Complete genome sequence of Rothia mucilaginosa D1, a genetically amenable strain

TL;DR

The complete genome of Rothia mucilaginosa D1, a strain useful for genetic studies, has been sequenced.

Contribution

This is the first complete genome sequence of a genetically amenable clinical isolate of Rothia mucilaginosa.

Findings

The genome sequence of R. mucilaginosa D1 is now available for further research.

This strain is genetically amenable, making it suitable for experimental studies.

The sequencing supports investigations into the pathophysiology of this emerging pathobiont.

Abstract

Here, we report the complete genome sequence of Rothia mucilaginosa D1. Unlike many other strains that have been sequenced, R. mucilaginosa D1, a clinical isolate, is genetically amenable. The complete sequencing of the genome provides potential for further studies on the genetics and pathophysiology of this emerging pathobiont.

Full text

ANNOUNCEMENT

Rothia mucilaginosa, one of the most abundant bacterial species in the oral cavity and the upper-respiratory tract, is associated with various acute and chronic diseases, including dental caries, endodontitis, candidiasis, and pneumonia (1), although its exact role in health and disease remains unclear. The lack of genetic transformability remains the major obstacle in the study of Rothia pathophysiology. Unlike many other strains that have been sequenced (www.homd.org) (2, 3), R. mucilaginosa D1 is genetically amenable.

R. mucilaginosa D1 was isolated from a patient’s saliva sample (LSUHSC-NO IRB# 7257) on selective medium as described by Tsuzukibashi et al. (4, 5). For genome sequencing, R. mucilaginosa D1 was grown in Brain Heart Infusion broth aerobically at 37°C and harvested when its optical density reached 0.4 at 600 nm. Its genomic DNA was extracted using a ZymoBIOMICS DNA Miniprep Kit. Sequencing and de novo assembly were carried out at the SeqCenter (Pittsburgh, PA) under default parameters for all software unless otherwise specified. Briefly, the genomic DNA was fragmented to 40–50 kb using a Megaruptor 3 instrument; sequencing library was prepared using the PacBio SMRTbell Prep Kit 3.0 (PacBio.com, PN: 102-182-700); post-library binding, cleanup, and polymerase treatment were performed with the Revio reagent kit (PacBio.com, PN: 102-739-100); and sequencing was performed using the PacBio Revio platform. Raw sequencing data underwent initial processing with Lima (https://github.com/PacificBiosciences/pbbioconda) for deduplication, adapter trimming, and quality control; BAM files were converted to FastQ format using SamTools (6). A total of 1,082,143 PacBio HiFi reads with an average length of 9,336.5 were analyzed via Flye (v2.9.2) with parameters set as -asm-coverage 50 -genome size 6 Mbp -pacbio hifi read-error 0.02 (7). Eligible contigs were circularized using Circulator (v1.5.5) using all pipeline and internal minimus2-based steps, followed by start position adjustment to dnaA to achieve a complete chromosome (8). The final assembly was functionally annotated using the NCBI Prokaryotic Genome Annotation Pipeline (v6.10) under default settings (9); assembly quality metrics, including contiguity and completeness, were assessed using QUAST (v5.2.0) and reported in a tsv format (10).

The genome of R. mucilaginosa D1 is 2,246,281 bp long with a GC content of 59.5%. It is predicted to encode a total of 1,787 coding sequences. It contains comEA with a potential role in DNA transport and probably competence development (11). It has multiple genes for phosphoenolpyruvate:sugar phosphotransferase systems, including lactose, glucose, beta-glucoside, mannose, and fructose, along with three non-specific sugar transporters. It contains a NAD-dependent glutamate dehydrogenase and two copies of type I glutamate-ammonia ligases, aka glutamine synthetases. R. mucilaginosa D1 is catalase-negative. The complete sequence and annotation presented here should augment future study of this organism in addition to providing resources for genetic manipulation.