**Bacteriophages of** *Bacillus subtilis* **(***natto***) and Their Contamination in Natto Factories**

Toshirou Nagai *National Institute of Agrobiological Sciences, Japan* 

#### **1. Introduction**

94 Bacteriophages

Svensson, V. & Christiansson, A. (1991). Methods for phage monitoring. *Bulletin FIL-IDF*,

Szczepańska, A.K.; Hejnowicz, M.S.; Kołakowski, P. & Bardowski, J. (2007). Biodiversity of

Ventura, M.; Canchaya, C.; Bernini, V.; Altermann, E.; Barrangou, R.; McGrath, S.; Claesson,

Ventura, M.; Zomer, A.; Canchaya, C.; O'Connell-Motherway, M.; Kuipers, O.; Turroni, F.;

Ventura, M.; Turroni, F.; Lima-Mendez, G.; Foroni, E.; Zomer, A.; Duranti, S.; Giubellini, V.;

Verreault, D.; Gendron, L.; Rousseau, G.M.; Veillette, M.; Massé, D.; Lindsley, W.G.;

Wilson, I.G. (1997). Inhibition and facilitation of nucleic acid amplication. *Applied and* 

Zago, M.; De Lorentiis, A.; Carminati, D.; Comaschi, L. & Giraffa, G. (2006). Detection and

Zago, M.; Rossetti, L.; Reinheimer, J.; Carminati, D. & Giraffa, G. (2008). Detection and

*Research*, Vol.75, No.2, (May 2008), pp. 196-201, ISSN 0022-0299.

*of Dairy Research*, Vol. 73, No.2, (May 2006), pp. 146-153, ISSN 0022-0299. Zago, M.; Suárez, V.; Reinheimer, J.A.; Carminati, D. & Giraffa, G. (2007). Spread and

*Polonica*, Vol. 54, No.1, (March 2007), pp. 151-158, ISSN 0001-527X.

*Lactococcus lactis* bacteriophages in Polish dairy environment. *Acta Biochimica* 

M. J.; Li, Y.; Leahy, S.; Walker, C.D.; Zink, R.; Neviani, E.; Steele, J.; Broadbent, J.; Klaenhammer, T.R.; Fitzgerald, G.F.; O'toole, P.W. & van Sinderen, D. (2006). Comparative genomics and transcriptional analysis of prophages identified in the genomes of *Lactobacillus gasseri*, *Lactobacillus salivarius*, and *Lactobacillus casei*. *Applied and Environmental Microbiology*, Vol.72, No.5, (May 2006), pp. 3130-3146,

Ribbera, A.; Foroni, E.; Buist, G.; Wegmann, U.; Shearman, C.; Gasson, M.J.; Fitzgerald, G.F.; Kok, J. & van Sinderen, D. (2007). Comparative analyses of prophage-like elements present in two *Lactococcus lactis* strains. *Applied and Environmental Microbiology,* Vol.73, No.23, (December 2007), pp. 7771-7780, ISSN

Bottacini, F.; Horvath, P.; Barrangou, R.; Sela, D.A.; Mills, D.A. & van Sinderen, D. (2009). Comparative analyses of prophage-like elements present in bifidobacterial genomes. *Applied and Environmental Microbiology,* Vol.75, No.21, (November 2009),

Moineau, S. & Duchaine, C. (2011). Detection of airborne lactococcal bacteriophages in cheese manufacturing plants. *Applied and Environmental Microbiology,* Vol.77,

*Environmental Microbiology,* Vol.63, No.10, (October 1997), pp. 3741–3751, ISSN

identification of *Lactobacillus delbrueckii* subsp. *lactis* bacteriophages by PCR. *Journal* 

variability of the integrase gene in *Lactobacillus delbrueckii* subsp. *lactis* strains and phages isolated from whey starter cultures. *Journal of Applied Microbiology*, Vol.102,

identification of *Lactobacillus helveticus* bacteriophages by PCR. *Journal of Dairy* 

Brussels, Belgium, Vol.263, pp. 29–39, ISSN 0250-5118.

ISSN 0099-2240.

0099-2240.

0099-2240.

pp. 6929-6936, ISSN 0099-2240.

No.2, (January 2011), pp. 491-497, ISSN 0099-2240.

No.2, (February 2007), pp. 344-351, ISSN 1364-5072.

Natto is a fermented soybean food, which is produced and consumed mainly in Japan (Nagai & Tamang, 2010). The Japanese usually eat natto with cooked rice, after mixing it with seasonings attached in a package or with soy sauce (Fig. 1). Natto has a characteristic odour of short-chain fatty acids and ammonia (Ikeda *et al.,* 1984; Kanno & Takamatsu, 1987), and a highly viscous polymer, poly-γ-glutamate (PGA, see Section 3).

*Bacillus natto,* named after "natto" when isolated from it for the first time, is the sole microorganism used for natto fermentation (Sawamura, 1906). However, the species was regarded as a "probable synonym" of *B. subtilis* in *Bergey's Manual of Determinative Bacteriology*, 8th Edition (Gibson and Gordon, 1974). This classification was supported by the fact that the chromosomal DNA of *B. natto* has a high level of homology with that of *B. subtilis* (Seki *et al.,* 1975). Phylogenetic analysis of *B. natto* (meaning *Bacillus* isolates from natto) and typical *B. subtilis* strains by sequencing of the 16S rRNA gene also showed that *B. subtilis* (*natto*) and *B. subtilis* are the same species (Tamang *et al.,* 2002). Although the scientific name "*B. natto*" was abandoned, the informal name "*B. subtilis* (*natto*)" is often used in the food industry, and even in the scientific field, to emphasize that *B. subtilis* (*natto*) isolates have the ability to produce natto unlike the type strain of *B. subtilis*.

Commercial *B. subtilis* (*natto*) starters for natto fermentation are sold by three companies (Miura, Naruse and Takahashi) in Japan. The key strains for natto fermentation were isolated from the starters and characterized (Kiuchi *et al.,* 1987; Sulistyo *et al.,* 1988), and the characteristics, including PGA production and flavor, were found to be very similar among the strains.

Until the early 20th century, natto had been produced by packing boiled soybeans in a bag made of rice straw, which *B. subtilis* (*natto*) inhabit as soil bacteria (Fig. 1 C). Since the discovery of *B. subtilis* (*natto*) by Sawamura (1906) and the development of sanitary containers (Fig. 1 A & B) as substitutes for bags made of rice straw, the process of natto fermentation has been modernized and automated.

Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 97

BN, *Bacillus subtilis* (*natto*). The production conditions shown on the left of the flow chart are cited from

The process of natto fermentation is so simple that very small factories, even homes, can produce natto in 2 or 3 days (Ueda, 1989; Kiuchi & Watanabe, 2004) (Fig. 2). After being selected and washed, soybeans are soaked in water at 10°C for 18 hr. Soybeans are cooked (steamed or boiled) and a suspension of spores of *B. subtilis* (*natto*) is sprayed on the boiled soybeans while they are hot to prevent the soybeans from being contaminated with other kinds of bacteria or phages. The soybeans are packed in containers made of polystyrene paper together with packed seasonings (typically, sauce and mustard; Fig. 1 A) and incubated at 50°C for 16–18 hr and then kept at 3–10°C for 8 hr to mature the natto further. Natto products are delivered to markets or stores through a cold chain system, keeping the products at a low temperature until consumers buy them. This simplicity of the process of natto fermentation, including a limited number of starters of natto, can result in devastating

reviews on natto fermentation (Ueda, 1989; Kiuchi & Watanabe, 2004).

damage to a factory that becomes contaminated with bacteriophages.

