Exploring the diversity and evolutionary strategies of prophages in Hyphomicrobiales, comparing animal-associated with non-animal-associated bacteria.

The Hyphomicrobiales bacterial order (previously Rhizobiales) exhibits a wide range of lifestyle characteristics, including free-living, plant-association, nitrogen-fixing, and association with animals (Bartonella and Brucella). This study explores the diversity and evolutionary strategies of bacteriophages within the Hyphomicrobiales order, comparing animal-associated (AAB) with non-animal-associated bacteria (NAAB). We curated 560 high-quality complete genomes of 58 genera from this order and used the PHASTER server for prophage annotation and classification. For 19 genera with representative genomes, we curated 96 genomes and used the Defense-Finder server to summarize the type of anti-phage systems (APS) found in this order. We analyzed the genetic repertoire and length distributions of prophages, estimating evolutionary rates and comparing intact, questionable, and incomplete prophages in both groups. Analyses of best-fit parameters and bootstrap sensitivity were used to understand the evolutionary processes driving prophage gene content. A total of 1860 prophages distributed in Hyphomicrobiales were found, 695 in AAB and 1165 in the NAAB genera. The results revealed a similar number of prophages per genome in AAB and NAAB and a similar length distribution, suggesting shared mechanisms of genetic acquisition of prophage genes. Changes in the frequency of specific gene classes were observed between incomplete and intact prophages, indicating preferential loss or enrichment in both groups. The analysis of best-fit parameters and bootstrap sensitivity tests indicated a higher selection coefficient, induction rate, and turnover in NAAB genomes. We found 68 types of APS in Hyphomicrobiales; restriction modification (RM) and abortive infection (Abi) were the most frequent APS found for all Hyphomicrobiales, and within the AAB group. This classification of APS showed that NAAB genomes have a greater diversity of defense systems compared to AAB, which could be related to the higher rates of prophage induction and turnover in the latter group. Our study provides insights into the distributions of both prophages and APS in Hyphomicrobiales genomes, demonstrating that NAAB carry more defense systems against phages, while AAB show increased prophage stability and an increased number of incomplete prophages. These results suggest a greater role for domesticated prophages within animal-associated bacteria in Hyphomicrobiales.

Host life-history traits influence the distribution of prophages and the genes they carry.

Bacterial strains with a short minimal doubling time-'fast-growing' hosts-are more likely to contain prophages than their slow-growing counterparts. Pathogenic bacterial species are likewise more likely to carry prophages. We develop a bioinformatics pipeline to examine the distribution of prophages in fast- and slow-growing lysogens, and pathogenic and non-pathogenic lysogens, analysing both prophage length and gene content for each class. By fitting these results to a mathematical model of the evolutionary forces acting on prophages, we predict whether the observed differences can be attributed to different rates of lysogeny among the host classes, or other evolutionary pressures. We also test for significant differences in gene content among prophages, identifying genes that are preferentially lost or maintained in each class. We find that fast-growing hosts and pathogens have a greater fraction of full-length prophages, and our analysis predicts that induction rates are significantly reduced in slow-growing hosts and non-pathogenic hosts. Consistent with previous results, we find that several proteins involved in the packaging of new phage particles and lysis are preferentially lost in cryptic prophages. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Evolutionary stability of the lysis-lysogeny decision: Why be virulent?

Lytic viruses infect and kill host cells, producing a large number of viral copies. Temperate viruses, in contrast, are able to integrate viral genetic material into the host cell DNA, leaving a viable host cell. The evolutionary advantage of this strategy, lysogeny, has been demonstrated in complex environments that include spatial structure, oscillating population dynamics, or periodic environmental collapse. Here, we examine the evolutionary stability of the lysis-lysogeny decision, that is, we predict the long-term outcome of the evolution of lysogeny rates. We demonstrate that viruses with high rates of lysogeny are stable against invasion by more virulent viral strains even in simple environments, as long as the pool of susceptible hosts is not unlimited. This mirrors well-known results in both r-K selection theory and virulence evolution: although virulent viruses have a faster potential growth rate, temperate strains are able to maintain positive growth on a lower density of the limiting resource, susceptible hosts. We then outline scenarios in which the rate of lysogeny is predicted to evolve either toward full lysogeny or full lysis. Finally, we demonstrate conditions under which intermediate rates of lysogeny, as observed in temperate viruses in nature, can be sustained long-term. In general, intermediate lysogeny rates persist when the coupling between susceptible host density and virus density is relaxed.

Prophage Provide a Safe Haven for Adaptive Exploration in Temperate Viruses.

Prophage sequences constitute a substantial fraction of the temperate virus gene pool. Although subject to mutational decay, prophage sequences can also be an important source of adaptive mutations for these viral populations. Here we develop a life-history model for temperate viruses, including both the virulent (lytic) and the temperate phases of the life cycle. We then examine the survival of mutations that increase fitness during the lytic phase (attachment rate, burst size), increase fitness in the temperate phase (increasing host survival), or affect transitions between the two phases (integration or induction probability). We find that beneficial mutations are much more likely to survive, ultimately, if they first occur in the prophage state. This conclusion applies even to traits that are only expressed during the lytic phase, and arises due to the substantially lower variance in the offspring distribution during the temperate cycle. This observation, however, is balanced by the fact that many more mutations can be generated during lytic replication. Overall we predict that the prophage state provides a refuge, relatively shielded from genetic drift, in which temperate viruses can explore possible adaptive steps.