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. 2012 Oct 10:13:542.
doi: 10.1186/1471-2164-13-542.

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

Jason J Gill  1 , Joel D Berry, William K Russell, Lauren Lessor, Diego A Escobar-Garcia, Daniel Hernandez, Ashley Kane, Jennifer Keene, Matthew Maddox, Rebecca Martin, Sheba Mohan, Ashlyn M Thorn, David H Russell, Ry Young
Affiliations

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

Jason J Gill et al. BMC Genomics. .

Abstract

Background: The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives.

Results: Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell's replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level.

Conclusions: Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus.

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Figures

Figure 1
Figure 1
Negative-stain transmission electron micrographs of the C. crescentus phage phiCbK and five phiCbK-like phages. All five exhibit Siphoviridae morphology and prolate heads. Scale bars are 100 nm.
Figure 2
Figure 2
Genomic map of C. crescentus phage phiCbK. Predicted genes are represented by boxes above and below the black line; boxes above the line are genes encoded on the forward strand, those below the line are on the reverse strand. Segments of heavier black line at each end of the genome represent the 10.3 kb terminal repeats present in the genome. Gene features (conserved, unique, hypothetical novel and virion-associated proteins; tRNA genes) and genome modules (assembly, lysis and DNA replication) are color-coded according to the legend below the figure. Selected genes and gene modules are annotated based on predicted function, as documented in Table S1 and the text. The ruler below the genomes indicates scale in kb.
Figure 3
Figure 3
Genomic map of C. crescentus phage Colossus. Predicted genes are represented by boxes above and below the black line; boxes above the line are genes encoded on the forward strand, those below the line are on the reverse strand. Gene features (conserved, unique, hypothetical novel and virion-associated proteins; tRNA genes) and genome modules (assembly, lysis and DNA replication) are color-coded according to the legend below the figure. Selected genes and gene modules are annotated based on predicted function, as documented in Table S6 and the text. The ruler below the genomes indicates scale in kb.
Figure 4
Figure 4
DNA sequence relatedness of six phiCbK-like phages. Upper section: pairwise percent DNA sequence identities between all six phages, as determined by BlastN analysis [37] followed by multiplication of the mean percent identity of matched segments by the percent length of the genomes matched. Lower section: dotplots visually representing DNA sequence homology between phages. For clarity, terminal repeat regions were removed from the DNA sequences prior to analysis.
Figure 5
Figure 5
Non-proportional synteny map showing the relationships between related C. crescentus phages at the protein level. The black blocks represent protein-coding genes in the order they appear in each phage genome, starting with gp1 at the top and running clockwise, with red tick marks indicating 10-gene intervals in phage phiCbK. Black lines connecting blocks indicate similarity of proteins between phages. From innermost to outermost, tracks represent phages phiCbK, Karma, Magneto, Swift and Rogue. Blue, green and purple arcs on the inside track indicate the boundaries of the phage morphogenesis, lysis and DNA replication modules, respectively. Terminal repeat regions were excluded from this figure for clarity.
Figure 6
Figure 6
Map showing the relationship between C. crescentus phages phiCbK and Colossus at the protein level. Both genomes start at position 0 at the top of the figure and move down, with Colossus in orange on the left and phiCbK in blue on the right. Black bands in each track denote the boundaries of protein-coding genes, grey areas represent non-coding regions. Grey tick marks on the outside edge represent 1 kb of sequence, with heavier ticks representing 10 kb. Proteins present in both phages are connected by red ribbons between the two genomes; green ribbons mark proteins with more than one homolog in the other phage, suggesting gene duplications. For clarity, terminal repeat regions were excluded from this figure.
Figure 7
Figure 7
The left and right genomic terminal repeat boundaries of phage phiCbK and four phiCbK-like phages. Terminal boundaries are indicated by the vertical red lines. Above: aligned DNA sequences 12 bp up- and downstream of each terminus are shown; alignments show that the experimentally confirmed boundary sequences of phiCbK are nearly identical to those found in the other four close phiCbK-like relatives. Below: average fold coverage at each base position for all five genomic sequences; note the coverage within the terminal repeats is approximately twofold greater than the surrounding genome, and the breakpoints are identical.
Figure 8
Figure 8
Coomassie-stained SDS-PAGE gel of purified phiCbK virions. The entire gel lane shown was segmented and subjected to proteomic analysis; band identities and predicted functions are annotated on the right side of the figure. While the entire gel lane was analyzed, only bands that returned conclusive peptide matches are annotated.
Figure 9
Figure 9
Schematic representations of the lysis genes and proteins of C. crescentus phages. Lysis components of phages phiCbK and Colossus are compared to those of other phages. A: Overall organization of the lysis cassettes of phages lambda, P2, phiCbK and Colossus; genes are represented by colored boxes, with gene functions labeled above each module and gene names below. Color indicates conserved function, not sequence similarity. B: Protein sequence alignment of the N-termini of the phages P1, 21, phiCbK and Colossus endolysins; the positions of equivalent E-D/C-T catalytic residues are highlighted in red, green and blue, transmembrane SAR domains are highlighted in grey. Identical residues in phiCbK gp104 and P1 Lyz are indicated by vertical bars, illustrating their relationship. C: The mature spanin proteins of phages lambda, P2 and phiCbK. Transmembrane domains are purple, predicted alpha-helical domains are blue, unstructured domains are black curves, and the positions of proline residues are represented by red dots. The spanin IM components are anchored to the inner membrane by a transmembrane helix, OM components are tethered to the outer membrane by a lipoylated (red bars) Cys residue (green dot). The P2 complex contains alpha-helical IM and OM components with proline-rich domains, and the phiCbK spanin complex more closely resembles that of P2. D: Possible membrane topologies of the phiCbK-like holin proteins. Left, three TMDs with N-in, C-out topology; right, two TMDs — including a 36-residue TMD2 — with N-in, C-in topology. Charge distributions of the protein and TMD length strongly favor the three TMD model.
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