Induction of multiple prophages from a marine bacterium: a genomic approach

Abstract

Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

FIG. 1.

Effect of mitomycin C treatment on growth of Silicibacter sp. strain TM1040. Cell densities were determined by measuring the optical densities at 600 nm in a culture treated with 0.5 μg/ml mitomycin C for 30 min and in a control culture without treatment.

FIG. 2.

Viral particle yield following mitomycin C induction of Silicibacter sp. strain TM1040. Microscopic counts of TM1040 cells and viral-like particles were done with (A) a mitomycin C-treated culture and (B) a control culture without mitomycin C.

FIG. 3.

TEM images of siphoviruses found in the lysate obtained from Silicibacter sp. strain TM1040 induced with 0.5 μg/ml mitomycin C. A P1-like siphovirus dominated the viral lysate (panels A and B), while other similar siphoviruses, such as P2 (panel C) and P3 (panel D), were found in the lysate at a low frequency.

FIG. 4.

PFGE analysis of viral genomic DNAs isolated from the induced viral lysate. From left to right, the lanes contain the following: 1, DNA obtained from the induced phage particles; 2, phage DNA with DNase treatment; 3, phage DNA with RNase treatment; 4, phage DNA with S1 nuclease treatment; and 5, size markers (New England Biolabs).

FIG. 5.

PCR detection of the five prophages. Silicibacter sp. strain TM1040 genomic DNA and DNAs obtained from the induced viral lysate were each used as templates for PCR amplification using five primer sets specific for the respective prophages (Table 1). Lanes 1 to 5 show the PCR products of prophages 1 to 5, respectively, amplified from the TM1040 genomic DNA, while lanes 10 to 14 show the results of PCR for prophages 1 to 5, respectively, amplified from CsCl-purified viral lysate. Positive PCR control reactions for host DNA contained Silicibacter sp. strain TM1040 genomic DNA as a template and PCR primer sets designed to amplify virD4 (lane 6) and the 16S rRNA gene (lane 7), respectively. Control reactions to ensure that viral lysates were free of host genomic DNA contamination contained viral lysate DNA as templates and either virD4 (lane 15) or 16S rRNA gene (lane 16) primers. Lanes 8 and 17 show the results of multiplex PCRs using all five prophage primer sets with host DNA and viral lysate, respectively. Lane 9 contains DNA size markers (2,178, 1,766, 1,230, 1,033, 653, 517, 453, 394, 298, 234/220, and 154 bp, from top to bottom of gel).

FIG. 6.

Maps of the five Silicibacter sp. strain TM1040 prophage genomes. The relative sizes and directions of transcription of the individual ORFs in each prophage genome are indicated by the arrows. The following colors are used to represent the potential functions of ORFs, as defined by BLASTP homology E values of e⁻³⁵ or less: blue, integrase; orange, DNA metabolism; green, regulation; red, terminase; yellow, lytic functions; and purple, structural components (head, tail, tail fibers, etc.). ORFs with E values of >e⁻³⁵ with homology to known phage genes in the database are indicated by gray arrows. White arrows indicate ORFs encoding hypothetical or conserved proteins without known functions. Vertical bars within the genome map symbolize small ORFs of unknown function. The genetic neighborhood surrounding each prophage genome contains the following flanking ORFs. The left side of prophage 1 is flanked by a hypothetical ORF separated from the prophage genome by 1.48 kb. The right side is flanked by three ORFs associated with purine metabolism (TPR protein gene, putR, and putA). The left side of prophage 2 is flanked by a hypothetical zinc-binding protein gene, while the right side is flanked by an ORF coding for a hypothetical protein. Prophage 3 is flanked on its left by an operon carrying genes involved in branched-chain amino acid ABC transport, and its right side lies adjacent to an ORF encoding 4-hydroxybenzoyl-coenzyme A thioesterase. Prophage 4 is flanked by a lon ortholog on its left side and an ortholog of the transcriptional regulatory protein gene tcsR on its right. A conserved hypothetical protein gene flanks the left side of prophage 5, while an ortholog of the gene for cold shock-like protein (CspE) flanks its right side. The prophage genomic maps were generated from the genomic sequences by using Vector NTI (Invitrogen) and Adobe Illustrator software.