192883-12-8Relevant articles and documents
Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria
Ziesche, Lisa,Rinkel, Jan,Dickschat, Jeroen S.,Schulz, Stefan
supporting information, p. 1309 - 1316 (2018/06/13)
N-Acylhomoserine lactones (AHLs) are important bacterial messengers, mediating different bacterial traits by quorum sensing in a cell-density dependent manner. AHLs are also produced by many bacteria of the marine Roseobacter group, which constitutes a large group within the marine microbiome. Often, specific mixtures of AHLs differing in chain length and oxidation status are produced by bacteria, but how the biosynthetic enzymes, LuxI homologs, are selecting the correct acyl precursors is largely unknown. We have analyzed the AHL production in Dinoroseobacter shibae and three Phaeobacter inhibens strains, revealing strain-specific mixtures. Although large differences were present between the species, the fatty acid profiles, the pool for the acyl precursors for AHL biosynthesis, were very similar. To test the acyl-chain selectivity, the three enzymes LuxI1 and LuxI2 from D. shibae DFL-12 as well as PgaI2 from P. inhibens DSM 17395 were heterologously expressed in E. coli and the enzymes isolated for in vitro incubation experiments. The enzymes readily accepted shortened acyl coenzyme A analogs, N-pantothenoylcysteamine thioesters of fatty acids (PCEs). Fifteen PCEs were synthesized, varying in chain length from C4 to C20, the degree of unsaturation and also including unusual acid esters, e.g., 2E,11Z-C18:2-PCE. The latter served as a precursor of the major AHL of D. shibae DFL-12 LuxI1, 2E,11Z-C18:2-homoserine lactone (HSL). Incubation experiments revealed that PgaI2 accepts all substrates except C4 and C20-PCE. Competition experiments demonstrated a preference of this enzyme for C10 and C12 PCEs. In contrast, the LuxI enzymes of D. shibae are more selective. While 2E,11Z-C18:2-PCE is preferentially accepted by LuxI1, all other PCEs were not, except for the shorter, saturated C10-C14-PCEs. The AHL synthase LuxI2 accepted only C14 PCE and 3-hydroxydecanoyl-PCE. In summary, chain-length selectivity in AHLs can vary between different AHL enzymes. Both, a broad substrate acceptance and tuned specificity occur in the investigated enzymes.
Identification and Quantification of N-Acyl Homoserine Lactones Involved in Bacterial Communication by Small-Scale Synthesis of Internal Standards and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
Leipert, Jan,Treitz, Christian,Leippe, Matthias,Tholey, Andreas
, p. 2538 - 2547 (2017/12/12)
N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. [Figure not available: see fulltext.].