Fig. 2. Process of natto fermentation

A) brown package, sauce; yellow package, mustard. B) Natto is very stringy because of PGA production (see Section 3). C) This natto is for a souvenir, so the straw is sterilized for hygien unlike the real classic type of natto. After sterilization, a spore suspension of *B. subtilis* (*natto*) is inoculated as shown in Fig. 2. Fig. 1. Modern natto (A and B) and a classic type of natto (C)

A) brown package, sauce; yellow package, mustard. B) Natto is very stringy because of PGA production (see Section 3). C) This natto is for a souvenir, so the straw is sterilized for hygien unlike the real classic type of natto. After sterilization, a spore suspension of *B. subtilis* (*natto*) is inoculated as shown in Fig. 2.

Fig. 1. Modern natto (A and B) and a classic type of natto (C)

BN, *Bacillus subtilis* (*natto*). The production conditions shown on the left of the flow chart are cited from reviews on natto fermentation (Ueda, 1989; Kiuchi & Watanabe, 2004).

Fig. 2. Process of natto fermentation

The process of natto fermentation is so simple that very small factories, even homes, can produce natto in 2 or 3 days (Ueda, 1989; Kiuchi & Watanabe, 2004) (Fig. 2). After being selected and washed, soybeans are soaked in water at 10°C for 18 hr. Soybeans are cooked (steamed or boiled) and a suspension of spores of *B. subtilis* (*natto*) is sprayed on the boiled soybeans while they are hot to prevent the soybeans from being contaminated with other kinds of bacteria or phages. The soybeans are packed in containers made of polystyrene paper together with packed seasonings (typically, sauce and mustard; Fig. 1 A) and incubated at 50°C for 16–18 hr and then kept at 3–10°C for 8 hr to mature the natto further. Natto products are delivered to markets or stores through a cold chain system, keeping the products at a low temperature until consumers buy them. This simplicity of the process of natto fermentation, including a limited number of starters of natto, can result in devastating damage to a factory that becomes contaminated with bacteriophages.

Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 99

Twenty *B. subtilis* (*natto*) phages were isolated from abnormally fermented natto, effluent from natto factories and soil of paddy fields in Kyushu Island, southern Japan (Fujii *et al.,* 1975). The phages, including PN-1, were classified into three groups based on host ranges, immunological reaction, and morphologies. All tested *B. subtilis* (*natto*) strains were infected by 21 *B. subtilis* (*natto*) phages, and some strains of *B. subtilis* also were infected by 17 phages. Three representative phages, PN-3, PN-6 and PN-19, from three serological groups were studied using an electron microscope. They had a head (diameter, 80–90 nm) and a contractile tail (length, 165–175 nm). Although PN-3 and PN-6 did not resemble each other in shape, judging from their morphologies on somewhat obscure photographs, they were

Nagai and Yamasaki (2009) classified 20 phages [deposited in the NIAS Genebank (Tsukuba, Japan) with accession numbers from MAFF 270101 to MAFF 270120 (Table 1)], mainly isolated from abnormally fermented natto, into two groups based on DNA-DNA hybridization (Fig. 3). No cross hybridized band is visible in the photograph, indicating that group I phages and group II phages are genetically independent of each other. Representative phages from the two groups were further characterized. Phage JNDMP (Group I) has a head (diameter, 60 nm) and a flexible tail (7 x 200 nm) (Fig. 4 A) and requires magnesium ions for amplification. Phage ONPA (Group II) has a head (diameter, 89 nm) and a contractile tail (9 x 200 nm) with a sheath (width, 23 nm) and does not require additional magnesium ions (Fig. 4 B). Plaques of ONPA were clearer than those of JNDMP.

Group type I II

Head (nm) 60 89 Tail (nm) 7 x 200 9 x 200

Host range2)

Marburg - - 1)Temperature at which about 1% of phage particles in suspension can survive after 10-min heating in a

Miura (MAFF 118100) - + Naruse (MAFF 118103) + + Takahashi (MAFF 118105) + +

2) Miura, Naruse and Takahashi are commercial starters for natto fermentation.

Table 2. Other characteristics of JNDMP and ONPA

water bath.

Genome DNA (kb) 42 91 Latent time (min) 35 50 Burst size 46 72 Heat stability (°C)1) 53 63 Mg ion requirement + -

JNDMP(MAFF 270105) ONPA (MAFF 270115)

found to belong to the same group (Nagai & Yamasaki, 2009).

Other characteristics of JNDMP and ONPA are summarized in Table 2.

#### **2. Classification of** *Bacillus subtilis* **(***natto***) phages**

The viscous polymer PGA on natto is an important characteristic. However, natto products without the polymer were often found in the market, despite normal synthesis of PGA just after fermentation in a factory. In some cases, the viscosity of natto decreased rapidly while mixing with seasonings. Fujii *et al.* (1967) found a bacteriophage from such an abnormal natto and named it PN-1. Fermentation of soybeans with *B. subtilis* (*natto*) and PN-1 resulted in the production of natto with no viscous polymer, indicating that PN-1 is attributed to a loss of polymer on natto. This was the first report on a *B. subtilis* (*natto*) phage.

Yoshimoto *et al.* (1970) surveyed contamination by *B. subtilis* (*natto*) phages in natto factories all over Japan. Among 60 factories, 28 were contaminated with phages at densities ranging from 5 PFU/cm3 sample to 2000. Forty-two phages were isolated from the samples and classified into 4 groups based on host ranges, and finally into two serological groups, NP-4 and NP-38 groups, using four anti-phage serums.


Note: These phage isolates are distributed by the NIAS Genebank (acronym, MAFF; web site, http://www.gene.affrc.go.jp/index\_en.php).

Table 1. *Bacillus subtilis* (*natto*) phages used in classification

The viscous polymer PGA on natto is an important characteristic. However, natto products without the polymer were often found in the market, despite normal synthesis of PGA just after fermentation in a factory. In some cases, the viscosity of natto decreased rapidly while mixing with seasonings. Fujii *et al.* (1967) found a bacteriophage from such an abnormal natto and named it PN-1. Fermentation of soybeans with *B. subtilis* (*natto*) and PN-1 resulted in the production of natto with no viscous polymer, indicating that PN-1 is attributed to a

Yoshimoto *et al.* (1970) surveyed contamination by *B. subtilis* (*natto*) phages in natto factories all over Japan. Among 60 factories, 28 were contaminated with phages at densities ranging from 5 PFU/cm3 sample to 2000. Forty-two phages were isolated from the samples and classified into 4 groups based on host ranges, and finally into two serological groups, NP-4

Group MAFF no. Strain Source & month and year of

270104 JNCHUP natto, Oct. 1980 270105 JNDMP natto, Feb. 1981 270106 JNHMP natto, Feb. 1981

270120 SUP-SS1P not recorded

Note: These phage isolates are distributed by the NIAS Genebank (acronym, MAFF; web site,

270101 P-1 abnormal natto, Jan. 1980 270102 DMP natto, May 1980 270103 MIP natto, May 1980 270107 MOP abnormal natto, Jul. 1981 270108 THP abnormal natto, Jul. 1981 270109 THAP abnormal natto, Dec. 1981 270110 SUP abnormal natto, Dec. 1981 270111 KKP abnormal natto, Mar. 1982 270112 KKP-GE industrial sewage, Mar. 1982 270113 SS1P abnormal natto, Dec. 1983 270114 SS2P abnormal natto, Dec. 1983 270115 ONPA abnormal natto, Aug. 1984 270116 ONPB abnormal natto, Aug. 1984 270117 ONPC abnormal natto, Sep. 1985 270118 FUKUSHOGUNP abnormal natto, Oct. 1985 270119 ONPD abnormal natto, Aug. 1986

isolation

loss of polymer on natto. This was the first report on a *B. subtilis* (*natto*) phage.

