Experimental bacteriophage addition to soil depresses bacterial respiration and alters nitrogen transformationsBenjamin D. Duval1*, Kurt E. Williamson2, Anika Baloun1, Linda C. DeVeaux11Biology Department, New Mexico Institute of Mining and Technology, Socorro, New Mexico, USA 878012Biology Department, The College of William and Mary, Williamsburg, Virginia, USA 23185*corresponding author:[email protected] ID: 0000-0001-7692-4400CRediT contributor roles:BDD: conceptualization, data curation, formal analysis, methodology, project administration, supervision, writing (draft), writing (editing)KEW: conceptualization, writing (draft), writing (review & editing)AB: investigation, writing (review & editing)LCD: methodology, resources, supervision, writing (review & editing)ABSTRACTSoil heterogeneity and complex host-phage relationships impede understanding phage influence on terrestrial nutrient cycles. We performed laboratory experiments quantifying phage effects on bacterial respiration and inorganic soil N and P changes. Soil microcosms under reducing conditions were inoculated with a single bacterial host (Gordonia rubripertincta ) or with the host + phage (φ Apollonia6). Respiration declined immediately following phage introduction, and was lower in the presence of phage through time. Phage additions increased variance in N transforms. NH4+ decline did not match NO3- gain, suggesting microbial immobilization in +phage microcosms. Net NO3- accumulation was observed with phage, suggesting viral interruption of bacterial NO3- reduction. Phosphate pools declined in both treatments, likely due to cell uptake and incorporation into phage. This single host-phage system highlights phage depression of bacterial respiration and altered nutrient transformation, and can be the basis for further investigations into phage-bacteria soil interactions and their impacts on terrestrial nutrient cycling.Keywords: Ammonium uptake, bacteriophage, biogeochemistry, Gordonia rubripertincta, nitrate reduction, respiration, soilINTRODUCTIONViruses that infect bacteria (bacteriophage or phage), are ubiquitous and numerous in soil systems, with abundances as high as reported for forest soils (1). There is a presumption of biogeochemical importance with such abundances, and phage are increasingly recognized as having a regulatory role within bacteria-driven portions of carbon (C), nitrogen (N) and phosphorus (P) cycles (2–4). However, there is tremendous uncertainty about the magnitude and direction of phage effects on most terrestrial biogeochemical processes, due to the physical and chemical complexity of soils, and the numerous types of potential phage-bacteria interactions in soil (5).Kuzyakov and Mason-Jones’ (2018) review of phage influence on soil processes arrives at two important conclusions: 1) soil bacterial mortality is principally caused by phage, and 2) nucleotide metabolism involved in phage genome replication creates high N and P demand which alters the stoichiometry of necromass relative to living biomass. Thus, phage-induced mortality is expected to release qualitatively different resources into the soil system than other mechanisms of bacterial mortality, e.g., protozoan grazing or from soil perturbations such as freeze-thaw cycles (7). However, variation in phage infection outcomes (e.g., lysis, lysogeny or chronic infection), from a population level, may also produce very different biogeochemical results with respect to both bacterial mortality and nutrient demands.Phage-induced mortality could simply lower host bacterial population activity and the rate and net products of any processes performed by that population (i.e., microbial N immobilization or NO3- reduction). This could be characterized as “kill the host, kill the process”. However, bacterial mortality also liberates cell components that are subsequently recycled as C and N sources promoting growth and activity in living microbial biomass (i.e., “eat the dead, change the product”). These possibilities, coupled with observations that phage lysate typically has higher C:N and lower phosphorus (P) than living biomass, suggest that phage mortality creates an intermediate biogeochemical scenario between process interruption from host death and process enhancement from necromass nutrient fertilization (6–8). Furthermore, a significant fraction of soil phages at any given time are likely lysogenic, and infect bacteria without immediately causing cell death via lysis (9,10). Physiological stress may skew temperate phages toward lysogenic replication, in which the phage genome takes up stable residence inside of its host cell instead of generating progeny particles and inducing cell lysis (11), and we expect those lysogenized cells to continue elemental uptake and transformation. It is unclear if element processing rates would be negatively impacted by the presence of a prophage, but prophage could slow bacterial element processing by reducing bacterial metabolism, and lysogeny prevalence can correlate with sub-optimal host conditions (12,13).Given the range of potential phage impacts on bacterial functional roles, microcosm experiments are well-suited to answer questions related to mortality effects and quantifying elemental flux changes as a result of single host-phage interactions in soil. To do so, we selected a heterotrophic (organic C processing) host bacterial strainGordonia rubritincta, that has known roles in organic compound degradation and synthesis (14,15), NO3- reduction, and has substantial P demand (16). Our group has recently isolated several phages from natural and anthropogenically influenced soils that infect Gordonia (17). From that group, we selected the phage φApollonia6 for its consistent infection rate in our lab trials (Duval and DeVeaux, pre-experimental observations). The influence of this phage on Gordonia functional ecology was quantified for two key ecosystem processes, organic C mineralization/respiration and inorganic nutrient (N and P) transformation. Microcosms were designed to promote NO3- reduction by this bacterial strain (high H2O/low O2) during a medium duration (~1 month) incubation experiment.We predict that sterile soils inoculated with Gordonia would have relatively high rates of CO2 respiration compared to soils that also had phage additions; these soils are also predicted to yield lower final pools of NO3- due to denitrification. Phage additions will result in lower CO2 fluxes due to cell mortality, and as a result, NO3- pools increase due to denitrification interruption due to phage. Because virion production is implicitly P limited, we expect greater reductions of inorganic P pools (as a result of P uptake and synthesis into organic structures) in soils with phage addition (16). Alternatively, a dip in respiration from mortality may be followed by a pulse of activity from cells utilizing lysis products as energy sources and possibly nutrients (7). In that instance, we expect higher rates of CO2 emissions and lower NO3- pools with phage addition compared to bacteria-only soils. Regardless of outcome, this experiment contributes to the growing literature of phage-influenced soil N and P transformations.METHODS