Phylotype-Level Characterization of Complex Lactobacilli Communities Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach

Abstract

The lactobacilli to date encompass more than 270 closely related species that were recently re-classified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related, yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA-based amplicon sequencing have significant limitations (high cost, low resolution, etc.). Here, we generated a lactobacilli phylogeny based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of lactobacilli community diversity and structure at the species-level. This framework is based on a total of 445 pheS gene sequences, including sequences of 277 validly described species and subspecies (out of a total of 283, coverage of 98%). It allows differentiation between 263 lactobacilli species-level clades out of a total of 273 validly described species (including the proposed species L. timonensis) and a further two subspecies. The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of [~]30,000 sequences, the minimum level of detection was approximately 0.02 pg per l DNA (equalling approximately 10 genome copies per {micro}l template DNA). The pheS approach along with parallel sequencing of partial 16S rRNA genes revealed a considerable lactobacilli diversity and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike. IMPORTANCESpecies within the former genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate for their exceptional functional diversity, the over 270 species were recently re-classified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost or copy number variations. The approach described here makes use of a single copy marker gene which outperforms other markers with regards to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address lactobacilli diversity and community structure in various environmental matrices. Such analyses can now be performed at broader scale to assess and monitor lactobacilli community assembly, structure and function at the species (in some cases even at sub-species) level across a wide range of academic and commercial applications.