**2. Classification of** *Bacillus subtilis* **(***natto***) phages** 

and NP-38 groups, using four anti-phage serums.

I

II

http://www.gene.affrc.go.jp/index\_en.php).

Table 1. *Bacillus subtilis* (*natto*) phages used in classification

Twenty *B. subtilis* (*natto*) phages were isolated from abnormally fermented natto, effluent from natto factories and soil of paddy fields in Kyushu Island, southern Japan (Fujii *et al.,* 1975). The phages, including PN-1, were classified into three groups based on host ranges, immunological reaction, and morphologies. All tested *B. subtilis* (*natto*) strains were infected by 21 *B. subtilis* (*natto*) phages, and some strains of *B. subtilis* also were infected by 17 phages. Three representative phages, PN-3, PN-6 and PN-19, from three serological groups were studied using an electron microscope. They had a head (diameter, 80–90 nm) and a contractile tail (length, 165–175 nm). Although PN-3 and PN-6 did not resemble each other in shape, judging from their morphologies on somewhat obscure photographs, they were found to belong to the same group (Nagai & Yamasaki, 2009).

Nagai and Yamasaki (2009) classified 20 phages [deposited in the NIAS Genebank (Tsukuba, Japan) with accession numbers from MAFF 270101 to MAFF 270120 (Table 1)], mainly isolated from abnormally fermented natto, into two groups based on DNA-DNA hybridization (Fig. 3). No cross hybridized band is visible in the photograph, indicating that group I phages and group II phages are genetically independent of each other. Representative phages from the two groups were further characterized. Phage JNDMP (Group I) has a head (diameter, 60 nm) and a flexible tail (7 x 200 nm) (Fig. 4 A) and requires magnesium ions for amplification. Phage ONPA (Group II) has a head (diameter, 89 nm) and a contractile tail (9 x 200 nm) with a sheath (width, 23 nm) and does not require additional magnesium ions (Fig. 4 B). Plaques of ONPA were clearer than those of JNDMP. Other characteristics of JNDMP and ONPA are summarized in Table 2.


1)Temperature at which about 1% of phage particles in suspension can survive after 10-min heating in a water bath.

2) Miura, Naruse and Takahashi are commercial starters for natto fermentation.

Table 2. Other characteristics of JNDMP and ONPA

Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 101

A) JNDMP, B) ONPA. Bar = 100 nm. (from Nagai and Yamasaki, 2009, with permission)

The phages of Yoshimoto's group (1970) had been discarded, and so genetic relationships among their two types of phages and ONPA or JNDMP could not be investigated. However, NP-4 and NP-38 had the same morphologies as JNDMP (Group I) and ONPA (Group II), respectively. Using DNA-DNA hybridization, type strains of Fujii *et al.* (1975), PN-3, PN-6 and PN-19, were found to belong to Group I, I and II, respectively (Nagai & Yamasaki,

*B. subtilis* (*natto*) produces a very viscous polymer of **DL**-glutamic acid, which has two carboxyl groups on α- and γ-carbons (Fig. 5A), with extremely high degrees of polymerization. Unlike proteins, in which amino acid residues bind via α-carboxyl groups and amino groups (Fig. 5B), the glutamic acids in this polymer, poly-γ-glutamate (PGA, Fig. 5C), are synthesized by binding a γ-carboxyl group and an amino group of an adjacent glutamic acid via a hyperphosphorylated intermediate (Fig. 5D) (Ashiuchi *et al.,* 2001). Genes related to the production of PGA were cloned and expressed well in *Escherichia coli* cells (Ashiuchi *et al.,* 1999). The gene *pgsBCA* is homologous with genes for capsular PGA production of *B. anthracis* and codes for a membranous enzyme complex (Ashiuchi *et al.,*

Fig. 4. Electron microscope photographs of *B. subtilis* (*natto*) phages

**3. Polyglutamate degrading enzyme** 

2009).

**3.1 Polyglutamate** 

A) An agarose gel electrophoresis of fragments of phage DNA after digestion with a restriction enzyme, *Hin*dIII. B and C) Southern hybridization of the gel using genomic DNA of JNDMP (B) or ONPA (C) as a probe. (from Nagai & Yamasaki, 2009, with permission)

Fig. 3. Analysis of Southern hybridization of phage genome DNA

A) JNDMP, B) ONPA. Bar = 100 nm. (from Nagai and Yamasaki, 2009, with permission) Fig. 4. Electron microscope photographs of *B. subtilis* (*natto*) phages

The phages of Yoshimoto's group (1970) had been discarded, and so genetic relationships among their two types of phages and ONPA or JNDMP could not be investigated. However, NP-4 and NP-38 had the same morphologies as JNDMP (Group I) and ONPA (Group II), respectively. Using DNA-DNA hybridization, type strains of Fujii *et al.* (1975), PN-3, PN-6 and PN-19, were found to belong to Group I, I and II, respectively (Nagai & Yamasaki, 2009).

#### **3. Polyglutamate degrading enzyme**

#### **3.1 Polyglutamate**

100 Bacteriophages

A) An agarose gel electrophoresis of fragments of phage DNA after digestion with a restriction enzyme, *Hin*dIII. B and C) Southern hybridization of the gel using genomic DNA of JNDMP (B) or ONPA (C) as

a probe. (from Nagai & Yamasaki, 2009, with permission)

Fig. 3. Analysis of Southern hybridization of phage genome DNA

*B. subtilis* (*natto*) produces a very viscous polymer of **DL**-glutamic acid, which has two carboxyl groups on α- and γ-carbons (Fig. 5A), with extremely high degrees of polymerization. Unlike proteins, in which amino acid residues bind via α-carboxyl groups and amino groups (Fig. 5B), the glutamic acids in this polymer, poly-γ-glutamate (PGA, Fig. 5C), are synthesized by binding a γ-carboxyl group and an amino group of an adjacent glutamic acid via a hyperphosphorylated intermediate (Fig. 5D) (Ashiuchi *et al.,* 2001). Genes related to the production of PGA were cloned and expressed well in *Escherichia coli* cells (Ashiuchi *et al.,* 1999). The gene *pgsBCA* is homologous with genes for capsular PGA production of *B. anthracis* and codes for a membranous enzyme complex (Ashiuchi *et al.,*

Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 103

the products of the reaction were di-γ-glutamate and tri-γ-glutamate (Hongo & Yoshimoto, 1970b). Optimal pH and temperature of the depolymerase were pH 6–8 and 40–50°C, respectively. The depolymerase was stable at pH values ranging from 4 to 9 and below 70°C.

