Structural Determination of a New Peptidolipid Family from Rhodococcus opacus and the Pathogen Rhodococcus equi by Multiple Stage Mass Spectrometry

Abstract

The cell walls of the genus Rhodococcus including the pathogenic bacterium Rhodococcus equi (R. equi) and biotechnologically important bacterium Rhodococcus opacus (R. opacus) contain an abundant peptidolipid (or termed lipopeptide) family whose structures have not been reported previously. Here, we describe a linear ion-trap multiple-stage mass spectrometric (LIT MSn) approach with high resolution mass spectrometry (HRMS), in conjunction with NMR spectroscopy, chemical reactions, and GC/MS analysis to define the structures of these compounds. We employed LIT MS n (n = 2–8) on the [M + Na]+ ion species to establish the peptide sequence, the identity of the fatty acyl substituent, and its location within the molecule, while NMR spectroscopy and GC/MS were used to recognize the Leu and Ile moieties. The major new lipopeptide found in R. opacus is defined as C17H35CH­(OH)­CH2CO-NHLeu-Ser-Leu-Ile-Thr-Ile-PheCOOH, where a β-OH fatty acyl (C18–C22) substituent is attached to the N-terminal of the LSLITIF peptide chain via a NH–CO bond. By contrast, the main peptidolipids found in R. equi belong to the cyclopeptidolipid family, which possesses the same peptide sequence and lipid chain, but the β-OH group of the fatty acyl moiety and the C-terminus of the peptide (i.e., the −COOH) are cyclized by an ester bond formation to a lactone, with a structure similar to iturin-A (Biochemistry19781739923996). The antibiotic activity test of these new lipids did not reveal an activity against any of seven microorganisms tested.

Identification of a Novel Biosurfactant with Antimicrobial Activity Produced by Rhodococcus opacus R7

“…The chemical characterization of the isolated biosurfactant revealed that the mass spectra assign a molecular weight (1292 Da) that is in the range of small biosurfactants structurally related to peptides (as lipopeptides and hydrophobic peptides) isolated from other bacteria [10,14,25,46]. The NMR spectra confirmed the presence of amino acids residues including aliphatic hydrophobic and hydrophilic amino acids: Ala, Gly, Asp, Thr, and Ser, and the analysis excluded the presence of a lipophilic component in the isolated compound.…”

“…A C domain subtype called the C-starter domain is usually the first in the assembly domain lines that drive the acylation of the first amino acid (with the presence of β-hydroxy or β-amino fatty acid) of the peptide moiety [1]. In general, lipopeptides or hydrophobic peptides from all bacteria are very interesting because of their high activity in lowering surface tension [9,10,[12][13][14][15] and because they often present antimicrobial activity [3,16].…”

Biosurfactant Production by Mycolic Acid-Containing Actinobacteria

“…A combination of Fourier transform infrared spectroscopy (FTIR), NMR, and liquid chromatography-mass spectrometry (LC-MS) enabled structural characterisation of a novel cyclic lipopeptide, Coryxin, produced by Corynebacterium xerosis NS5 [48]. Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization has been used to determine the structure of trehalose monomycolate (TMM) and trehalose dimycolate (TDM) in the cell wall of Rhodococcus equi and R. opacus [49]. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been utilised successfully for the purification and characterisation of sophorolipids and rhamnolipids in Pseudomonas aeruginosa [50] and could be applied to similar compounds produced by mycolate species.…”