be detected. AHLs were regarded as being present only if the HPLC retention time, the full-scan MS, and subsequent fragmentation analysis were in agreement with those of the reported AHLs (Sharif et al., 2008).

Morphological observation of M. aeruginosa cells

Algal cells for SEM observation were collected from the abovementioned 1000-mL culture of M. aeruginosa at 10, 20, 30, and 40 days after inoculation and prepared according to the method described by Chen & Yeh (2005). Basically, the algae cells were filtered through a 0.45-micron nylon membrane filter. Then, the membrane filters were fixed with 2.5% gluteraldehyde at 4 °C overnight, washed with PBS buffer (pH7.0), and dehydrated with successive increasing different concentrations of ethanol. The dried samples were mounted on copper stubs and sputter coated with gold–palladium and observed using a scanning electron microscope (JEOL-JSM-6490, Japan).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bioassay of QS

Detection with three bioreporters showed that both C. violaceum CV026 and V. harveyi BB170 exhibited a negative reaction, while A. tumefaciens KYC55 revealed a positive reaction when they were cultured with the addition of the dichloromethane extracts of M. aeruginosa at 10, 20, and 30 days after inoculation. Based on specific targets of the three biosensors, the results demonstrated that M. aeruginosa could produce QS signals that belonged to an autoinducer-1 (AI-1) with a long chain.

The relative concentrations of QS signals in the metabolites of M. aeruginosa at given growth phases were measured based on the β-galactosidase activity of strain KYC55 when they were treated with AHL compound N-3-oxo-octanoyl homoserine lactones (OOHL). Results showed that the concentration of QS signals in the metabolites of M. aeruginosa increased in a density-dependent manner and reached its highest concentration when the OD680 nm was about 1.57 (1.03 × 107 cells mL−1), 30 days after inoculation, and then the concentration declined rapidly (Fig. 1). The highest concentration of signaling molecules in the culture was about 18 nM relative to the reference OOHL based on the β-galactosidase activity.

image

Figure 1. Relationship between cell growth (■: OD680) and the production of QS signals (▲: β-galactosidase activity) in the metabolites of M. aeruginosa PCC-7820.

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Mass spectrometric identification of AHLs

Mass scan analysis from 50 to 800 Da showed that three compounds in the metabolites of M. aeruginosa possessed the characteristic lactone moiety at m/z 102 of AHL-like molecules at retention time of 25.7, 27.7, and 39.2 min (Fig. 2). One of the compounds eluted at 39.2 min exhibited a quasi-molecular ion peak at m/z 256, in addition to the typical ion at m/z 101.8 that is characteristic of an AHL fragment (Shaw et al., 1997). The ion at m/z 238 owing to [M +H−18]+ was produced by the AHLs because of the loss of water from the alkyl chain (Morin et al., 2003). These common features disclosed that this compound also belonged to the C4–14 AHL series. However, the strongest product of ions at m/z 88.1 is quite different from either the 3-oxo-C4–14 AHL compounds whose putative diagnostic ions often appeared at m/z 98 (Ortori et al., 2007) or the 3-hydroxy-AHLs series whose diagnostic ions appeared at m/z values of 55, 69, 83, 97, etc., according to different alkyl chain length (Shaw et al., 1997). As for the unsubstituted acyl side chains systems, the diagnostic ions at m/z values of 95, 109, 123, and 137 become more prevalent (Ortori et al., 2007). This observation proved the existence of a CH3CH(OH)CH2CO-unit in the alkyl chain. Moreover, the quasi-molecular ion peak at m/z 256, along with the AHL moiety led to the deduction of the structure (Fig. 2).

image

Figure 2. MS/MS spectrum of the peak eluted at 27.68, 33.86, and 39.2 min in HPLC, respectively.

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Morphological observation

SEM photographs of M. aeruginosa showed that the algal cells seemed to be experiencing free-living (< 20 day), aggregation (20–40 days), and disintegration (> 40 days) growth phases under laboratory culture conditions (Fig. 3). In addition, a biofilm-like membrane layer formed at 30 days after inoculation, which accompanied a strong aggregation of the cells (Fig. 3c1 and c2).

image

Figure 3. SEM photographs of M. aeruginosa PCC-7820 during growth periods. (a), (b1), (c1) and (d) indicate cell morphology at 10, 20, 30, and 40 days, respectively, after inoculation in fresh BG-11, while (b2) and (c2) indicate that at 20 and 30 days after inoculation in BG-11 medium containing AHL extracts.

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To test the biological effects of QS signal, algal cells were cultured in BG-11 medium containing AHLs extracts (about 20 nM relative to the reference OOHL), which was obtained from the culture of M. aeruginosa at 30 days after inoculation. Compared with those in the fresh BG-11, the AHLs extracts could promote the formation of a biofilm-like membrane in M. aeruginosa, which appeared at 20 days (Fig. 3b2) and became thicker at 30 days (Fig. 3c2) after inoculation.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

