GroEL Gene-Based Phylogenetic Analysis of Lactobacillus Species by High-Throughput Sequencing.

Xie Xie,Chen Chen,Lu Lu,Jiang Jiang,Zhang Zhang,Liu Liu,Zhao Zhao,Pan Pan

doi:10.3390/genes10070530

Xie Xie, Chen Chen + Show 6 more

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https://doi.org/10.3390/genes10070530

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Abstract

Lactobacillus is a fairly diverse genus of bacteria with more than 260 species and subspecies. Many profiling methods have been developed to carry out phylogenetic analysis of this complex and diverse genus, but limitations remain since there is still a lack of comprehensive and accurate analytical method to profile this genus at species level. To overcome these limitations, a Lactobacillus-specific primer set was developed targeting a hypervariable region in the groEL gene—a single-copy gene that has undergone rapid mutation and evolution. The results showed that this methodology could accurately perform taxonomic identification of Lactobacillus down to the species level. Its detection limit was as low as 104 colony-forming units (cfu)/mL for Lactobacillus species. The assessment of detection specificity using the Lactobacillus groEL profiling method found that Lactobacillus, Pediococcus, Weissella, and Leuconostoc genus could be distinguished, but non-Lactobacillus Genus Complex could not be detected. The groEL gene sequencing and Miseq high-throughput approach were adopted to estimate the richness and diversity of Lactobacillus species in different ecosystems. The method was tested using kurut (fermented yak milk) samples and fecal samples of human, rat, and mouse. The results indicated that Lactobacillus mucosae was the predominant gut Lactobacillus species among Chinese, and L. johnsonii accounted for the majority of lactobacilli in rat and mouse gut. Meanwhile, L. delbrueckii subsp. bulgaricus had the highest relative abundance of Lactobacillus in kurut. Thus, this groEL gene profiling method is expected to promote the application of Lactobacillus for industrial production and therapeutic purpose.

Highlights

Lactobacillus genus is a Gram-positive, non-spore-forming, catalase-negative, rhabditiform, facultative anaerobic or microaerophilic group of bacteria [1]
The results showed the following distinct types of core genes: first, core genes without highly variable region sequences and low resolution that could not be used to distinguish between Lactobacillus species; second, core genes with highly variable regions and high resolution that could distinguish between different Lactobacillus species but their ends did not contain relatively conserved sequences, which restrict the designing of primers; third, core genes with highly variable regions, high resolution to distinguish between Lactobacillus species, and relatively conserved sequences at their ends, e.g., the groEL gene, suitable for designing primers
The groEL and 16S rRNA gene sequences of all Lactobacillus species that deposited in the EMBL and National Center for Biotechnology Information (NCBI) databases were downloaded and pairwise sequence similarity using BLAST were performed

Summary

Introduction

Lactobacillus genus is a Gram-positive, non-spore-forming, catalase-negative, rhabditiform, facultative anaerobic or microaerophilic group of bacteria [1]. According to the fermentation products, Lactobacillus species can be segmented into homofermentative and heterofermentative lactobacilli. Due to fastidious nutritional requirement, Lactobacillus lives in nutrient-rich environment, such as fermented or spoiled foods, animal feed, plant and soil surfaces, and bodies of invertebrate and. Lactobacillus species are indigenous to human digestive, urinary, and genital systems [3]. They usually protect human from pathogen invasion, they can be occasional opportunistic pathogens [4]. Assessment of the diversity of Lactobacillus species in different ecological environment is expected to provide valuable reference to promote the application of the bacteria for industrial production and therapeutic purpose

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