Bacterial 'Grounded' Prophages: Hotspots for Genetic Renovation and Innovation

Abstract

Bacterial genomes are highly plastic allowing the generation of variants through mutations and acquisition of genetic information. The fittest variants are then selected by the econiche thereby allowing the bacterial adaptation and colonization of the habitat. Larger genomes, however, may impose metabolic burden and hence bacterial genomes are optimized by the loss of frivolous genetic information. The activity of temperate bacteriophages has acute consequences on the bacterial population as well as the bacterial genome through lytic and lysogenic cycles. Lysogeny is a selective advantage as the prophage provides immunity to the lysogen against secondary phage attack. Since the non-lysogens are eliminated by the lytic phages, lysogens multiply and colonize the habitat. Nevertheless, all lysogens have an imminent risk of lytic cycle activation and cell lysis. However, a mutation in the attachment sites or in the genes that encode the specific recombinase responsible for prophage excision could result in 'grounding' of the prophage. Since the lysogens with grounded prophage are immune to respective phage infection as well as dodge the induction of lytic cycle, we hypothesize that the selection of these mutant lysogens is favored relative to their normal lysogenic counterparts. These grounded prophages offer several advantages to the bacterial genome evolution through propensity for genetic variations including inversions, deletions, and insertions via horizontal gene transfer. We propose that the grounded prophages expedite bacterial genome evolution by acting as 'genetic buffer zones' thereby increasing the frequency as well as the diversity of variations on which natural selection favors the beneficial variants. The grounded prophages are also hotspots for horizontal gene transfer wherein several ecologically significant genes such as those involved in stress tolerance, antimicrobial resistance, and novel metabolic pathways, are integrated. Moreover, the high frequency of genetic changes within prophages also allows proportionate probability for the de novo genesis of genetic information. Through sequence analyses of well-characterized E. coli prophages we exemplify various roles of grounded prophages in E. coli ecology and evolution. Therefore, the temperate prophages are one of the most significant drivers of bacterial genome evolution and sites of biogenesis of genetic information.

Keywords: bacterial ecology; bacteriophage; genome evolution; genome plasticity; horizontal gene transfer.

FIGURE 1

Bacterial genome evolution. Based on the habitat, bacterial genomes acquire DNA (genetic information) from the environment. Genetic information from the phages, plasmids, and dead organisms maybe integrated into the genome by homologous or non-homologous recombination.

FIGURE 2

Mechanisms of bacteriophage genome integration into bacterial chromosome. (A) Site-specific recombination mediated-lysogenization is well characterized for lambdoid phages. Site-specific recombination is catalyzed by the integrase through phage and bacterial attachment sites (attP and attB, respectively) resulting in recombinant attL and attR sites (Landy and Ross, 1977; Menouni et al., 2015). Prophage induction is mediated by excisase and integrase complex. (B) Transposition based integration of Mu phage into the target site in the host genome requires MuA transposase. The integration results in target site duplication (TSD) (Allet, 1979; Harshey, 2014).

FIGURE 3

A hypothetical model to explain the prevalence of cryptic prophages in bacterial genomes. A typical temperate phage injects its genome in the bacterial cells that may undergo either lytic cycle or lysogeny. In the lytic cycle, more copies of phages are produced, the host bacterium is lysed, and a multitude of new phages are released into the surrounding. The lytic cycle repeats with each bacterium, which almost annihilates the bacterial population. In some bacteria, the injected genome undergoes lysogeny by integrating into the bacterial genome. Lysogeny allows vertical propagation of prophage along with that of the bacterial genome. By means of the prophage, the lysogens attain immunity to the infecting secondary phages thereby preventing infection/lysis. Apart from other mutants, the lysogens are the only surviving kin and hence will multiply well due to lack of intraspecies competition. Lysogeny, upon reversal, will ultimately lead to a lytic cycle leading to the death of the host bacterium. In some of the prophages, mutations in the recombinase recognition sites or recombinase genes occur, which prevent the elicitation of the lytic cycle: a phenomenon referred to as ‘grounding of prophages.’ The bacteria with grounded prophages have two advantages; immunity from attack by related secondary phages and irreversible lysogeny that prevents phage-mediated cell death. Eventually, other lysogens undergo lytic cycle upon induction by various stimuli resulting in lysis of the host bacterium. The bacterium with cryptic prophages will survive and have a growth advantage due to reduced competitive pressure from kin.