Culture supernatant of *B. subtilis* (*natto*) infected with a phage ΦNIT1, which had been isolated from natto containing a small amount of PGA, caused the viscosity of PGA to decrease rapidly, and degraded PGA with a molecular weight of 106 Daltons to oligo-γglutamyl peptides (Kimura & Itoh, 2003). From the culture supernatant, a 25-kDa monomeric enzyme was purified through five column chromatographic steps and was named PghP for "γ-PGA hydrolase of phage". Analysis of the products of enzymatic reaction on PGA showed that they were tri-γ-glutamate, tetra-γ-glutamate and penta-γglutamate. PghP was inhibited with monoiodoacetate and EDTA and the activity inhibited by EDTA was restored by adding Zn2+ or Mn2+, indicating that a cisteine residue(s) of PghP

The gene for PghP was cloned based on a nucleotide sequence predicted from the Nterminal amino acid sequence of purified PghP, and sequenced (Kimura & Itoh, 2003). The predicted PghP was a 22.9-kDa protein with 203 amino acid residues, in which the first methionine was eliminated posttranslationally. PghP was a unique protein: similar proteins were not detected in the database by a BLAST search program. PghP is distributed in a variety of *B. subtilis* (*natto*) phages including some phages isolated from *B. subtilis* strains which produce no PGA. PghP of ΦNIT1 has a different substrate specificity from the PGA

ΦNIT1 could amplify in both encapsulated and non-encapsulated *B. subtilis* (*natto*). On the other hand, *B. subtilis* phage BS5 (Ackermann *et al.,* 1995), which produced no PghP, could amplify only in non-encapsulated *B. subtilis* (*natto*). BS5, however, could amplify in encapsulated *B. subtilis* (*natto*) in the presence of additive PghP. These results indicate that *B. subtilis* (*natto*) produces PGA for physical protection from attacks by phages (Kimura & Itoh,

Apparently opposite results were also reported: that PGA production made the cells susceptible to *B. subtilis* (*natto*) phages (Hara *et al.,* 1984). In their study, *B. subtilis* (*natto*) phages could infect *B. subtilis* (*natto*) producing PGA. After curing of plasmid pUH1, which harboured genes controlling PGA production, of *B. subtilis* (*natto*) strains, the cured strains could no longer be infected by the phages. *B. subtilis* Marburg, which was not infected by the *B. subtilis* (*natto*) phages and did not produce PGA by nature, and the cured *B. subtilis* (*natto*) strains became susceptible to the phages after transformation with DNA from *B. subtilis* (*natto*). Hara *et al.* thought that "PGA might be associated with phage absorption" from the results. However, a cured *B. subtilis* (*natto*) strain was susceptible to phage ΦBN100, indicating basically that the plasmid did not control phage absorption (Nagai & Itoh, 1997). The experiments conducted by Hara *et al.* examined three factors: the existence of pUH1 type plasmids, PGA productivity and γ-glutamyl transpeptidase (γ-GTP) (at the time, γ-GTP and pUH1 were thought to be a PGA synthesizing enzyme and a plasmid coding γ-GTP

The depolymerase was not linked to phage particles.

and these ions participated in the hydrolase reaction.

depolymerase in Section 3.2 (Hongo & Yoshimoto, 1970b).

2003).

**3.3 Poly-γ-glutamate hydrolase, PghP** 

2001). On the other hand, a regulatory gene for PGA production was cloned and found to be *comP*, which codes for a sensor protein kinase of the ComP-ComA two-component signal transduction system (Nagai *et al.,* 2000). Recently, PGA cross-linked by γ-radiation was found to hold a large quantity of water and to be useful for greening of desert areas by scattering the PGA resin in which seeds (e.g. soybeans) are embedded (Hara, 2006). Thus, PGA is becoming an important industrial material.

Fig. 5. Glutamic acid (A), tri-α-glutamic acid (B), tri-γ-glutamic acid (C), and the mechanism of synthesis of poly-γ-glutamic acid (D)

#### **3.2 Polyglutamate depolymerase**

It was found that a *B. subtilis* (*natto*) lysogenic strain could not accumulate PGA in the culture, whereas a nonlysogenic strain accumulated it under the same conditions (Hongo & Yoshimoto, 1968). In the culture of the lysogenic strain, bacteriophages were induced at a high density of 109 PFU/ml from the early stage of the experiment. Around the peak of production of phages, PGA depolymerase appeared to be released from the lysogenic strains to the culture. The enzyme was also produced extracellularly, when *B. subtilis* (*natto*) was infected by phages. After infection with phage NP-1 cl (Yoshimoto & Hongo, 1970), depolymerase was synthesized in parallel with the production of phage particles in host cells (Hongo & Yoshimoto, 1970a). The depolymerase digested PGA by endopeptidase-type action, resulting in the rapid loss of viscosity of PGA. Chromatographic studies showed that the products of the reaction were di-γ-glutamate and tri-γ-glutamate (Hongo & Yoshimoto, 1970b). Optimal pH and temperature of the depolymerase were pH 6–8 and 40–50°C, respectively. The depolymerase was stable at pH values ranging from 4 to 9 and below 70°C. The depolymerase was not linked to phage particles.

#### **3.3 Poly-γ-glutamate hydrolase, PghP**

102 Bacteriophages

2001). On the other hand, a regulatory gene for PGA production was cloned and found to be *comP*, which codes for a sensor protein kinase of the ComP-ComA two-component signal transduction system (Nagai *et al.,* 2000). Recently, PGA cross-linked by γ-radiation was found to hold a large quantity of water and to be useful for greening of desert areas by scattering the PGA resin in which seeds (e.g. soybeans) are embedded (Hara, 2006). Thus,

Fig. 5. Glutamic acid (A), tri-α-glutamic acid (B), tri-γ-glutamic acid (C), and the mechanism

It was found that a *B. subtilis* (*natto*) lysogenic strain could not accumulate PGA in the culture, whereas a nonlysogenic strain accumulated it under the same conditions (Hongo & Yoshimoto, 1968). In the culture of the lysogenic strain, bacteriophages were induced at a high density of 109 PFU/ml from the early stage of the experiment. Around the peak of production of phages, PGA depolymerase appeared to be released from the lysogenic strains to the culture. The enzyme was also produced extracellularly, when *B. subtilis* (*natto*) was infected by phages. After infection with phage NP-1 cl (Yoshimoto & Hongo, 1970), depolymerase was synthesized in parallel with the production of phage particles in host cells (Hongo & Yoshimoto, 1970a). The depolymerase digested PGA by endopeptidase-type action, resulting in the rapid loss of viscosity of PGA. Chromatographic studies showed that

PGA is becoming an important industrial material.

of synthesis of poly-γ-glutamic acid (D)

**3.2 Polyglutamate depolymerase** 

Culture supernatant of *B. subtilis* (*natto*) infected with a phage ΦNIT1, which had been isolated from natto containing a small amount of PGA, caused the viscosity of PGA to decrease rapidly, and degraded PGA with a molecular weight of 106 Daltons to oligo-γglutamyl peptides (Kimura & Itoh, 2003). From the culture supernatant, a 25-kDa monomeric enzyme was purified through five column chromatographic steps and was named PghP for "γ-PGA hydrolase of phage". Analysis of the products of enzymatic reaction on PGA showed that they were tri-γ-glutamate, tetra-γ-glutamate and penta-γglutamate. PghP was inhibited with monoiodoacetate and EDTA and the activity inhibited by EDTA was restored by adding Zn2+ or Mn2+, indicating that a cisteine residue(s) of PghP and these ions participated in the hydrolase reaction.

The gene for PghP was cloned based on a nucleotide sequence predicted from the Nterminal amino acid sequence of purified PghP, and sequenced (Kimura & Itoh, 2003). The predicted PghP was a 22.9-kDa protein with 203 amino acid residues, in which the first methionine was eliminated posttranslationally. PghP was a unique protein: similar proteins were not detected in the database by a BLAST search program. PghP is distributed in a variety of *B. subtilis* (*natto*) phages including some phages isolated from *B. subtilis* strains which produce no PGA. PghP of ΦNIT1 has a different substrate specificity from the PGA depolymerase in Section 3.2 (Hongo & Yoshimoto, 1970b).

ΦNIT1 could amplify in both encapsulated and non-encapsulated *B. subtilis* (*natto*). On the other hand, *B. subtilis* phage BS5 (Ackermann *et al.,* 1995), which produced no PghP, could amplify only in non-encapsulated *B. subtilis* (*natto*). BS5, however, could amplify in encapsulated *B. subtilis* (*natto*) in the presence of additive PghP. These results indicate that *B. subtilis* (*natto*) produces PGA for physical protection from attacks by phages (Kimura & Itoh, 2003).

