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Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria

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.


S5
Fatty acid methyl esters: A few colonies of bacteria were transferred with a loop from an agar plate or a cell pellet, obtained by centrifugation from a well-grown liquid culture, to a 1 mL GC vial filled with 10 µL distilled water. The bacteria were directly lysed by adding 30 µL methanolic trimethylsulfonium hydroxide (TMSH) solution (0.25 M) that concomitantly converted bound and free fatty acids to FAMEs [2]. The mixture was dried in a nitrogen stream and the residue was dissolved in 200 µL diethyl ether/methanol (10:1). This solution was analyzed by a GC/MS. Gene cloning: Genomic DNA was isolated from liquid cultures of P. inhibens DSM17395 and D. shibae DFL-12 in half strength MB medium grown for 7 days using a standard protocol based on phenol/chloroform/isoamyl alcohol extraction [5].
Using these templates, the target AHL synthase genes pgaI 2 , luxI 1 and luxI 2 (for accession numbers, see Table S1) were amplified by PCR using Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) and the primers without homology arms listed in  (Table S2) with the reaction conditions described above. These fragments were added to linearized yeast-E. coli shuttle vector pYE-express [6] (HindIII and EcoRI digested) and used for homologous recombination in yeast by the LiOAc/SS DNA/PEG protocol [7].
Transformed yeast colonies were grown on SM-URA medium to isolate the product plasmids by Plasmid Miniprep II kit (Zymo Research, Irvine, USA). The obtained plasmid mixtures were shuttled to E. coli BL21(DE3) cells by electroporation. The transformed cells were grown on selective LB agar medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 18 g/L agar, pH 7.2) supplied with kanamycin (50 g/mL) to pick single colonies, which were grown in liquid LB medium (50 g/mL kanamycin) at 37 °C. Isolated plasmids were checked for correct insertion of the target gene by analytical digest and sequencing of the positive samples.  by ultra-sonication on ice (50% power, 5 × 30 s, 4 °C) and the cell debris was removed by centrifugation (5500g, 10 min, 4 °C) to yield the soluble protein fraction, which was applied to Ni 2+ -NTA affinity chromatography using Ni-NTA superflow (Qiagen, Venlo, Netherlands). Washing was done by binding buffer (3 × 2 mL) and the target protein was isolated by elution buffer (

(Z)-7-Tetradecenoic acid
To a solution of 7-tetradecynoic acid (0.22 g, 0.98 mmol) and methanol (5 mL) Lindlar's catalyst (0.02 g) was added. The mixture was stirred for 20 minutes at room temperature under a hydrogen atmosphere. The catalyst was removed by filtration through a short pad of silica and the solvent was evaporated. The product was obtained as colorless oil without the need of further purification (205 mg, 0.90 mmol, 92%) [11]. All analytical data were in agreement with those previously reported [4].

S-Acyl-N-pantothenoyl acetonide derivatives (9)
To a stirred solution of an acid in dry DCM, p-dimethylaminopyridine (DMAP) and 8 were added. N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) was added at 0 °C, the solution was stirred for 5 minutes at 0 °C and overnight at room temperature [12]. 1N HCl was added to the reaction mixture followed by extraction with DCM three times. The combined organic phases were washed with sat. NaHCO 3 sol. and brine, dried with MgSO 4 , filtered and concentrated to give an oil.

(R)-S-Acyl-N-pantothenoylcysteamines (10-13)
The thioesters 9 were dissolved in AcOH/H 2 O 2:1 (2 mL) and stirred for 5 h at room temperature. The solvent was evaporated in vacuo and the crude product was used for incubation experiments without further purification [13].