Novel Assay To Assess Permissiveness of a Soil Microbial Community toward Receipt of Mobile Genetic Elements

Abstract

There is a wealth of evidence indicating that mobile genetic elements can spread in natural microbial communities. However, little is known regarding the fraction of the community that actually engages in this behavior. Here we report on a new approach to quantify the fraction of a bacterial community that is able to receive and maintain an exogenous conjugal plasmid termed community permissiveness. Conjugal transfer of a broad-host-range plasmid labeled with a zygotically inducible green fluorescent protein (RP4::gfp) from a donor strain (Pseudomonas putida) to a soil bacterial suspension was examined. The mixture of cells was incubated on membrane filters supported by different solid media. Plasmid transfer was scored by in situ visualization of green fluorescent transconjugant microcolonies, and host range was determined by traditional plating or microcolony isolation by using a micromanipulator. Among the conditions tested, the highest plasmid transfer incidence (approximately 1 transfer per 10 4 soil bacteria) was measured after 48 h of incubation on either a 10% soil extract or a 10-fold diluted R2A medium. Stereomicroscopy combined with image analysis allowed easy examination and enumeration of green fluorescent microcolonies. In all experiments, however, stereomicroscopy consistently underestimated the number of conjugation events (approximately 10-fold) in comparison to confocal laser scanning microscopy. The plasmid host range was broad and included bacteria belonging to the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria classes of proteobacteria. The isolation of transconjugant microcolonies by micromanipulation greatly extended the estimated plasmid host range among soil bacteria. The new approach can be applied to examine the permissiveness of various communities toward receipt of different mobile elements.

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

“…Unlike in Musovic et al (2010), we did not use additional nutrient additions, but only relied on soil-extracted nutrients to support activity during the mating incubations. Successful conjugation was checked after 48 h by epifluorescence stereomicroscopy and confocal laser scanning microscopy ( Figure 1) (Musovic et al, 2010).…”

“…Plasmids RP4 and pIPO2tet have been constructed earlier (Musovic et al, 2010(Musovic et al, , 2014. The 54 kbp IncP-1 Figure 1 Typical transconjugal microcolonies for plasmid pKJK5::gfp introduced through E. coli MG1655::lacI q -pLppmCherry-Km R .…”

“…As a growth medium, we used a 10% soil extract medium as described by Musovic et al (2010) buffered at pH 7.2 with 5 mM 3-(N-morpholino)propanesulfonic acid and supplemented with 20 mg ml À 1 nystatin to avoid fungal growth. Unlike in Musovic et al (2010), we did not use additional nutrient additions, but only relied on soil-extracted nutrients to support activity during the mating incubations. Successful conjugation was checked after 48 h by epifluorescence stereomicroscopy and confocal laser scanning microscopy ( Figure 1) (Musovic et al, 2010).…”

“…Solid surface filter mating assay The extracted recipient community was challenged with exogenous plasmids via solid surface filter matings (Musovic et al, 2010) modified to an initial ratio of donor-to-recipient bacteria of 1:1 at a density of B30 000 bacteria mm À 2 on the filter. As a growth medium, we used a 10% soil extract medium as described by Musovic et al (2010) buffered at pH 7.2 with 5 mM 3-(N-morpholino)propanesulfonic acid and supplemented with 20 mg ml À 1 nystatin to avoid fungal growth.…”

“…The resulting soil volume was sieved and homogenized to obtain a representative sample. From a total of 30 g of the homogenized chosen soils, indigenous bacterial communities were isolated by Nycodenz extraction (Musovic et al, 2010) and used as recipients in the mating assay. Donor strains were grown overnight in LB medium supplemented with the plasmid-specific antibiotics (Table 1) and were harvested by centrifugation.…”