Apparently opposite results were also reported: that PGA production made the cells susceptible to *B. subtilis* (*natto*) phages (Hara *et al.,* 1984). In their study, *B. subtilis* (*natto*) phages could infect *B. subtilis* (*natto*) producing PGA. After curing of plasmid pUH1, which harboured genes controlling PGA production, of *B. subtilis* (*natto*) strains, the cured strains could no longer be infected by the phages. *B. subtilis* Marburg, which was not infected by the *B. subtilis* (*natto*) phages and did not produce PGA by nature, and the cured *B. subtilis* (*natto*) strains became susceptible to the phages after transformation with DNA from *B. subtilis* (*natto*). Hara *et al.* thought that "PGA might be associated with phage absorption" from the results. However, a cured *B. subtilis* (*natto*) strain was susceptible to phage ΦBN100, indicating basically that the plasmid did not control phage absorption (Nagai & Itoh, 1997). The experiments conducted by Hara *et al.* examined three factors: the existence of pUH1 type plasmids, PGA productivity and γ-glutamyl transpeptidase (γ-GTP) (at the time, γ-GTP and pUH1 were thought to be a PGA synthesizing enzyme and a plasmid coding γ-GTP

Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 105

Four independent factories (indicated by different color bars) were surveyed for *B. subtilis* (*natto*) phages in several facilities in the process of natto fermentation (the right flow chart, also see Fig. 2) and

Fig. 6. Contamination by *B. subtilis* (*natto*) phages in natto factories (adapted from Nakajima,

The following disinfectants were effective against *B. subtilis* (*natto*) phages: benzalkonium chloride, chloramine-T, sodium hypochlorite, TEGO-51 and Vantocil IB (Fujii *et al.,* 1983). The most important measure is cleaning the machines and floors of natto factories to remove

For genetic transfer of DNA between *B. subtilis* (*natto*) strains by transduction, a phage ΦBN100 was screened in laboratory stock strains (Nagai & Itoh, 1997). The phage could transduce prototroph genes (for adenine, uracil or leucine requirement) to auxotrophs at rates ranging from 3.8 x 10-8 to 1.6 x 10-6 (number of transductants per phage particle). The phage was also used for analysis of transposon insertional mutagenesis on a gene responsible for the regulation of PGA production (Nagai & Itoh, 1997) and construction of

the floors near the facilities. BN, *Bacillus subtilis* (*natto*)

1995)

Detection rate (%) = no. of detections / no. of samples x 100

**5. Other topics on** *B. subtilis* **(***natto***) phages** 

**5.1 Generalized transducing phage for** *B. subtilis* **(***natto***)** 

soybean debris on which *B. subtilis* (*natto*) and phages can propagate.

genes, respectively [Aumayr *et al.,* 1981; Hara *et al.,* 1981], but pUH1 was found to harbour no genes for PGA production [Nagai *et al.,* 1997]), so the situation might make the results difficult to interpret accurately. This discrepancy remains to be elucidated.

Natto without PGA has no commercial value as a food on the market, but might have a value as an ingredient of natto fried rice or natto snack foods because of its ease of manufacturing in food factories (Kimura, 2008). Thus, PghP could be useful in the food industry.

### **4. Phage contamination in natto factories**

The first report on contamination of natto by phages was reported by Fujii *et al.* (1967). At that time, the authors did not search the factory for phages where the contaminated natto had been made. In 1975, the authors investigated contamination by phages in the same factory. Phages were not detected in the factory, but in effluent from it because of modernization of the factory (Fujii *et al.,* 1975).

Yoshimoto *et al.* (1970) searched natto factories throughout Japan for contamination by phages. Phages were detected in 28 factories (47%) among 60 factories at a density ranging from 5 to over 1000 PFU/cm3 of sample. Phages were very often detected in old factories, the walls of which were made of clay (common in old Japanese buildings). The surfaces of clay walls have too many asperities to clean off soybean debris perfectly, resulting in amplification of phages in their host cells on soybean debris. The walls of modern factories are made of clean stainless steel, so phages were rarely detected. In factories contaminated by phages, the phages were detected most frequently in the fermentation rooms. Fujii *et al.*  also reported that 25% of factories (2/8 factories in Kyushu Island, west Japan) were polluted with phages (Fujii *et al.,* 1975).

Nakajima (1995) investigated contamination by *B. subtilis* (*natto*) phages in four natto factories in Ibaraki prefecture, central Japan (Fig. 6). Before inoculation of natto starter to soybeans, phages were detected only on the floor of the washing room in a factory and its detection rate was very low. The phages might have been brought in with raw soybeans or dust in the air. After inoculation, the detection rate rose to 100%. These results indicate that perfect cleaning to ensure that no soybean debris remains in machines or on floors is essential, especially after soybeans have been sprayed with a spore suspension of *B. subtilis* (*natto*). The author did not mention contamination by phages of natto products made by the four factories. Another report showed that phage contamination was not detected in any natto made in factories in Iwate prefecture, northern Japan (Yamamoto, 1986). In total, contamination of natto products by phages has decreased drastically, but *B. subtilis* (*natto*) phages still exist in natto factories, fields, and waste water.

Improvement of a factory highly polluted with phages and bacteria was reported (Takiguchi *et al.,* 1999). When phages and bacteria were detected in natto made by the factory, a manual was compiled to ensure strict separation of the entrance and exit, hand-washing, removal of abnormal natto from the factory as soon as possible, dilution of starter with sterilized water, installation of UV lighting, replacement of wooden parts with those made of stainless steel, including periodical cleaning and hygiene education. After these efforts, no contamination was detected in the natto.

Four independent factories (indicated by different color bars) were surveyed for *B. subtilis* (*natto*) phages in several facilities in the process of natto fermentation (the right flow chart, also see Fig. 2) and the floors near the facilities.

BN, *Bacillus subtilis* (*natto*)

104 Bacteriophages

genes, respectively [Aumayr *et al.,* 1981; Hara *et al.,* 1981], but pUH1 was found to harbour no genes for PGA production [Nagai *et al.,* 1997]), so the situation might make the results

Natto without PGA has no commercial value as a food on the market, but might have a value as an ingredient of natto fried rice or natto snack foods because of its ease of manufacturing in food factories (Kimura, 2008). Thus, PghP could be useful in the food

The first report on contamination of natto by phages was reported by Fujii *et al.* (1967). At that time, the authors did not search the factory for phages where the contaminated natto had been made. In 1975, the authors investigated contamination by phages in the same factory. Phages were not detected in the factory, but in effluent from it because of

Yoshimoto *et al.* (1970) searched natto factories throughout Japan for contamination by phages. Phages were detected in 28 factories (47%) among 60 factories at a density ranging from 5 to over 1000 PFU/cm3 of sample. Phages were very often detected in old factories, the walls of which were made of clay (common in old Japanese buildings). The surfaces of clay walls have too many asperities to clean off soybean debris perfectly, resulting in amplification of phages in their host cells on soybean debris. The walls of modern factories are made of clean stainless steel, so phages were rarely detected. In factories contaminated by phages, the phages were detected most frequently in the fermentation rooms. Fujii *et al.*  also reported that 25% of factories (2/8 factories in Kyushu Island, west Japan) were

Nakajima (1995) investigated contamination by *B. subtilis* (*natto*) phages in four natto factories in Ibaraki prefecture, central Japan (Fig. 6). Before inoculation of natto starter to soybeans, phages were detected only on the floor of the washing room in a factory and its detection rate was very low. The phages might have been brought in with raw soybeans or dust in the air. After inoculation, the detection rate rose to 100%. These results indicate that perfect cleaning to ensure that no soybean debris remains in machines or on floors is essential, especially after soybeans have been sprayed with a spore suspension of *B. subtilis* (*natto*). The author did not mention contamination by phages of natto products made by the four factories. Another report showed that phage contamination was not detected in any natto made in factories in Iwate prefecture, northern Japan (Yamamoto, 1986). In total, contamination of natto products by phages has decreased drastically, but *B. subtilis* (*natto*)

Improvement of a factory highly polluted with phages and bacteria was reported (Takiguchi *et al.,* 1999). When phages and bacteria were detected in natto made by the factory, a manual was compiled to ensure strict separation of the entrance and exit, hand-washing, removal of abnormal natto from the factory as soon as possible, dilution of starter with sterilized water, installation of UV lighting, replacement of wooden parts with those made of stainless steel, including periodical cleaning and hygiene education. After these efforts, no contamination

difficult to interpret accurately. This discrepancy remains to be elucidated.

