Functional metagenomic analysis of quorum sensing signaling in a nitrifying community

Chuan Hao Tan,Yee Phan Yeo,Muhammad Hafiz,Scott A Rice,Noele Kai Jing Ng,Staffan Kjelleberg,Martin Tay,Shireen Taj,Xie Chao,Sujatha Subramoni

doi:10.1038/s41522-021-00250-3

Abstract

Quorum sensing (QS) can function to shape the microbial community interactions, composition, and function. In wastewater treatment systems, acylated homoserine lactone (AHL)-based QS has been correlated with the conversion of floccular biomass into microbial granules, as well as EPS production and the nitrogen removal process. However, the role of QS in such complex communities is still not fully understood, including the QS-proficient taxa and the functional QS genes involved. To address these questions, we performed a metagenomic screen for AHL genes in an activated sludge microbial community from the Ulu Pandan wastewater treatment plant (WWTP) in Singapore followed by functional validation of luxI activity using AHL biosensors and LC–MSMS profiling. We identified 13 luxI and 30 luxR homologs from the activated sludge metagenome. Of those genes, two represented a cognate pair of luxIR genes belonging to a Nitrospira spp. and those genes were demonstrated to be functionally active. The LuxI homolog synthesized AHLs that were consistent with the dominant AHLs in the activated sludge system. Furthermore, the LuxR homolog was shown to bind to and induce expression of the luxI promoter, suggesting this represents an autoinduction feedback system, characteristic of QS circuits. Additionally, a second, active promoter was upstream of a gene encoding a protein with a GGDEF/EAL domain, commonly associated with modulating the intracellular concentration of the secondary messenger, c-di-GMP. Thus, the metagenomic approach used here was demonstrated to effectively identify functional QS genes and suggests that Nitrospira spp. maybe QS is active in the activated sludge community.

Highlights

Quorum sensing (QS) is a bacterial communication system that involves production, secretion, and response to signal molecules known as autoinducers and QS has been shown to regulate many bacterial behaviors[1,2,3]
Phylogenetic tree of the putative luxIR genes and their taxonomic relationships To investigate the phylogenetic relationship between the putative luxIR from sludge community and the characterized luxIR, we compared the amino acid sequence of the putative luxIR genes to 80 and 85 verified luxI and luxR homologs, respectively, which derived from pure cultures and metagenomic studies, as well as four luxI/R pairs obtained from Nitrospira (Supplementary Table 2)
We have previously shown that the accumulation of specific acylated homoserine lactone (AHL) was correlated with the granulation process and EPS production[13]

Summary

INTRODUCTION

Quorum sensing (QS) is a bacterial communication system that involves production, secretion, and response to signal molecules known as autoinducers and QS has been shown to regulate many bacterial behaviors[1,2,3]. The formation of activated sludge granules was correlated with an increased in AHL concentration[13] and was accompanied by a shift in microbial community species composition This change in community members was associated with a reduction in the organisms encoding quorum quenching functions to be dominated by those that encoded QS systems[14]. A total of 569 luxI sequences were found from the analysis of 68 metagenome samples collected from the Global Ocean Sampling[24], while another in-silico study identified 31 luxI sequences from 14 environmental metagenomes[25] These results highlight the power of metagenomic approaches in the search for AHL QS genes in complex communities. Phylogenetic tree of the putative luxIR genes and their taxonomic relationships To investigate the phylogenetic relationship between the putative luxIR from sludge community and the characterized luxIR, we compared the amino acid sequence of the putative luxIR genes to 80 and 85 verified luxI and luxR homologs, respectively, which derived from pure cultures and metagenomic studies, as well as four luxI/R pairs obtained from Nitrospira (Supplementary Table 2)

RESULTS

99 SmeSl TfrreaRTraR

DISCUSSION

CODE AVAILABILITY