THE LC-MS/MS PROFILING OF AHLs PRODUCED IN SINORHIZOBIUM MELILOTI NODULATING ALYSICARPUS BUPLEURIFOLIUS (original) (raw)
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The aim of this research was to analyze the Quorum sensing (QS) and Quorum quenching (QQ) mechanisms based on N-acyl-L-homoserine lactones (AHLs) in A. brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions. Methods and Results We performed an in silico and in vitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained in vitro by the use of the reporter strains C. violaceum and A. tumefaciens and Liquid Chromatography coupled to Mass-Mass Spectrometry (LC-MS/MS) analysis shown that although Az39 does not produce molecules AHL, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated to the bacterial cell. In Az39 inoculated cultures incubated with 500 nmol l-1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than non-inoculated LB media controls. 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The appearance of LuxR-solos regulators indicates that these protein families could be involved in intra-kingdom or inter-kingdom signaling systems through the detection of different compounds produced by other prokaryotes or eukaryotes organisms (Patankar and Gonzalez 2009, Patel et al. 2013). In nature, there are also bacterial mechanisms that inactivate quorum signals called Quorum Quenching (QQ) (Zhang 2003). These can generally act both at the level of signal generation and reception. Although there are several QS mechanisms involving inhibitory proteins and/or AHL antagonist molecules, the mechanisms that involve enzymes are widespread in different environments. Three main enzymatic QQ mechanisms have been clearly described: (1) hydrolysis of the lactone ring (AHL lactonase activity), (2) hydrolysis of the amide bound (AHL acylase activity), and (3) modification of the acyl chain (AHL oxidase and reductase activity) (Uroz et al. 2009), but they have not been studied in depth in soil bacteria. As occurs in the QS system, QQ mechanisms can serve in particular environments to modulate the interaction between a bacterial community and eukaryotic organisms (Tait et al. 2009). Soil bacteria living in the rhizosphere, or rhizobacteria, have the ability to associate with numerous plant species. If this association is beneficial for plant growth or development, they are called Plant Growth Promoting Rhizobacteria or PGPR (Kloepper et al. 1989). Among the most successful associations and therefore the most studied in nature, are those related to the genus Azospirillum sp. The ability of these rhizobacteria to promote plant growth depends mainly on the presence of one or more mechanisms that might act individually or in synch on the physiology or metabolism of the colonized plant (Bashan and de-Bashan 2010) A. brasilense Az39 was isolated in 1982 from surface-sterilized wheat seedlings in Marcos Juarez, Córdoba, Argentina, evaluated under agronomic conditions and selected based on its ability to 4 increase crop yields of maize and wheat under agronomic conditions (Díaz-Zorita and Canigia 2009). A. brasilense Az39 has been widely used in agriculture in America during the last 40 years (Cassán and Díaz Zorita 2016). The potential mechanisms responsible for growth promotion in this strain have been partially unraveled (Perrig et al. 2007, Cassán et al. 2009). Despite its agroeconomic importance and the fact that several genomes from this genus have been sequenced, such as those belonging to Azospirillum sp. B510, A. lipoferum 4B, A. brasilense Sp245, CBG497 and Az39 (Kaneko et al. 2010, Wisniewski-Dyé et al. 2011, Wisniewski-Dyé et al. 2012, Rivera et al. 2014), there are few reports related to bacterial capacity to produce AHL-like molecules and/or other phenomena associated with quorum mechanisms. Therefore, there is little understanding about the Azospirillum-Azospirillum, Azospirillum-bacteria and Azospirrillum-plant interactions mediated by quorum mechanisms, highlighting the need for a more exhaustive genomic-functional analysis of these bacteria due their agricultural and economic interest. Considering this background, the main objective of this work was to analyze both in silico and in vitro the quorum sensing and quorum quenching phenomenon mediated by AHLs in the model strain A. brasilense Az39. 2. Material and Methods 2.1. Bacterial strains and growth conditions A. brasilense Az39 was obtained from the Bacterial Culture Collection at the INTA-IMYZA, Castelar, Buenos Aires, Argentina (WDCM31). Pure cultures of A. brasilense Az39 were obtained in Petri dishes containing Luria-Bertani medium (Miller 1972) modified by the addition of 15 ml l-1 Congo Red (LB-RC) or MMAB minimal medium (Vanstockem et al. 1987). Typical colonies from such media were used to inoculate LB liquid medium in 100 ml flasks and cultured at 37°C with 240 rpm shaking until late exponential growth phase was reached. Chromobacterium violaceum CV026 (McClean et al. 1997) grew in LB medium supplemented with 25 μg ml-1 kanamycin (Km). Agrobacterium tumefaciens NTL4/pZLR4 (Cha et al. 1998) was cultured in AT medium (Morton and Fuqua, 2013) supplemented with 50 μg ml-1 gentamicin (Gm). These two strains were used as reporter strains in the bioassays described below. 6 the reproducibility of the methodology and the stability of the AHL molecules in the Petri dish during incubation. Experiments were carried out in triplicate. 2.3.1.2. Evaluation of production of AHLs by Az39 using Liquid Chromatography coupled to Mass-Mass Spectrometry (LC-MS/MS) analysis 2.3.1.2.1. Extraction of AHLs from Az39 cultures Typically, A. brasilense AZ39 colonies grown on LBRC medium were used to inoculate 250 ml of LB medium and incubated at 37°C, with shaking (200 rpm) until stationary growth phase had been reached. Aliquots (100 ml) of centrifuged (5 min at 10000 rpm), and sterile filtered supernatant (0.22 µm, Millipore Express PLUS) were acidified to pH 2 with the addition of HCl. Supernatant samples were extracted three times by liquid-liquid extraction using an equal volume of acidified ethyl acetate (1% (v/v) AcOH in EtOAc). Combined extracts were dried under vacuum and stored at-80° C prior to analysis. 2.3.1.2.2. LC-MS/MS analysis The LC-MS/MS analysis of extracted samples was conducted as previously described (Ortori et al. 2011) with minor modification. Dried extracts were re-dissolved in 50 µl of 0.1% (v/v) formic acid in MeOH. The chromatography column used was a Phenomenex Gemini C18 (3.0 µm, 150 x 3.0 mm), and the mobile phases used were 0.1 % (v/v) formic acid and 0.1% (v/v) formic acid in methanol. The analysis was conducted with the MS operating in multiple reaction monitoring (MRM) mode, simultaneously screening the LC eluent for all specific AHLs, comparing the retention time of detected analytes with authentic synthetic standards. For each detected chromatographic peak a mean peak area was calculated from three biological replicates. 2.3.2. Quorum quenching 2.3.2.1. Evaluation of degradation of AHLs by Az39 by LC-MS/MS analysis