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

Lisa Ziesche,Stefan Schulz,Jan Rinkel,Jeroen S Dickschat

doi:10.3762/bjoc.14.112

Abstract

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.

Highlights

The Roseobacter group, a subgroup of the Rhodobacteraceae family, constitutes an important class of Gram-negative marine bacteria, occurring in many different habitats [1,2], in fresh water as well as on surfaces [3]
The bacteria were isolated from different habitats: D. shibae DFL-12 was isolated from the dinoflagellate Prorocentrum lima [29], P. inhibens T5 was collected from a water sample of the German Wadden Sea [30], P. inhibens DSM17395 was isolated from seawater of larval cultures of the scallop Pecten maximus in Spain [25] and P. inhibens 2.10 stemmed from the surface of the green macroalga Ulva australis in Australia [31]
The results showed that P. inhibens 2.10 and P. inhibens DSM17395 produce the same four Acylhomoserine lactones (AHLs), 3-OH-C10:0-homoserine lactone (HSL) as major components and known from previous analyses of P. inhibens [9,32], C16:0-HSL, C16:1-HSL, and C18:1-HSL (Table 1)

Summary

Introduction

The Roseobacter group, a subgroup of the Rhodobacteraceae family, constitutes an important class of Gram-negative marine bacteria, occurring in many different habitats [1,2], in fresh water as well as on surfaces [3]. The differences between the AHLs in terms of chain length and degree of unsaturation prompted us to investigate the acyl-chain selectivity of LuxI-type enzymes in roseobacters. LuxI-type enzymes were heterologously expressed in E. coli and the purified recombinant enzymes were tested with different precursors to probe their selectivity Both model organisms of the Roseobacter group, P. inhibens (formerly P. gallaeciensis [25]) DSM17395 and Dinoroseobacter shibae DFL-12, were investigated, together with closely related P. inhibens strains T5 and Scheme 1: Biosynthesis of AHLs by ACP-dependent LuxI type enzymes. We report on the characterization of PgaI2 from P. inhibens and of LuxI1 and LuxI2 from D. shibae by in vitro incubation experiments

Results and Discussion

2.10 DSM17395

Conclusion