Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach

Abstract

A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-L-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/ AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-L-homoserine lactone (C 12 -HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria. Bacteria interact with one another using chemical molecules as sensing signals. Detection of the molecules allows bacteria to distinguish between low and high cell population densities and to control gene expression in response to changes in cell number (46) and local environment (12). This process, referred to as quorum sensing (QS), allows a population of bacteria to coordinately control gene expression. Several types of QS signals have been found, including N-acyl-L-homoserine lactone (AHL) in the Proteobacteria (5,45,46). AHL-producing bacteria have been identified in over 37 genera within the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria (9,11,14,32) and the Cyanobacteria (10). In these bacteria, AHL-dependent QS systems have been shown to regulate many bacterial behaviors, such as virulence (8, 48) and biofilm formation (24, 29), mainly in response to cell densities. Therefore, AHL-dependent QS systems are now recognized for playing important roles in the regulation of bacterial behavior.The AHL-based QS systems usually contain a luxI gene homologue, responsible for the synthesis of AHLs, and a luxR gene homologue, an AHL-dependent transcriptional regulator (19). The LuxI/LuxR-type QS systems have been experimentally identified and studied in more than 70 different species in the phylum Proteobacteria (6, 15)...

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

“…We cannot rule out the possibility that the broad-range bioassay may not be able to detect autoinducers produced by the LuxI homologs of N. hamburgensis or that acyl-HSLs may be produced under different growth conditions. There is metagenomic evidence that other NOB may produce acyl-HSLs (20), but a broad survey of acyl-HSL production by AOB and NOB is necessary to establish if acyl-HSL production is ubiquitous in environmental nitrifying bacteria, particularly when they are actively growing together in coculture. Future transcriptome analyses of N. winogradskyi, possibly by measuring transcriptome changes in response to autoinducer addition or depletion, may shed light on the purpose of QS in this NOB.…”

“…Two AOB may produce acyl-HSLs (17)(18)(19), and a metagenomic clone from the phylum Nitrospirae and potentially from the NOB "Candidatus Nitrospira defluvii" contained LuxI and LuxR homologs (20). There is precedent for acyl-HSL QS in mixed populations of bacteria carrying out anaerobic ammonium oxidation (anammox) (21,22); however, we are focused on aerobic nitrifying bacteria.…”

A NewN-Acyl Homoserine Lactone Synthase in an Uncultured Symbiont of the Red Sea Sponge Theonella swinhoei

“…To detect any additional unmapped/unassembled reads of luxI origin, a reciprocal BLASTX search was performed as follows: (i) read pairs were merged into single ϳ105-to 185-bp segments using PANDAseq 2.5 (35) and queried against a target protein database of ϳ50 LuxI representatives (see Table S2 in the supplemental material), obtained from a study by Nasuno et al (36); (ii) query sequences with hit E values of Ͻ10 Ϫ2 were retrieved and then compared to the NCBI nonredundant (nr) protein database using a cloud server (http://www.diagcomputing.org). The investigation of AHL produced by other AHL synthases (e.g., LuxM) was beyond the scope of this study.…”

Metagenomic approaches to understanding phylogenetic diversity in quorum sensing

“…Using liquid cell lysates of metagenomic clones in a microtiter-plate format is one strategy for increasing the screening sensitivity. 99 The biosensor responded with a signal compound producing from a metagenome clone could be sensitively detected using a spectrofluorometer based on the GFP fluorescence. Crude cell lysates were prepared by either chemical 99,100 or physical 101 procedures.…”

“…99 The biosensor responded with a signal compound producing from a metagenome clone could be sensitively detected using a spectrofluorometer based on the GFP fluorescence. Crude cell lysates were prepared by either chemical 99,100 or physical 101 procedures. The cells are lysed by addition of a protein extraction reagent in the chemical procedure, while the cells are broken in a vibrating crusher using glass beads in the physical procedure.…”