Identification of unsaturated N‐acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by liquid chromatography coupled to electrospray ionization‐hybrid linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometry

Abstract

The identification of two unsaturated N-acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Besides the confirmation of the signaling molecule already described in the literature, i.e. (Z)-N-tetradec-7-enoyl-homoserine lactone (C(14:1)-HSL), we have discovered the occurrence, at low, yet significant levels, of another monounsaturated compound, C(12:1) -HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high-resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion into bromohydrins. Collision-induced dissociation (CID) spectra were then collected on the LTQ mass analyzer. A careful comparison of tandem MS spectra of monounsaturated (i.e., C(12:1)-HSL and C(14:1)-HSL) and saturated AHLs (i.e. C(12)-HSL and C(14)-HSL) led to the emphasis of two series of product ions, exhibiting 14 Da spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e. CH(2)=CH(CH(2))(n)H). In particular, the series located at the higher end of the explored m/z range (>200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain.

Isolation of Marine Paracoccus sp. Ss63 from the Sponge Sarcotragus sp. and Characterization of its Quorum‐Sensing Chemical‐Signaling Molecules by LC‐MS/MS Analysis

“…The same difference was found in the alkyl chain fragments of each AHL therefore located on this moiety. Owing to the slightly higher polarity of the double bonds in the alkyl chains, the putative unsaturated AHLs exhibited a shorter retention time than the saturated counterparts 34. The position and the configuration of double bonds were not determined in this work.…”

Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N -Acyl-Homoserine Lactone Autoinducers

“…Product ion parameters were set at a collision energy (CE) of 30 and a declustering potential (DP) of 80. Product ion spectra were searched for marker fragment products at m/z (mass/ charge) 102.055 since acyl-HSLs consistently fragment to a protonated ( The position of the CϭC double bond on the acyl chain of C 10:1 -HSL was determined based on the ion fragmentation series of unsaturated acyl-HSLs reported previously (40). Briefly, unsaturated acyl-HSL ion fragmentation suggests that if the double bond were placed on the (7 Ϯ x)th carbon distant from the amidic group, then the series of ions with even-numbered m/z ratios would end at m/z (198 Ϯ 14x) (40).…”

“…In addition, we identified the position of the CϭC bond in the acyl chain between carbon 9 and carbon 10 based on the ion fragmentation model presented in a previous study on unsaturated acyl-HSLs by Cataldi et al (Fig. 5C, chemical structure) (40). Unfortunately, our analysis was unable to clarify the isomeric form of the CϭC bond of C 10:1 -HSL.…”

“…Finally, to further confirm the identity of an unsaturated acyl-HSL, acyl-HSL extracts were treated with bromine aqueous solution and examined by UPLC-IDA-MS. Bromohydrins are readily formed by a water nucleophilic attack to the reactive bridged bromonium ion formed by the reaction of bromine with the CϭC bond (40). After bromine treatment, the [MϩH] ϩ m/z of 254.1729 corresponding to C 10:1 -HSL was not detected, but the corresponding isotopologues and XIC fragmentation pattern of the oxybrominated OHBrC 10 -HSL were detected (Fig.…”

Identification of Quorum Sensing Activators and Inhibitors in The Marine Sponge Sarcotragus spinosulus

“…The difference of 2 amu m/z ratio of pseudomolecular ions with the saturated AHLs and the molecular formula determination with a difference of 2 hydrogen atoms confirmed the presence of unsaturated acyl chain AHLs. Higher polarity due to unsaturation of acyl chain among unsaturated AHLs exhibited a shorter retention time when compared to its saturated counterparts ( Figure S1) [20,42]. The location of the carbonyl group has only been assumed to be at position 3 due to biosynthetic origins, as for the most AHLs, but it remained unassigned [43].…”