Improved complete genome sequence of Bdellovibrio bacteriovorus 109J, a widely studied laboratory strain of predatory bacteria

ABSTRACT The complete genome sequence of Bdellovibrio bacteriovorus 109J, a well-studied laboratory strain of predatory bacteria, first determined in 2014. Here we report an improved complete genome sequence of B. bacteriovorus 109J, incorporating 16 assembly and 87 nucleotide corrections. This revised genome will be helpful to studies on the predatory bacteria.

consuming the cellular contents of other Gram-negative bacteria as its source of nourishment (1).The laboratory strain 109J (ATCC 43826, US sewage isolate), in addition to the type strain HD100 (ATCC15356), is extensively employed in both basic and applied microbial studies (2,3).The complete genome sequence of 109J was deposited in 2014 in NCBI database (4).Nonetheless, our dot-plot analysis of the 109J chromosome in comparison with the HD100 chromosome unveiled structural anomalies encompassing several large translocations therein (Fig. 1a).Furthermore, the 109J chromosome showed two unusual GC-skew shifts in addition to those at the replication origin and terminus regions (Fig. 1b).Another noteworthy consideration is that the deposited 109J genome was determined solely from low coverage (12×) of Illumina short-read data.Consequently, we performed the resequencing of the 109J genome, employing a combination of short-and long-read sequencing.
To extract 109J gDNA, a coculture of 109J, which has undergone eight-times passages from frozen stock, and E. coli BW25113, serving as host cells, was established by inoculation of 109J plaque (a 1.5 × 1.5 cm piece) and BW25113 overnight culture (10 9 CFU/mL) into 50 mL of 50-fold diluted nutrient broth.Following a 48 h incubating at 37°C with shaking at 200 rpm, the coculture was filtered twice through a 0.45-µm pore size filter to remove E. coli cells.Subse quently, genomic DNA extraction from the filtrated 109J was performed using the DNeasy Blood & Tissue Kit (Qiagen).The library for short-read sequencing was prepared using the xGen DNA Library Prep EZ Kit (Integrated DNA Technologies) and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (New England BioLabs) according to the manufacturer's instructions.The library was subjected to sequencing on an Illumina NovaSeq X plus platform to generate 151 bp paired-end reads.For long-read sequencing, following cutoff of ~10 k-bp DNA fragments using the Short Read Eliminater XS kit (PacBio), the library was prepared using Rapid Barcoding Sequencing kit SQK-RBK004 (Oxford Nanopore Technologies).The library was loaded onto the R9.4.1 flow cell on a MinION Mk1C platform.The row data were base called with Guppy 6.5.7.Illumina reads were trimmed with Platanus_trim 1.1.0(6) and Nanopore reads were filtered with NanoFilt 2.8.0 (7) to remove <5 kb reads.A hybrid assembly of both generated sequencing data was conducted by Unicycler v0.5.0 (8).Annotation was performed using Prokka 1.14.6 (9).Default parameters were used for all software unless otherwise specified.
The improved 109J genome sequence consists of a 3,836,928 bp chromosome, exhibiting an enlargement of 6,501 bp in contrast to the original sequence (Table 1).Sequence comparison using MUMmer3.23 (10) unveiled 16 structural differences and 87 nucleotide substitutions between the original and improved genomes (Supple mentary data).The chromosomal synteny has become well conserved between the HD100 and improved 109J genomes.Furthermore, the GC skew pattern of the improved 109J chromosome has assumed a normative configuration (Fig. 1c).The improved 109J genome sequence will be helpful to studies of B. bacteriovorus.

FIG 1
FIG 1 Dot plot comparison and circular map of B. bacteriovorus strains HD100 and 109J.(a) Dot plot matrix of the HD100, original 109J and improved 109J chromosomes.The start positions of their chromosome sequences were rearranged to the dnaA gene.The matrix was depicted by an in-house Python script based on the results of NCBI BLASTN search.(b and c): Circular maps of the original (b) and improved (c) B. bacteriovorus 109J chromosome generated using Proksee server (https://proksee.ca/)(5) are shown.

TABLE 1
Summary of the comparison of the original and improved B. bacteriovorus 109J genomes a ND: No data.