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. 2022 Apr 27;7(2):e0093021.
doi: 10.1128/msphere.00930-21. Epub 2022 Mar 21.

Plasmid-Mediated Stabilization of Prophages

Matthew J Tuttle  1 , Frank S May  1 , Jonelle T R Basso  1 , Eric R Gann  1 , Julie Xu  2 , Alison Buchan  1
Affiliations

Plasmid-Mediated Stabilization of Prophages

Matthew J Tuttle et al. mSphere. .

Abstract

Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In natural systems, bacteria simultaneously possess or encounter multiple MGEs, yet their combined influences on microbial communities are poorly understood. Here, we investigate interactions among MGEs in the marine bacterium Sulfitobacter pontiacus. Two related strains, CB-D and CB-A, each harbor a single prophage. These prophages share high sequence identity with one another and an integration site within the host genome, yet these strains exhibit differences in "spontaneous" prophage induction (SPI) and consequent fitness. To better understand mechanisms underlying variation in SPI between these lysogens, we closed their genomes, which revealed that in addition to harboring different prophage genotypes, CB-A lacks two of the four large, low-copy-number plasmids possessed by CB-D. To assess the relative roles of plasmid content versus prophage genotype on host physiology, a panel of derivative strains varying in MGE content were generated. Characterization of these derivatives revealed a robust link between plasmid content and SPI, regardless of prophage genotype. Strains possessing all four plasmids had undetectable phage in cell-free lysates, while strains lacking either one plasmid (pSpoCB-1) or a combination of two plasmids (pSpoCB-2 and pSpoCB-4) produced high (>105 PFU/mL) phage titers. Homologous plasmid sequences were identified in related bacteria, and plasmid and phage genes were found to be widespread in Tara Oceans metagenomic data sets. This suggests that plasmid-dependent stabilization of prophages may be commonplace throughout the oceans. IMPORTANCE The consequences of prophage induction on the physiology of microbial populations are varied and include enhanced biofilm formation, conferral of virulence, and increased opportunity for horizontal gene transfer. These traits lead to competitive advantages for lysogenized bacteria and influence bacterial lifestyles in a variety of niches. However, biological controls of "spontaneous" prophage induction, the initiation of phage replication and phage-mediated cell lysis without an overt stressor, are not well understood. In this study, we observed a novel interaction between plasmids and prophages in the marine bacterium Sulfitobacter pontiacus. We found that loss of one or more distinct plasmids-which we show carry genes ubiquitous in the world's oceans-resulted in a marked increase in prophage induction within lysogenized strains. These results demonstrate cross talk between different mobile genetic elements and have implications for our understanding of the lysogenic-lytic switches of prophages found not only in marine environments, but throughout all ecosystems.