**4. Phage contamination in natto factories** 

modernization of the factory (Fujii *et al.,* 1975).

polluted with phages (Fujii *et al.,* 1975).

was detected in the natto.

phages still exist in natto factories, fields, and waste water.

industry.

Detection rate (%) = no. of detections / no. of samples x 100

Fig. 6. Contamination by *B. subtilis* (*natto*) phages in natto factories (adapted from Nakajima, 1995)

The following disinfectants were effective against *B. subtilis* (*natto*) phages: benzalkonium chloride, chloramine-T, sodium hypochlorite, TEGO-51 and Vantocil IB (Fujii *et al.,* 1983). The most important measure is cleaning the machines and floors of natto factories to remove soybean debris on which *B. subtilis* (*natto*) and phages can propagate.

#### **5. Other topics on** *B. subtilis* **(***natto***) phages**

#### **5.1 Generalized transducing phage for** *B. subtilis* **(***natto***)**

For genetic transfer of DNA between *B. subtilis* (*natto*) strains by transduction, a phage ΦBN100 was screened in laboratory stock strains (Nagai & Itoh, 1997). The phage could transduce prototroph genes (for adenine, uracil or leucine requirement) to auxotrophs at rates ranging from 3.8 x 10-8 to 1.6 x 10-6 (number of transductants per phage particle). The phage was also used for analysis of transposon insertional mutagenesis on a gene responsible for the regulation of PGA production (Nagai & Itoh, 1997) and construction of

Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 107

*B. subtilis* (*natto*) phages that have been isolated in Japan are classified into two groups (Groups I and II), which are genetically independent of each other judging from DNA-DNA hybridization analysis. Phage JNDMP (Group I) has a head (diameter, 60 nm) and a flexible tail (7 x 200 nm) and requires magnesium ions for amplification. Phage ONPA (Group II) has a head (diameter, 89 nm) and a contractile tail (9 x 200 nm) with a sheath (width, 23 nm) and does not require additional magnesium ions. JNDMP was found to be a generalized transducing phage for *B. subtilis* (*natto*). Natto contaminated with phages is not covered with PGA, which is an important factor of the quality of natto. The loss of PGA is attributed to PGA hydrolase, PghP, or its relevant enzyme, which is expressed from a gene on phage genomic DNA in infected host cells. The enzymes digest PGA by endopeptidase-type action, resulting in a rapid loss of viscosity of PGA. Contamination of natto products by phages can be prevented by cleaning the facilities and floors of natto factories. Until 1980, contamination by phages had caused devastating damage to natto factories, but such trouble is now rare thanks to the modernization of natto factories and hygiene education for

Ackermann, H., Azizbekyan, R., Bernier, R., de Barje, H. , Saindouk, S. , Valéro, J. & Yu, M.

Anderson, L.& Bott, K. (1985). DNA Packaging by the *Bacillus subtilis* Defective

Ashiuchi, M., Soda, K. & Misono, H. (1999). A poly-γ-glutamate synthetic system of *Bacillus* 

Aumayr, A. , Hara, T. & Ueda, S. (1981). Transformation of *Bacillus subtilis* in polyglutamate

Fujii, H. , Oki, M. , Makihara, M. , Keshino, J. & Takeya, R. (1967). On the Formation of

Fujii, H. , Shiraishi, A. , Kaba, K , Shibagaki, M. , Takahashi, S. & Honda, A. (1975).

*Microbiology*, Vol. 27, No. 2, (1981), pp. 115-123, ISSN 0022-1260

Vol. 146, Issue 8, (October 1995), pp. 643–657, ISSN 0923-2508

20, (October 2001), pp. 5321-5328, ISSN 0014-2956

(1995). Phage typing of *Bacillus subtilis* and *B. thuringensis*, *Research in Microbiology*,

Bacteriophage PBSX, *Journal of Virology,* Vol. 54, No.3, ( June 1985), pp. 773-780,

*subtilis* IFO 3336: Gene cloning and biochemical analysis of poly-γ-glutamate produced by *Escherichia coli* clone cells, *Biochemical and Biophysical Research Communications,* Vol.263, Issue 1, (September 1999), pp. 6-12, ISSN 0006-291X Ashiuchi, M., Nawa, C., Kamei, T., Song, J., Hong, S., Sung, M., Soda, K. , Yagi, T. & Misono,

H. (2001). Physiological and Biochemical Characteristics of Poly γ-glutamate synthetase complex of *Bacillus subtilis*, *European Journal of Biochemistry,* Vol. 268, No.

production by deoxyribonucleic acid from *B. natto*. *Journal of General and Applied* 

Mucilage by *Bacillus natto* ; Part VII. Isolation and Characterization of a Bacteriophage Active against "Natto"-Producing Bacteria, *Journal of the Agricultural Chemical Society of Japan,* Vol.41, No.1, (January 1967), pp. 39-43, ISSN 0002-1407 (in

Abnormal Fermentation in Natto Production and *Bacillus natto* Phages, *Journal of Fermentation Technology,* Vol.53, No.7, (July 1975), pp. 424-428, ISSN 0367-5963 (in

**6. Conclusion** 

workers.

**7. References** 

ISSN 0006-2960

Japanese)

Japanese)

mutants on production of branched short-chain fatty acids for preparation of odorless natto (Takemura *et al.,* 2000). ΦBN100 is a synonym for JNDMP (see Section 2).

#### **5.2** *B. subtilis* **(***natto***) phage PM1 and a phage detection system by PCR**

A phage was newly isolated from natto producing no PGA and characterized (Umene *et al.,* 2009). The morphology of the phage, PM1, was very similar to that of JNDMP, and the size of its genomic DNA was found to be 50 kb using field inversion gel electrophoresis, 10 kb smaller than that of JNDMP. The genome of PM1 was a linear double-stranded DNA, and might be circularly permuted and have no definite termini, like T4 phage.

Based on a sequence of a 1.1-kb *Eco*RI fragment of genomic DNA, which did not have significant homology with any sequences deposited at the DNA database so far, the following pair of primers for PCR to amplify a 0.53-kb region in the 1.1-kb *Eco*RI fragment was designed:

5'-CGCACTGGAAGCAATCAAGTCGG-3' (corresponding to nt 33–55)

5'-CAACCCTCTGACCGACTTTTCCC-3' (corresponding to nt 538–560)

Among ten *B. subtilis* (*natto*) phage isolates in the authors' laboratory, eight were target sequences of amplified with the primer set, suggesting that PM1 phages are distributed over a large area of Japan.

#### **5.3** *Bacillus* **phage isolated from chungkookjang**

Chungkookjang is a Korean soybean food fermented by *Bacillus subtilis*. From the fermented soybeans, a virulent *Bacillus* phage was isolated and named Bp-K2 (Kim *et al.,* 2011). Bp-K2 resembled ONPA in morphology, but had a smaller head (width, 80 nm) and genomic DNA (21 kb). Bp-K2 had a contractile tail with a sheath (85–90 nm x 28 nm), a tail fiber (80–85 nm x 10 nm) and a basal plate (29 nm x 47 nm). Bp-K2 could develop plaques on not only *B. subtilis* strains isolated from chungkookjang but also *B. subtilis* (*natto*).