FIGURE 4

Genetic variations caused by prophage integration in E. coli MG1655. The examination of specific integration loci of each of the five prophages (DLP12, e14, Rac, CPZ-55, and Qin) concerning E. coli MG1655 among the strains with and without corresponding prophages uncovered the genetic variations caused post integration event as shown above. (A) Integration of DLP12 via homologous recombination near the 3′ end of the argU gene. The 47 bp region similar to 3′ end of argU gene is located on the other end of the DLP12. (B) Integration of e14 at the 3′ end of icd gene causing the formation of pseudo icd’ (163 bp) gene as a duplicated region. (C) Site-specific integration of Rac at the 5′ end of ttcA gene. The variation in the N-terminus of TtcA protein was observed. (D) The transposon-like integration of CPZ-55 next to the stop codon of the eutA gene causing duplication of 8 bp region. No variations were seen in the bacterial flanking genes (eutB and eutA). (E) The site-specific integration of Qin at the 5′ end of ydfJ gene resulting in promoter loss and truncation (lacking 28 codons at the N terminal) of YdfJ protein.

FIGURE 5

Locus-specific distribution of the five prophages (with reference to E. coli MG1655 strain) in 638 completely sequenced E. coli strains. The cryptic prophages (DLP12, e14, Rac, CPZ-55, and Qin) of MG1655 strain were analyzed for their distribution within 638 completely sequenced E. coli strains taking the bacterial and prophage borders sequences as a query in BLASTn. Only cryptic prophages of MG1655 strain with location specificity were analyzed. Other strains may have various other prophages at multiple locations. (A) The prevalence of each cryptic prophage within various Escherichia and Shigella strains. The distribution was determined by curating the BLAST hits obtained in completely sequenced strains showing >85% sequence identity. (B) The prevalence of various permutations of prophage co-occurrence within various Escherichia and Shigella strains. (C) The diversity in the locus-specific prophage distribution across Escherichia and Shigella species. Only representative strains are indicated for illustration purposes.

FIGURE 6

The genetic diversity of Qin prophage in various E. coli strains. Correlation between the prophage size and the number of genes carried within the prophage is studied using BLAST tool and curated in excel. For each strain, the length of site- specific Qin prophage and the number of prophage encoding proteins were obtained using BLASTn tool taking gene sequence of each gene as query and hits are curated using excel. The dependence of prophage size and the flux of genes are represented in a scatter plot as shown taking prophage size on primary Y-axis, the number of genes on the prophage as secondary Y-axis and 189 strains on the X-axis. For Qin prophage analyses, the prophage coordinates were used to estimate the prophage size. Having E. coli MG1655 as the reference strain, the number of genes carried on Qin prophage among 189 strains was noted. The number of DUFs (Domain of Unknown Function) and hypothetical proteins in Qin prophage of each strain was fetched using a JavaScript to search the GenBank files of each prophage for “DUF” and “hypothetical proteins.” The graph was plotted to present the diversity of prophage in terms of its size, the number of genes carried, and novel genes in each of the strains.

FIGURE 7

A genetic history and advantages of cryptic prophages. The genome of temperate phages integrates with the bacterial chromosome through site-specific recombination between sites similar to those of attP and attB. Occasionally, mutations in the attL, attR, recombinase genes, and genes encoding other essential accessory factors would result in a ‘grounded’ prophage. Eventually but gradually, more mutations, usually deletions, accumulate to ease the metabolic and genetic burden imposed by the cryptic prophage. The cryptic prophage offers several advantages to the host bacterium. The genetic variations caused in the host genome due to integrations, inversions involving prophage sequences, and the action of their recombinases may have a selective advantage. The prophage confers immunity from secondary phage infection and acts as hotspots for gene influx through horizontal gene transfer of important genes such as those involved in metabolism, antibiotic resistance, virulence, and stress tolerance. More importantly, the cryptic prophage is a site for the emergence of new ORFs that may have novel and useful functions.