Complete genomic sequence and mass spectrometric analysis of highly diverse, atypical Bacillus thuringiensis phage 0305ϕ8–36

Thomas, Julie A.; Hardies, Stephen C.; Rolando, Mandy; Hayes, Shirley J.; Lieman, Karen; Carroll, Christopher A.; Weintraub, Susan T.; Serwer, Philip

doi:10.1016/j.virol.2007.06.043

Cited by 64 publications

(95 citation statements)

References 102 publications

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“…The phage genome was purified by phenol-chloroform extraction with protease K-sodium dodecyl sulfate (SDS) treatment (19). For other analyses, the phage was purified by sucrose density gradient centrifugation as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

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

Yuan

Qin

et al. 2015

Appl Environ Microbiol

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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...

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Section: Methodsmentioning

confidence: 99%

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

Yuan

Qin

et al. 2015

Appl Environ Microbiol

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“…SDS-PAGE was performed as described (19) with the exception that the phage was first heated to 75°C for 4 min and then treated with 0.1 g/ml DNase. Preliminary experiments were conducted to determine the maximum SDS gel protein load that did not overtly smear the banding pattern.…”

Section: Methodsmentioning

confidence: 99%

Proteome of the Large Pseudomonas Myovirus 201φ2-1

Thomas¹,

Weintraub

Hakala

et al. 2010

Molecular & Cellular Proteomics

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Pseudomonas chlororaphis phage 2012-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 2012-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions. Molecular & Cellular Proteomics 9: 940 -951, 2010.There has been a resurgence in interest in bacteriophages because of their potential for use in phage therapy (1, 2), their role at the base of the food chain (3-5), their abundance in metagenomic data (6), and their content of the last great unexplored repository of genetic diversity (7) and as models of complex macromolecular assemblies (8, 9). In bacteriophage genomics, there is a bewildering array of different genomes to characterize. Biochemical or genetic data are available for only a few prototypical phages. Most of the characterization of new phage genomes is derived by sequence comparison with other phages. This is complicated by the fact that phages distribute into a dozen or more groups with intergroup divergence of phage genes being greater than interkingdom divergence of cellular genes (10). Hence, phage-to-phage sequence comparison is often at or beyond the limit of the most advanced profile methods. Yet progress can still be made in modeling the biology of new viruses through observation of structural homology and seeking analogous features between new phage forms and established prototypes. The most abundant bacteriophages are the tailed phages, and the most divergent and challenging of them to characterize at the sequence level have been the...

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“…This assignment was aided by having an HHpred-style HMM library derived from previously compiled extensive alignments of homologs of proteins from the prototypical myovirus P2 (51). Searching an HMM constructed from OBP-Gp132 and its orthologs against all P2 HMMs produced a top scoring match to P2-gpQ, with an E value of 0.005.…”

Section: Figmentioning

confidence: 99%

Complete Genome Sequence of the Giant Virus OBP and Comparative Genome Analysis of the Diverse ϕKZ-Related Phages

Cornelissen

Hardies

Shaburova

et al. 2012

J Virol

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The 283,757-bp double-stranded DNA genome of Pseudomonas fluorescens phage OBP shares a general genomic organization with Pseudomonas aeruginosa phage EL. Comparison of this genomic organization, assembled in syntenic genomic blocks interspersed with hyperplastic regions of the KZ-related phages, supports the proposed division in the "EL-like viruses," and the "phiKZ-like viruses" within a larger subfamily. Identification of putative early transcription promoters scattered throughout the hyperplastic regions explains several features of the KZ-related genome organization (existence of genomic islands) and evolution (multi-inversion in hyperplastic regions). When hidden Markov modeling was used, typical conserved core genes could be identified, including the portal protein, the injection needle, and two polypeptides with respective similarity to the 3=-5= exonuclease domain and the polymerase domain of the T4 DNA polymerase. While the N-terminal domains of the tail fiber module and peptidoglycan-degrading proteins are conserved, the observation of C-terminal catalytic domains typical for the different genera supports the further subdivision of the KZ-related phages. P seudomonas fluorescens phage OBP (vB_PflM_OBP) (30, 47) is member of a growing group of giant phages, isolated, to date, only from Pseudomonas species and represented by the completely sequenced and well-studied Pseudomonas aeruginosa myovirus KZ (9,18,19,[31][32][33]36,42). Three other phages encoding many proteins with similarity to KZ proteins have been completely sequenced: EL (23), 2012-1 (52), and PA3 (43). Krylov et al. (33) assigned 19 unsequenced Pseudomonas phages to this group. We refer to the group as KZ-related phages. Lavigne et al. (35) have argued that EL should be classified as a genus separate from KZ and 2012-1 based on its less extensive levels of similarity. By that criterion, PA3 belongs to the "phiKZ-like viruses" and the results reported here classify OBP as the second member of the possible genus EL-like viruses.OBP shares a number of definitive properties with the other KZ-related phages. Typically, KZ-related phages have a very large icosahedral head, ϳ122 nm in diameter, and a long (ϳ190-nm) contractile tail surrounded by fibers. A KZ-related phage head contains a proteinic inner body, which has been speculated to organize the packaged DNA (18, 32). The exceptionally large genomes of the KZ-related phages (between 211 and 317 kb of nonredundant sequence) are composed of circularly permuted and terminally redundant linear double-stranded DNAs as determined for KZ and 2012-1 (42, 52). The KZ-related genomes display a pronounced difference in GϩC content (between 36.8 and 48%) and all have markedly lower GϩC content than the chromosomes of their GC-rich Pseudomonas hosts (60 to 66% GϩC).This group of phages has numerous genes involved in nucleotide metabolism (e.g., thymidylate synthase, thymidylate kinase, ribonucleoside diphosphate reductase, subunit beta [NrdB], and dihydrofolate reductase) and at least six genes enco...

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Complete genomic sequence and mass spectrometric analysis of highly diverse, atypical Bacillus thuringiensis phage 0305ϕ8–36

Cited by 64 publications

References 102 publications

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

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

Proteome of the Large Pseudomonas Myovirus 201φ2-1

Complete Genome Sequence of the Giant Virus OBP and Comparative Genome Analysis of the Diverse ϕKZ-Related Phages

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