Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase

Abstract

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

A Survey of Phage Contamination in Natto-producing Factories and Development of Phage-resistant <i>Bacillus subtilis</i> (natto) Strains

“…Some B. subtilis strains produce an extracellular slimy glutamic acid polymer (poly-γ-glutamic acid, γPGA) and fructose polymer (levan). These compounds surrounding the cells are assumed to work as a barrier against phage attack and progeny expansion (Kimura and Itoh, 2003;Ozaki et al, 2016). On the contrary, phages appear to have evolutionally acquired enzymatic counter levers, hydrolases for the γPGA and the levan (Ozaki et al, 2016).…”

“…These compounds surrounding the cells are assumed to work as a barrier against phage attack and progeny expansion (Kimura and Itoh, 2003;Ozaki et al, 2016). On the contrary, phages appear to have evolutionally acquired enzymatic counter levers, hydrolases for the γPGA and the levan (Ozaki et al, 2016). We examined the presence of the γPGA hydrolase gene (pghP) and levanase gene (levP) among isolated phages (Fig.…”

“…B. subtilis NFRI8322 was isolated from Tua Nao (a natto-like food of Chiang Rai, Thailand) (Inatsu et al, 2002;Kamada et al, 2015). A commercial nattofermenting strain Miyagino and φNIT1 phage were used as reference strains (Kimura and Itoh, 2003;Nishito et al, 2010;Ozaki et al, 2016). Phages JNDMP and ONPC previously isolated from spoiled natto were obtained from the Genebank of the National Agriculture and Food Research Organization (i).…”

“…To date, the phages found in spoiled natto belong to the Myoviridae family and can be classified into two groups (group I and II) based on the similarity of their genomes (Nagai and Yamasaki, 2009). Among them the entire genome information of φNIT1 phage of the group II is available (Ozaki et al, 2016).…”

“…It is known that some Bacillus phages have pghP and levP genes that encode enzymes that break down extracellular sugar and amino acid polymers, fructooligosaccharide (levan) and poly-γ-glutamic acid (γPGA), respectively, to facilitate infection and propagate phage progenies (Kimura and Itoh, 2003;Ozaki et al, 2016). The appearance of pghP and levP in the genome of the contaminated phage was also examined.…”