Effects of Actin-Like Proteins Encoded by Two Bacillus pumilus Phages on Unstable Lysogeny, Revealed by Genomic Analysis

Abstract

We characterized two newly isolated myoviruses, Bp8p-C and Bp8p-T, infecting the ginger rhizome rot disease pathogen Bacillus pumilus GR8. The plaque of Bp8p-T exhibited a clear center with a turbid rim, suggesting that Bp8p-T could transform into latent phage. Lysogeny assays showed that both the two phages could form latent states, while Bp8p-T could form latent phage at a higher frequency and stability than Bp8p-C. The genomes of Bp8p-C and Bp8p-T were 151,417 and 151,419 bp, respectively; both encoded 212 putative proteins, and only differed by three nucleotides. Moreover, owing to this difference, Bp8p-C encoded a truncated, putative actin-like plasmid segregation protein Gp27-C. Functional analysis of protein Gp27 showed that Gp27-T encoded by Bp8p-T exhibited higher ATPase activity and assembly ability than Gp27-C. The results indicate that the difference in Gp27 affected the phage lysogenic ability. Structural proteome analysis of Bp8p-C virion resulted in the identification of 14 structural proteins, among which a pectin lyase-like protein, a putative poly-gamma-glutamate hydrolase, and three proteins with unknown function, were firstly identified as components of the phage virion. Both phages exhibited specific lytic ability to the host strain GR8. Bp8p-C showed better control effect on the pathogen in ginger rhizome slices than Bp8p-T, suggesting that Bp8p-C has a potential application in bio-control of ginger rhizome rot disease. Bacteriophages (phages) are viral predators of bacteria that propagate by infecting the host (1) and, based on the mode of replication of the phage genome, they are classified as lytic or lysogenic phages. To maintain the lysogenic state, phage gene expression is regulated by the phage lysogenic regulatory system. The lytic switch mechanism of the temperate phage has been well studied (2), which illustrates the classical lysogen conversion mechanism of and similar phages. Several other strategies for phage lysogenic conversion have also been observed. For example, a single nucleotide mutation in the LexA-binding site of phage SPC32H induces phage SPC32H to maintain the lysogenic state (1). According Yoshihiko et al., a temperate phage, c-st, with a linear genome can transform into a circular plasmid prophage in the host, Clostridium botulinum, which provides phage c-st the ability to exhibit unstable lysogeny (3). Meanwhile, the TubZ-like plasmid segregation system protein encoded by c-st is essential for partition of the prophage plasmid (4).Plasmid segregation systems are essential for accurate partition of large and essential plasmids to daughter cells during cell division (5). The plasmid segregation system typically consists of three components: a centromere-like DNA region, a small DNA-binding adaptor protein, and a nucleotide-driven motor protein with NTPase activity. Based on the nature of the motor proteins, three major classes of segregation systems have been identified to date and can be described as ParA-like system (type I, ParABS system), ParM-like system (type...

Genomic analysis of a ginger pathogen Bacillus pumilus providing the understanding to the pathogenesis and the novel control strategy

“…The R-M system recognized the specific sites on the foreign DNA and cleaved the DNA without modification 41 . According to previous reports, strain GR8 could be lyzed by kinds of phages 42 . The reason might be the lack of phage defense system in the GR8 genome.…”

Isolation and Characterization of a Novel Phage for Controlling Multidrug-Resistant Klebsiella pneumoniae

“…To observe the virion morphology of the phage, the phage plaques formed on the agar plate were washed off by using an SM buffer (10 mM Tris, 100 mM NaCl, and 10 mM MgSO 4 , pH 7.5) and further filtered through a 0.22 µm pore-size membrane filter for use. After negative staining with 2% potassium phosphotungstate (pH 7.2), the phage morphology was imaged by transmission electron microscopy (TEM, G20, Tecnai, FEI, Hillsboro, OR, USA) at an acceleration voltage of 200 kV [36].…”

“…The function of these two gene products is to degrade the bacterial cell wall for the release of mature phage virions [51]. The phage host lysis gene module usually contains additional holin protein, whose function is to form pores on the bacterial cell membrane for the bypass of lysin [36]. However, no holin gene was identified in the genome of the vB_KleS-HSE3 phage, suggesting that the lysin of the vB_KleS-HSE3 phage passes through the bacterial cell membrane via another strategy.…”

“…The chosen phage plaque was further purified by using the double-ager overlay method at last 5 times until the formation of uniform phage plaques. The efficiency-of-plating was also tested by using the double-agar overlay method [36].…”

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

“…The genomic structure of the Bacillus Myoviridae phages phiAGATE 43) and Bp8p-c 44) carrying a PghPlike gene was similar to that of ϕNIT1, but the existence of TRs in their genomes was not investigated (Fig. 9(A)).…”