Keywords: lysogenic-lytic switch; marine; mobile genetic elements; plasmids; spontaneous prophage induction; temperate phages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Genomic composition of Sulfitobacter pontiacus CB-D. (A) Circular maps of chromosome and plasmids. Regions of interest are highlighted in yellow. From the outside inward are the following tracks: (1) genomic location in kb, (2) forward strand genes (colored according to broad gene functional categories based on KEGG Orthology), (3) circular chromosome or plasmid DNA (dark gray), (4) reverse-strand genes (same colors as forward-strand genes), (5) GC content (light gray), and (6) GC skew (blue and orange). (B) Percentage of genes on each genomic element belonging to broad gene functional categories based on KEGG Orthology.
FIG 2
FIG 2
Organization of CB-D plasmid replication modules. (A) Plasmid replicon regions with arrows indicating genes involved in plasmid replication and partitioning. Vertical black lines indicate locations of palindromic incompatibility regions. (B) Identified palindromic sequences unique to each plasmid. (C) Plasmid compatibility group based on genetic organization and palindromic sequences identified (compared to those previously described; 25, 69).
FIG 3
FIG 3
Growth dynamics of parental and derivative S. pontiacus strains. (A and B) Prophage-A-lysogenized derivative strains (A) and prophage-D-lysogenized derivative strains (B) compared to parental strains CB-D (blue) and CB-A (red) in liquid culture. Points denote the mean of biological triplicates, and error bars indicate the standard deviation from the mean at each time point.
FIG 4
FIG 4
Relative biofilm formation of parental and derivative S. pontiacus strains. (A and B) Crystal violet biofilm assays of prophage-A-lysogenized derivative strains (A) and prophage-D-lysogenized derivative strains (B) compared to parental strains CB-D (blue) and CB-A (red). Plots depict the median (bold line), 25th and 75th percentiles (box), 1.5 times the interquartile ranges (whiskers), and outliers (black dots) with all replicates overlaid (transparent circles). For each panel, pairwise Wilcoxon tests were used to determine significant differences between derivatives and their parental strain (CB-D for panel A; CB-A for panel B). Significant differences are denoted by asterisks (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).
FIG 5
FIG 5
S. pontiacus strains lacking plasmids have high free-phage titers in the absence of induction. (A) Phage dilution assay showing titers of phage from cultures after 24 h growth in SMM broth without exogenous induction. Phage serial dilutions (10 μL) were inoculated onto host organisms susceptible to lysis by the respective phage types (CB-A for φ-D and CB-D for φ-A). Controls represent phage-free medium inoculated onto hosts (left, CB-D; right, CB-A). See Fig. S4E for images of all replicates. (B) Presence of plasmids pSpoCB-1 through pSpoCB-4 within strains. Plasmid presence was determined via amplification of genomic DNA from strains using at least three primer sets unique to each plasmid.
FIG 6
FIG 6
Plasmids appear stable after multiple passages on agar medium, except for pSpoCB-1. (A) Diagram of serial passaging and screening of colonies. (B) The parental strains CB-D (top) and CB-A (bottom) were serially passaged in triplicate (Rep 1 to 3) on agar plates for a total of 20 passages. Every 5 passages, the presence of individual plasmids was assessed via PCR amplification of genomic DNA with at least two primer sets that amplify unique regions of their respective plasmid. (C) Fold change of pSpoCB-1 abundance with serial passaging on agar as determined by qPCR. Data were normalized to zero passages on agar and represent mean values of pSpoCB-1-specific qPCRs relative to single-copy chromosomal gene qPCRs (map and alaS). ND (no detection), indicating samples with plasmid copies below the assay limit of detection. Significant differences (Student’s t tests) in ΔCT values compared to zero passages for each replicate are denoted by asterisks (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Phage dilution assay showing titers of free phage from cultures after 24 h growth in broth culture in the absence of induction. Phage dilutions were inoculated onto host organisms susceptible to lysis by the respective phage types (CB-A for φ-D and CB-D for φ-A). For each strain, biological triplicates (columns) and technical triplicates (rows) are shown. Controls represent phage-free medium inoculated onto hosts (left, CB-D; right, CB-A).
FIG 7
FIG 7
pSpoCB-1 and pSpoCB-2 share a high degree of synteny with other sequenced Sulfitobacter plasmids. (A) pSpoCB-1 and (B) pSpoCB-2 compared to other sequenced roseobacter plasmids. Blue arrows represent ORFs for individual plasmids. Gray bars indicate long-range homology between plasmids with ≥75% nucleotide sequence similarity. Plasmid sequences were downloaded from NCBI. The accession numbers for panel A are CP072614 (CB-D), CP049345 (S1704), JACIFR010000004 (N5S), CP025810 (SK025). The accession numbers for panel B are CP025811 (SK025), CP072615 (CB-D), CP018078 (AM1-D1), CP040823 (D4M1).
FIG 8
FIG 8
Biogeographical distribution and relative abundance of host and phage genes in the oceans. Each column represents a single gene located on either a plasmid or the chromosome or within phage genomes. Each row represents a different sampling depth from the Tara Oceans Expedition (SRF, surface ocean; DCM, deep chlorophyll maximum; MES, mesopelagic; MIX, marine epipelagic mixed layer; ZZZ, marine water layer). All Tara Oceans sampling stations and depths are indicated with an X. Blue circles represent the abundance of BLAST hits of genes to the OM-RGCv2 database with a cutoff threshold of 10−10. Abundance is normalized as a percentage of the total reads within that sample. Maps are omitted for depths at which no BLAST hits to genes were detected (i.e., for the φ-A major capsid protein). Individual maps were generated by the Ocean Gene Atlas online server (77).
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