#### **5.4 Defective phage of** *B. subtilis* **(***natto***)**

As *B. subtilis* Marburg strain produces a defective phage PBSX (Seaman *et al.,* 1964; Anderson & Bott, 1985; Zahler, 1993 for a review), which cannot amplify in host cells, *B. subtilis* (*natto*) IAM 1207 produces defective phage PBND8 after induction with bleomycin (Tsutsumi *et al.,* 1990). Although PBND8 resembled PBSX in morphology, the size of DNA contained in heads of PBND8 was 8 kb, 5 kb smaller than that of PBSX (13 kb). SDSpolyacrylamide gel electrophoresis of component proteins of the phage particles showed that PBND8 was clearly distinct from PBSX and PBSY, a defective phage from *B. subtilis* W23.

Seaman *et al.* (1964) also showed the production of PBSX-like particles from *B. natto*. The particles neutralized antiserum against PBSX particles, indicating that the PBSX-like particles from *B. natto* were very closely related to PBSX. At least two kinds of defective phages might be produced by strains belonging to a *B. subtilis* (*natto*) group (i.e., PBND8 and PBSX-like defective phage).

#### **6. Conclusion**

106 Bacteriophages

mutants on production of branched short-chain fatty acids for preparation of odorless natto

A phage was newly isolated from natto producing no PGA and characterized (Umene *et al.,* 2009). The morphology of the phage, PM1, was very similar to that of JNDMP, and the size of its genomic DNA was found to be 50 kb using field inversion gel electrophoresis, 10 kb smaller than that of JNDMP. The genome of PM1 was a linear double-stranded DNA, and

Based on a sequence of a 1.1-kb *Eco*RI fragment of genomic DNA, which did not have significant homology with any sequences deposited at the DNA database so far, the following pair of primers for PCR to amplify a 0.53-kb region in the 1.1-kb *Eco*RI fragment

5'-CGCACTGGAAGCAATCAAGTCGG-3' (corresponding to nt 33–55)

5'-CAACCCTCTGACCGACTTTTCCC-3' (corresponding to nt 538–560) Among ten *B. subtilis* (*natto*) phage isolates in the authors' laboratory, eight were target sequences of amplified with the primer set, suggesting that PM1 phages are distributed over

Chungkookjang is a Korean soybean food fermented by *Bacillus subtilis*. From the fermented soybeans, a virulent *Bacillus* phage was isolated and named Bp-K2 (Kim *et al.,* 2011). Bp-K2 resembled ONPA in morphology, but had a smaller head (width, 80 nm) and genomic DNA (21 kb). Bp-K2 had a contractile tail with a sheath (85–90 nm x 28 nm), a tail fiber (80–85 nm x 10 nm) and a basal plate (29 nm x 47 nm). Bp-K2 could develop plaques on not only *B.* 

As *B. subtilis* Marburg strain produces a defective phage PBSX (Seaman *et al.,* 1964; Anderson & Bott, 1985; Zahler, 1993 for a review), which cannot amplify in host cells, *B. subtilis* (*natto*) IAM 1207 produces defective phage PBND8 after induction with bleomycin (Tsutsumi *et al.,* 1990). Although PBND8 resembled PBSX in morphology, the size of DNA contained in heads of PBND8 was 8 kb, 5 kb smaller than that of PBSX (13 kb). SDSpolyacrylamide gel electrophoresis of component proteins of the phage particles showed that PBND8 was clearly distinct from PBSX and PBSY, a defective phage from *B. subtilis* W23.

Seaman *et al.* (1964) also showed the production of PBSX-like particles from *B. natto*. The particles neutralized antiserum against PBSX particles, indicating that the PBSX-like particles from *B. natto* were very closely related to PBSX. At least two kinds of defective phages might be produced by strains belonging to a *B. subtilis* (*natto*) group (i.e., PBND8 and

(Takemura *et al.,* 2000). ΦBN100 is a synonym for JNDMP (see Section 2).

**5.2** *B. subtilis* **(***natto***) phage PM1 and a phage detection system by PCR** 

might be circularly permuted and have no definite termini, like T4 phage.

was designed:

a large area of Japan.

**5.3** *Bacillus* **phage isolated from chungkookjang** 

**5.4 Defective phage of** *B. subtilis* **(***natto***)** 

PBSX-like defective phage).

*subtilis* strains isolated from chungkookjang but also *B. subtilis* (*natto*).

*B. subtilis* (*natto*) phages that have been isolated in Japan are classified into two groups (Groups I and II), which are genetically independent of each other judging from DNA-DNA hybridization analysis. Phage JNDMP (Group I) has a head (diameter, 60 nm) and a flexible tail (7 x 200 nm) and requires magnesium ions for amplification. Phage ONPA (Group II) has a head (diameter, 89 nm) and a contractile tail (9 x 200 nm) with a sheath (width, 23 nm) and does not require additional magnesium ions. JNDMP was found to be a generalized transducing phage for *B. subtilis* (*natto*). Natto contaminated with phages is not covered with PGA, which is an important factor of the quality of natto. The loss of PGA is attributed to PGA hydrolase, PghP, or its relevant enzyme, which is expressed from a gene on phage genomic DNA in infected host cells. The enzymes digest PGA by endopeptidase-type action, resulting in a rapid loss of viscosity of PGA. Contamination of natto products by phages can be prevented by cleaning the facilities and floors of natto factories. Until 1980, contamination by phages had caused devastating damage to natto factories, but such trouble is now rare thanks to the modernization of natto factories and hygiene education for workers.

#### **7. References**


Bacteriophages of *Bacillus subtilis* (*natto*) and Their Contamination in Natto Factories 109

Kiuchi, K. & Watanabe, S. (2004). Industrialization of Japanese Natto, In : *Industrialization of* 

Nagai, T. & Itoh, Y. (1997). Characterization of a Generalized Transducing Phage of Poly-γ-

Nagai, T. , Koguchi, K. & Itoh, Y. (1997). Chemical analysis of poly-γ-glutamic acid

Nagai, T. , Tran, L. , Inatsu,Y. & Itoh, Y. (2000). A New IS*4* Family Insertion Sequence,

Nagai, T. & Yamasaki, F. (2009). *Bacillus subtilis* (*natto*) Bacteriophages Isolated in Japan, *Food* 

Nagai, T. & Tamang, J. (2010). Fermented Legumes : Soybean and Non-Soybean Products, In

Nakajima, J. (1995). Natto hishitsu Furyo no Kaizen : Phage Osen Tyosa (Inprovement of

Sawamura, S. (1906) On the Micro-organisms of Natto, B*ulletin of the College of Agriculture,* 

Seaman, E. , Tarmy, E. & Marmur, J. (1964). Inducible Phages of *Bacillus subtilis*, *Biochemistry,*

Seki, T. , Oshima, T. & Oshima, Y. (1975). Taxonomic Study of *Bacillus* by Deoxyribonucleic

Sulistyo, J. , Taya, N. , Funane, K. & Kiuchi, K. (1988) Production of Natto Satarter, *Nippon Shokuhin Kogyo Gakkaishi*, Vol. 35, No. 4, (April 1987), pp. 278-283, ISSN 0029-0394 Takemura, H. , Ando, N. & Tsukamoto, Y. (2000). Breeding of Branched Short-Chain Fatty

Takiguchi, T. , Yoshino, I. , Yuasa, H. , Kawano, I. & Aoki, Y. (1999). Pollution of

Tamang, J. , Thapa, S. , Dewan, S. , Jojima, Y. , Fudou, R. & Yamanaka, S. (2002).