QS that involves AHLs has been described in more than 70 different Gram-negative species of bacteria. All AHLs are composed of the conserved homoserine lactone ring and an amide (N)-linked acyl side chain that varies in the range of 4–18 carbons, may be saturated or unsaturated and be with or without the substitution at the third position (usually hydroxy- or oxo-) (Czajkowski & Jafra, 2009). However, to date, there have been only a few studies of QS occurrence in cyanobacteria, which are an important group of photosynthetic Gram-negative prokaryotes (Bachofen & Schenk, 1998; Braun & Bachofen, 2004). Even the reported results also suffered from the same deficiency in that samples used to detect AHLs were obtained from an open lake, which certainly contained numerous other AHL-producing bacteria. Only in 2008 did Sharif et al. show for the first time that the cyanobacterium Gloeothece could produce C8-AHL QS signal in axenic culture. In this study, M. aeruginosa PCC-7820 was cultured axenically during the whole growth period and was tested for the presence of other microorganisms periodically by microscopic observation and culture detection on LB plates. Other microorganisms were not found in these two detection methods throughout the M. aeruginosa growth process. Therefore, it is the first report to detect the production of AHLs in the cyanobacterium M. aeruginosa in axenic cultures by both bioreporters assay and LC-MS technique.

The bioassay strain C. violaceum CV026 has high sensitivity to short-chain unsubstituted AHLs such as C4-AHL and C6-AHL, but not C8-AHL or longer, while A. tumefaciens KYC55 has the broadest range of AHL detection including short-chain, long-chain, substituted, and unsubstituted AHLs (Steindler & Venturi, 2007). Vibrio harveyi BB170 is another type of bioreporter that is applied widely to detect AI-2-like molecules (DeKeersmaecker & Vanderleyden, 2003). Based on the characteristics of the three bioreporters and the results of the biosensors assay, A. tumefaciens KYC55 showed a positive reaction but C. violaceum CV026 and V. harveyi BB170 did not; we suggest that M. aeruginosa could synthesize AHL-like molecules with long acyl side chains. Moreover, the concentration of these signaling molecules increased in a density-dependent manner and reached its highest concentration of 18 nM relative to the reference OOHL when the cell density was about 1.03 × 107 cells mL−1, 30 days after inoculation (Fig. 1). Such concentration might be sufficient to trigger a QS-related response in M. aeruginosa. However, the AHLs concentration of M. aeruginosa declines sharply at day 30 when the alga moves to the late growth phase (Fig. 1). Similar phenomenon has been observed in other bacteria such as A. tumefaciens, Erwinia carotovora, and Xanthomonas campestris, the QS signal of the bacteria accumulates in early stationary phase and its level subsequently declines sharply when bacteria move into stationary phase (Barber et al., 1997; Holden et al., 1998; Zhang et al., 2002). This phenomenon might be controlled by quorum-sensing signal-turnover systems in the bacteria (Zhang et al., 2002) or AHLs alkaline hydrolysis with the pH increase in the cultures (Gao et al., 2005).

LC-MS has been used to identify the structure of AHL-like molecules in microbial metabolites, because all AHLs are composed of the conserved homoserine lactone ring and an amide (N)-linked acyl side chain that varies in the range of 4–18 carbons. The structure of the characteristic lactone ring will not be destroyed in the MS process to produce a characteristic fragment of m/z 102, which corresponds to the homoserine lactone moiety (Bruhn et al., 2004). Based on the characteristic ion peak m/z 102, 3 AHL candidates have been detected at retention time 25.7, 27.7, and 39.2 min. One of them has been identified possibly to be a AHL with a CH3CH(OH)CH2CO- unit in the alkyl chain. However, the precise structure of the deduced compound has not been fully elucidated because of the limited amount of the metabolites in M. aeruginosa. The method of synthetic the compound has should be researched to further verify the accuracy of deduced compound and its function.

SEM photographs of M. aeruginosa revealed that the algal cells experienced free-living within 20 days and appeared a biofilm-like membrane at 30 days after inoculation, which led to a strong aggregation of the cells (Fig. 3). The coincident appearance of the biofilm-like membrane and the AHL indicates that QS might play an important role in morphological changes in M. aeruginosa for environmental adaptation. Compared with those in the fresh BG-11, algal cells cultured in BG-11 medium containing AHLs extracts (about 20 nM relative to the reference OOHL) had an earlier and thicker formation of biofilm-like membrane, which provided strong evidence that M. aeruginosa had a QS system regulating colony formation because of the biofilm-like membranes. In fact, many reports indicate that the biofilm is regulated by QS. For instance, Davies et al. (1998) reported that Pseudomonas aeruginosa formed undifferentiated and thin biofilms in comparison with the wild type when the QS system–encoding genes of lasR-lasI and rhlR-rhlI had mutated. Similar phenomena have been observed in the species of Burkholderia cepacia (Huber et al., 2001) and Aeromonas hydrophila (Lynch et al., 2002). Therefore, the formation of a biofilm-like membrane, an important physiological characteristic of Microcystis, can not only help Microcystis acquire a better niche (Cheng & Qiu, 2006) and capture plenty of light and nutrients in the aquatic ecosystem, but also play an important role in resistance to zooplankton prey (Lynch & Shapiro, 1981), which is important for Microcystis to stay as the dominant species and for outbreak of blooms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the National Basic Research Program of China (2008CB418004), the Jiangsu Science and Technology Support Program (BE2011355, BE2012372), the Special Fund for the Public Service Sector of the National Environmental Protection Ministry (201009023), the Fundamental Research Funds for the Central Universities (1082020803, 1092020804), and the National Training Program for Fundamental Scientists (J1103512).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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