Exchange of a Conjugative Plasmid At Different Soil Moisture Levels Between Streptomyces Species Colonizing Artificial Soil Aggregates

摘要

Spores of a Streptomyces lividans strain (donor) bearing the recombinant conjugative plasmid pIJ303, which codes for thiostrepton resistance, and spores of a plasmid-free Streptomyces parvulus strain (recipient) were added together to finely-sieved, sterile silt loam soil. This bulk soil was adjusted to either 20%, 35% or 45% of the soil’s water holding capacity. To this inoculated bulk soil were added sterile, nutrient- amended, artificial soil aggregates. After incubation to allow streptomycete growth and aggregate colonization, both the sieved bulk soil and the aggregates were assayed for numbers of transconjugants by spread- planting on a thiostrepton – agar selective medium. This allowed estimation of parental ability to colonize nutrient – rich soil sites at different soil moisture levels, and comparison of plasmid exchange frequency in the nonamended bulk soil versus the nutrient – rich soil aggregates. Plasmid exchange was detected only on aggregates of about 20% water holding capacity or less (4.2% wt/wt H20). The soil moisture threshold for heterotrophic streptomycete activity appeared to lie between 2.87% and 4.2% wt/wt H20 for the nutrient- amended soil aggregates. Introduction The ability to colonize and occupy solid substrates in soil, such as plant or animal remains, is vital to the growth and existence of soil microorganisms (Hissett and Gray, 1976). Genetically-engineered microorganisms (GEMs) which are released into the soil environment have to compete with native soil microorganisms during colonization and occupation of soil microhabitats. The outcome of such competitive colonization helps determine whether a GEM persists in the environment or dies off, influencing whether the GEM is able to manifest effects in the soil environment, or is able to transfer its DNA to native soil microorganisms (Stotzky and Babich, 1986). Gene transfer events between soil prokaryotes have effects on phenomena including microbial evolution in general, and more specifically the spread of antibiotic resistance genes to microorganisms affecting human and animal health (reviewed by Thomas and Nielsen, 2005). Conjugation is one of the major types of gene transfer events, and is known to occur between many types of prokaryotes, including Streptomyces (reviewed by Marsh and Wellington, 1994; and Strap and Crawford, 2007).The filamentous growth habit of some microorganisms (such as Streptomyces spp.) allows them to bridge air-filled pores in dry soil and to penetrate solid substrates (Griffin, 1981). Studies have been conducted on colonization of plant materials (Keinath and Loria, 1989; Lewis, 1970; Mayfield et al., 1972; Watson and Williams, 1974) and of autoclaved soil or of nutrients in amended soil (Bleakley and Crawford, 1989; Lloyd, 1969; Mayfield et al., 1972) by streptomycetes. Conjugation-mediated plasmid exchange has been observed between streptomycetes as they colonize autoclaved soil ( Bleakley and Crawford, 1989; Rafii and Crawford, 1988; and reviewed by Strap and Crawford, 2007). Plasmid transfer frequency between certain streptomycete strains has been found to be highest when nutrients are added to relatively dry soil (Bleakley and Crawford, 1989). The nutrients in that previous study were mixed throughout the soil matrix, probably affording multiple nutritional “hot-spots” at which streptomycete hyphae met, fused and participated in conjugation-mediated plasmid exchange.The purpose of the present study was to see if larger, macroscopic “hot-spots” in the form of artificially molded, nutrient-amended soil aggregates would result in the same pattern of plasmid exchange as previously observed between a Streptomyces lividans donor and a Streptomyces parvulus recipient. The hypothesis was that plasmid exchange would be most frequent between hyphae on nutrient-amended, relatively dry soil aggregates. Heterotrophic microbial activity in bulk soil and aggregates was assayed using a fluorescein diacetate (FDA) hydrolysis assay,