Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

Shinya Watanabe,Aa Haeruman Azam,Xin-Ee Tan,Yusuke Sato’O,Bintao Cui,Tanit Boonsiri,Kanate Thitiananpakorn,Yoshifumi Aiba,Moriyuki Kawauchi,Kotaro Kiga,Yumi Nakada,Longzhu Cui,Teppei Sasahara,Fen-Yu Li,Yusuke Taki

doi:10.3389/fmicb.2019.02838

Shinya Watanabe, Aa Haeruman Azam + Show 13 more

Open Access

https://doi.org/10.3389/fmicb.2019.02838

Copy DOI

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Highlights

Leptotrichia species, which are strict anaerobic or facultative anaerobic Gram-negative rods found in the oral cavity, intestines, female genital tract and urogenital system of both humans and animals, can ferment carbohydrates to generate multiple products, including lactic acid, and traces of acetic, formic or succinic acid, depending on the substrates and species (Eribe and Olsen, 2017)
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a systems are broadly distributed among Leptotrichia species
The estimation of the occurrence, composition, and diversity of CRISPR-Cas13a systems in the genus Leptotrichia is challenging because the complete whole-genome information is limited

Summary

Introduction

Leptotrichia species, which are strict anaerobic or facultative anaerobic Gram-negative rods found in the oral cavity, intestines, female genital tract and urogenital system of both humans and animals, can ferment carbohydrates to generate multiple products, including lactic acid, and traces of acetic, formic or succinic acid, depending on the substrates and species (Eribe and Olsen, 2017). Some Leptotrichia species cause opportunistic infectious diseases, including gingivitis, periodontitis, infectious endocarditis and bacteraemia (Eribe and Olsen, 2008, 2017). With the recent progress and spread of modern techniques, the involvement of Leptotrichia species in various infectious diseases has gained considerable attention (Eribe and Olsen, 2017). The exact clinical impact of Leptotrichia species relevant to infectious diseases remains unclear due to difficulties in bacterial culture, isolation and identification. Many clinical Leptotrichia isolates have remained unclassified due to the lack of the whole-genome sequences and biochemical properties of these isolates (Eribe and Olsen, 2017). Leptotrichia genome data are very limited as there are only one complete whole genome sequences of species-identified strain and three unidentified strains available in the GenBank database (Ivanova et al, 2009; Gupta and Sethi, 2014)

Methods

Results

Conclusion