Characterization of a novel mucolytic bacterium, Prevotella mucinisolvens sp. nov., isolated from rumen epithelium of Korean cattle

Abstract

Mucin, a heavily glycosylated protein produced by epithelial cells, plays an integral role in lubrication to chemical barriers and protection against pathogens. Although commensal mucolytic bacteria have been reported that they improve host health, there is a lack of research on mucolytic bacteria. In this study, mucolytic bacteria were isolated from the rumen epithelial tissue of the dorsal sac of Korean cattle steer using a targeted cultivation on a mucin defined medium as a sole carbon source in anaerobic conditions. A novel bacterium, designated strain Rep29T, was isolated and characterized in terms of phenotypic, biochemical, and chemotaxonomic characteristics and phylogenetic position based on 16S rRNA sequence analysis. The 16S rRNA sequence analysis showed that strain Rep29T belonged to a member of the genus Prevotella and was most closely related to Prevotella brevis GA33T with 89.8% similarity. Strain Rep29T was Gram-negative, obligately anaerobic, and non-motile coccus. Growth of strain Rep29T was observed at 35-45°C (optimum, 39°C), at pH 6.0-7.5 (optimum, 7.0), and in the presence of 0.0–1.5% (w/v) NaCl (optimum, 0.0–0.5%). The major cellular fatty acids of strain Rep29T were C16:0, C18:1 ω9c, and iso-C15:0. The G+C content of genomic DNA was 47.7 mol%. Analysis of enzyme profiles using API Rapid ID 32A and API ZYM identification kit revealed that this strain gave positive reactions for α-galactosidase, β-galactosidase, α-glucosidase, and N-acetyl-β-glucosaminidase related to mucin degradation. In addition, functional annotation of the whole genome sequences of strain Rep29T showed that this bacterium had a putative mucolytic pathway. Strain Rep29T is expected to utilize mucins by Sus-like systems and had the metabolism of galactose, GlcNAc, sialic acid (Neu5Ac), and mannose. Strain Rep29T was identified the presence of putative extracellular polysaccharide biosynthesis mechanisms via Wzx/Wzy-dependent pathway and synthase-dependent pathway and virulence factors for bacterial adhesion. On the basis of the above-mentioned findings, strain Rep29T represents a novel species of the genus Prevotella, the name Prevotella mucinisolevens sp. nov. is proposed; the type strain is Rep29T (= KCTC 15980T).

본 연구는 한우의 반추위벽으로부터 당단백질인 뮤신을 분해하는 박테리아를 분리하여 특성을 규명하기 위해 수행하였다. 혐기적 조건에서 유일한 탄소원으로 뮤신을 첨가한 배지를 통한 표적 배양으로 도축된 한우 거세우의 등 쪽 반추위벽으로부터 뮤신 분해 박테리아를 분리하였다. 총 40개의 단일 콜로니 중에서 12개가 뮤신만 첨가된 배지에서 성장하였다. 분리된 균주의 16S rRNA 유전자 분석을 통해 2종의 이미 밝혀진 박테리아 (Paraclostridium benzoelyticum와 Citrobacter murliniae)와 3종의 신종 박테리아로 확인되었다. 그리고 분리된 신종 균주 중에서 strain Rep29의 표현형, 생화학적, 화학 분류상 및 계통학적 분석을 하였다. strain Rep29는 그람 음성, 운동성이 없는 구형 혐기성 균으로, 35-45°C (최적 39°C), pH 6.0-7.5 (최적 7.0) 및 0.0–1.5 % NaCl (최적 0.0-0.5%)에서 성장을 확인하였다. 주요 지방산은 C16:0, C18:1 ω9c와 iso-C15:0으로 확인하였다. API Rapid ID 32A와 API ZYM identification kit (bioMe´rieux)를 이용한 효소 분석을 통해 strain Rep29는 뮤신 분해 효소인 α-galactosidase, β-galactosidase, α-glucosidase, N-acetyl-β-glucosaminidase 활성을 보였다. 또한 전체 유전자 서열 분석을 통해 Sus-like 시스템을 이용하여 뮤신을 에너지원으로 이용할 것으로 예상되며, galactose, GlcNAc, Neu5Ac 및 mannose 대사를 확인하였다. 그리고 반추위벽에 부착하기 위한 세포 외 다당류 생합성 경로 (Wzx/Wzy-dependent pathway와 synthase-dependent pathway)와 독성인자를 확인하였다. 16S rRNA 유전자의 서열을 기반으로 시행한 계통 분석을 통해 strain Rep29는 Prevotella brevis GA33T와 가장 가까운 유사성을 보이며, genus Prevoetlla에 속하는 새로운 species임을 확인하였다. 균주의 이름은 Prevotella mucinisolevens라 명명하였으며 type strain은 Rep29T이다 (= KCTC 15980T).