Restriction and modification of SP10 phage byBsuM ofBacillus subtilisMarburg

Abstract

Bacillus subtilis Marburg has only one intrinsic restriction and modification system BsuM that recognizes the CTCGAG (XhoI site) sequence. It consists of two operons, BsuMM operon for two cytosine DNA methyltransferases, and BsuMR operon for a restriction nuclease and two associated proteins of unknown function. In this communication, we analyzed the BsuM system by utilizing phage SP10 that possesses more than twenty BsuM target sequences on the phage genome. SP10 phages grown in the restriction and modification-deficient strain could not make plaques on the restriction-proficient BsuMR(+) indicator strain. An enforced expression of the wild type BsuMM operon in the BsuMR(+) indicator strain, however, allowed more than thousand times more plaques. DNA extracted from SP10 phages, thus, propagated became more but not completely refractory to XhoI digestion in vitro. Thus, the SP10 phage genome DNA is able to be nearly full-methylated but some BsuM sites are considered to be unmethylated.

Inhibitory effect of prophage SP.BETA. fragments on phage SP10 ribonucleotide reductase function and its multiplication in Bacillus subtilis

“…After injection of phage DNA into bacterial cells, phage DNA is restricted by well-known endonuclease restriction/modification systems (Bickle and Kruger, 1993). In this context, the phage SP10 and how its genome is subject to the endogenous restriction system based on the nonB gene in B. subtilis has previously been investigated (Matsuoka et al, 2005). A large group of phages (the lamboid phages in particular) shares a very similar genome organization, the so-called lambda immunity model (Ptashne, 2004).…”

“…BsuMR recognizes the CTC-GAG sequence (the XhoI site). It has been surmised that the SP10 genome possesses more than twenty XhoI sites, possible targets of BsuMR (Matsuoka et al, 2005). In fact, the recently determined SP10 genome sequence possesses 36 XhoI sites, a clear indication of the susceptibility of the SP10 genome to BsuM attack in B. subtilis 168 (Yee et al,manuscript in preparation).…”

Methylation-dependent DNA restriction in Bacillus anthracis

“…B. subtilis 168 possesses only one intrinsic DNA MTase, viz., BsuM, which recognizes CTCGAG (XhoI) (Matsuoka et al, 2005). Of the two plasmids used for transformation in the prior study (Marrero and Welkos, 1995), pHV33 does not have any XhoI sites (Guha, 1985; Primrose and Ehrlich, 1981) and pLTV1 has only a single XhoI site (Camilli et al, 1990).…”

SP10 Infectivity Is Aborted after Bacteriophage SP10 Infection Induces nonA Transcription on the Prophage SPβ Region of the Bacillus subtilis Genome

“…The target sequence of BsuMR is identical to that of XhoI (CTCGAG) (28,29). Since 36 BsuMR recognition sites are located in the SP10 genome, BsuMR cleaves SP10 genomic DNA after DNA injection, thus restricting SP10 multiplication (26,30). In contrast to nonB, the function of nonA has remained unknown.…”

“…B. subtilis was cultured in 300-ml conical flasks containing 60 ml of LB medium at 37°C to an OD 600 of 0.5 and then harvested before infection (0 min) and at 15,30,45, and 60 min after infection with SP10 at an MOI 10. The cells harvested from 1.0-ml cultures were washed once with an equal volume of 10 mM Tris-HCl buffer (pH 7.4), pelleted, frozen, and stored at Ϫ80°C.…”