ABSTRACT
We report on the complete genome sequences of strongly hemolytic Brachyspira hyodysenteriae strain B204 and weakly hemolytic Brachyspira hyodysenteriae strain JR80 as an important contribution for studying the genetic basis for these different phenotypes. The basionym for both strains is Treponema hyodysenteriae, and the homotypic synonym is Serpulina hyodysenteriae.
KEYWORDS: Brachyspira hyodysenteriae, hemolysis
ANNOUNCEMENT
Strongly hemolytic Brachyspira hyodysenteriae strains are causative for severe swine dysentery, an economically important enteric disease in fattening pigs. Here, we provide the complete genome of the strongly hemolytic Brachyspira hyodysenteriae strain B204, a former ATCC reference strain. Many research groups have used this strain as a virulent prototype of this bacterial species. The strain was isolated in the 1970s at the Institute of Veterinary Microbiology and Preventive Microbiology of Iowa State University, Ames, USA, from a pig with swine dysentery (1) and acquired from T.B. Stanton and S.B. Humphrey, National Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, USA.
Furthermore, the first complete genome sequence of a weakly hemolytic isolate of this species, Brachyspira hyodysenteriae strain JR80, is provided. It was isolated in 2015 at the Institute for Microbiology, University of Veterinary Medicine, Hannover, Germany, from a pig with mild non-bloody diarrhea (2). The strain was identified as Brachyspira hyodysenteriae by nox-gene sequencing, cpn60 sequencing, and whole-genome sequencing (2, 3). Hemolytic activity was quantified in relation to the strongly hemolytic type strain of the species, strain B78T (2).
For DNA extraction, bacteria were grown anaerobically at 40°C for 22 h in Trypticase Soy broth (Oxoid, Wesel, Germany) with yeast extract (Oxoid, Wesel, Germany) (4), and DNA was extracted using the Wizard HMW DNA Extraction Kit (Promega, Walldorf, Germany) following the protocol for gram-negative bacteria. DNA quality was checked using a Bioanalyzer (Agilent).
DNA libraries for Illumina sequencing were prepared using the NEBNext Ultra II FS DNA Library Prep Kit. Library for ONT system was prepared using Ligation Kit (SQK-LSK 109) without fragmentation and size selection. Sequencing of the respective library followed on MiSeq (2 × 300 bp) with a 330×/220× fold coverage per sample and on ONT MinION MK1B Flongle (FLG001) with 1G/680Mbp with a 52×/93× fold coverage per sample. Illumina read quality was assessed using FastQC v.0.12.0 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc), while ONT reads were base called with Dorado v0.7.0 model dna_r9.4.1_e8_hac@v3.3 (https://github.com/nanoporetech/dorado) and underwent filtering with a minimum subread length of 1000 bp and quality via Filtlong v.0.2.1 (https://github.com/rrwick/Filtlong).
Hybrid assembly of both read types was performed using Unicycler 0.5.0 (5). The resulting contigs were also circularized by Unicycler, adjusted to start consistently at a designated gene, and represented the complete genome’s chromosome and plasmid for both strains. Genome annotation was conducted through the NCBI Prokaryotic Genome Annotation Pipeline (6), utilizing the best-placed reference protein set, GeneMarkS-2+. Unless noted otherwise, default parameters were applied for all software tools. Sequencing statistics and annotation details are shown in Table 1.
TABLE 1.
Genome assembly statistics for Brachyspira hyodysenteriae strains B204 and JR80
| Sequencing method or annotation | Data for B. hyodysenteriae strain | |
|---|---|---|
| B204 | JR80 | |
| ONT MinION sequencing | MK1B (FLG001) | MK1B (FLG001) |
| No. of subreads | 36.812 | 16.047 |
| Avg read length (bp) | 5,146 | 9,628 |
| Avg quality score | 15% > Q20 | 18% > Q20 |
| Sequencing throughput (bp) | 157M | 117M |
| Avg coverage depth (×) | 52 | 39 |
| SRA accession no. | SRR30838155 | SRR30837070 |
| MiSeq sequencing | MiSeq (2 × 300) | MiSeq (2 × 300) |
| No. of reads | 3,327,930 | 2,261,570 |
| Avg read length (bp) | 301 | 301 |
| Avg quality score | 86% > Q30 | 87% > Q30 |
| Sequencing throughput (bp) | 1.0G | 680M |
| Avg coverage depth (×) | 330 | 220 |
| SRA accession no. | SRR30838156 | SRR30837071 |
| PGAP annotation | 2024-04-27.build7426 | 2024-07-18.build7555 |
| chromosome | B204 | JR80WH |
| GenBank accession no. | CP170528 | CP171216 |
| Length (bp) | 3.004.220 | 3.041.621 |
| No. of rRNAs | 1,1,1 (5S, 16S, 23S) | 1,1,1 (5S, 16S, 23S) |
| No. of tRNAs | 34 | 34 |
| No. of genes | 2,662 | 2,708 |
| No. of coding sequences (CDSs) | 2,622 | 2,668 |
| No. of protein coding genes | 2,601 | 2,619 |
| GC content (%) | 27 | 27 |
| plasmid | pBHB204 | pBHJR80 |
| GenBank accession no. | CP170529 | CP171217 |
| Genome length (bp) | 35.914 | 32.585 |
| No. of coding sequences | 33 | 30 |
| No. of protein coding genes | 33 | 30 |
| GC content (%) | 27 | 27 |
ACKNOWLEDGMENTS
This work was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) based on a decision of the Parliament of the Federal Republic of Germany, granted by the Federal Office for Agriculture and Food (BLE; grant number 28N-2-071-00).
Contributor Information
Ralph Goethe, Email: ralph.goethe@tiho-hannover.de.
John J. Dennehy, Queens College Department of Biology, Queens, New York, USA
DATA AVAILABILITY
This whole-genome project at GenBank can be found under the accession numbers given in Table 1. The version described in this paper is the first version.
In addition to the deposition of the sequencing data, we also deposited both strains at the German Cell Culture Collection DSMZ, Braunschweig, Germany, with the accession numbers DSM 118648 for strain B204 and DSM 118649 for strain JR80.
REFERENCES
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Associated Data
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Data Availability Statement
This whole-genome project at GenBank can be found under the accession numbers given in Table 1. The version described in this paper is the first version.
In addition to the deposition of the sequencing data, we also deposited both strains at the German Cell Culture Collection DSMZ, Braunschweig, Germany, with the accession numbers DSM 118648 for strain B204 and DSM 118649 for strain JR80.