*Microbiology*, Vol. 43, No. 3, (1997), pp. 139-143, ISSN 0022-1260

191-224, CRC Press, ISBN 978-1-4200-9495-4, Boca Raton, FL

*Tokyo Imperial University*, Vol. 7, (1906), pp. 107-110

Vol. 3, No. 5, ( May 1964), 607-613, ISSN 0006-2960

2000), pp. 773-779, ISSN 1341-027X (in Japanese)

0-8247-4784-4, New York, NY

0099-2240

ISSN 0021-9193

59, ISSN 0912-9936 (in Japanese)

270, ISSN 1466-5026

ISSN 1341-0245 (in Japanese)

6606

*Indigenous Fermented Foods*, K. Steinkraus, (Ed.), pp. 193-246, Marcel Dekker, ISBN

Glutamic Acid-Producing *Bacillus subtilis* and Its Application for Analysis of Tn*917*- LTV1 Insertional Mutants Defective in Poly-γ-Glutamic Acid Production, *Applied and Environmental Microbiology,* Vol.63, No.10, (October 1997), pp. 4087-4089, ISSN

produced by plasmid-free *Bacillus subtilis* (*natto*) : Evidence that plasmids are not involved in poly-γ-glutamic acid production. *Journal of General and Applied* 

IS*4Bsu1*, Responsible for Genetic Instability of Poly-γ-Glutamic Acid Production in *Bacillus subtilis*, *Journal of Bacteriology,* Vol.182, No.9, (May 2000), pp. 2387-2392,

*Science and Technology Research,* Vol.15, No.3, (May 2009), pp. 293-298, ISSN 1344-

: *Fermented Foods and Beverages of the World*, J. Tamang & K. Kailasapathy, (Eds.), pp.

Quality of Natto: Research on Contamination of *B. subtilis* (*natto*) Phages in Natto Factories), *Reports of the Ibaraki Prefectural Industrial Technology Center,* Vol.23, (1995),

Acid-Deoxyribonucleic Acid Hybridization and Interspecific Transformation. International Journal of Systematic Bacteriology, Vol. 25, No. 3, (July, 1975), pp.258-

Acids Non-Producing Natto Bacteria and its Application to Production of Natto with Light Smells, *Nippon Shokuhin Kagaku Kogaku Kaishi,* Vol.47, No.10, (October

Fermentation Process of Natto by Some Kind of Bacteriophage, *Reports of Gunma Prefectural Industrial Technology Research Laboratory,* Vol. 1999, (1999), pp. 35-39,

Phylogenetic Analysis of *Bacillus* Strains Isolated from Fermented Soybean Foods of


Fujii, H. , Shiraishi, A. , Kiryu, K. & Fujimoto, Y. (1983). Isolation and some characteristics of

*University,* Vol. 14, ( January 1983), pp. 1-5, ISSN 0288-3953 (in Japanese) Gibson, T. & Gordon, R. (1974). Genus I. *Bacillus* Cohn 1872, In : *Bergey's Manual of* 

The Williams & Wilkins Company, ISBN 0-683-01117-0, Baltimore, Md Hara, T. , Aumayr, A. & Ueda, S. (1981). Characterization of plasmid deoxyribonucleic acid

26, No. 7, (July 2005), pp. 14-18, ISSN 0286-4835 (in Japanese)

(July 1970), pp. 1055-1063, ISSN 0002-1369

1984), pp. 19-24, ISSN 0289-3827 (in Japanese)

5435

*Chemistry,* Vol.32, No.4, (April 1968), pp. 525-527, ISSN 0002-1369

*and Applied Microbiology*, Vol. 27, No. 4, (1981), pp. 299-305, ISSN 0022-1260 Hara, T. , Shiraishi, A. , Fujii, H. & Ueda, S. (1984). Specific Host Range of Bacillus subtilis

Hongo, M. & Yoshimoto, A. (1968). Formation of Phage-Induced γ-Polyglutamic Acid

Hongo, M. & Yoshimoto, A. (1970a). Bacteriophages of *Bacillus natto*. Part II. Induction of γ-

*Biological Chemistry,* Vol.34, No.7, (July 1970), pp. 1047-1054, ISSN 0002-1369 Hongo, M. & Yoshimoto, A. (1970b). Bacteriophages of *Bacillus natto*. Part III. Action of

Ikeda, H. & Tsuno, S. (1984) The componential changes during the manufacturing process of

Kanno, A. &Takamatsu, H. (1987) Changes in the volatile components of "Natto" during

Kim, E. , Hong, J. , Yun, N. & Lee, Y. (2011). Characterization of *Bacillus* Phage-K2 Isolated

Kimura, K. & Itoh, Y. (2003). Characterization of Poly-γ-Glutamate Hydrolase Encoded by a

Kimura, K. (2008). γ-PGA Hydrolase of Phage, In : *Advanced Science on Natto : Japanese* 

Kiuchi, K. , Taya, N. , Sulistyo, J. & Funane, K. (1987). Isolation and Identification of Natto

*Gakkaishi*, Vol. 34, No. 5, (May 1987), pp. 330-335, ISSN 0029-0394

Vol.69, No. 5, (May 2003), pp. 2491-2497, ISSN 0099-2240

Kenpakusha, ISBN 978-4-7679-6123-1, Tokyo, Japan (in Japanese)

Vol.50, ( March 1987), pp. 18-21, ISSN 0301-9780 (in Japanese)

*Bacillus natto* phage PM, *Bulletin of the Faculty of Home Life Science, Fukuoka Women's* 

*Determinative Bacteriology, 8th ed.,* Buchanan, R. & Gibbons, N., (Ed.), pp. 529-550,

in *Bacillus natto*: Evidence for plasmid-linked PGA production. *Journal of General* 

(natto) Phages Associated with Polyglutamate Production, *Agricultural and Biological Chemistry,* Vol.48, No.9, (Augast 1984), pp. 2373-2374, ISSN 0002-1369 Hara, T. (2005) Desert Greening with Natto Resin – From Dream to Reality, *Kino Zairyo* , Vol.

Depolymerase in Lysogenic Strain of *Bacillus natto*, *Agricultural and Biological* 

Polyglutamic Acid Depolymerase Following Phage Infection, *Agricultural and* 

Phage-induced γ-Polyglutamic Acid Depolymerase on γ-Polyglutamic Acid and the Enzymatic Hydrolyzates, *Agricultural and Biological Chemistry,* Vol.34, No.7,

Natto (Part 1): On the amino nitrogen, ammonia nitrogen, carbohydrates and vitamin B2, *Journal of Food Science, Kyoto Women's University*, Vol. 39, ( December

manufacturing and storage (Studies on "Natto", Part IV), *Nippon Shokuhin Kogyo* 

from Chungkookjang, A Fermented Soybean Foodstuff, *Journal of Industrial Microbiology & Biotechnology,* Vol. 38, No. 1, ( January 2011), pp. 39-42, ISSN 1367-

Bacteriophage Genome: Possible Role in Phage Infection of *Bacillus subtilis*  Encapsulated with Poly-γ-Glutamate, *Applied and Environmental Microbiology,*

*Soybean Fermented Foods,* K. Kiuchi, T. Nagai & K. Kimura, (Ed.), pp. 268-270,

Bacteria from Market-Sold Natto Starters, *Report of National Food Research Institute,*


**Part 3** 

**Bacteriopgahes as Tools** 

**and Biological Control Agents** 

Asia : Kinema, Chungkokjang and Natto, *Journal of Hill Resarch,* Vol.15, No.2, (2002), pp. 56-62, ISSN 0970-7050

