Genetic cargo and bacterial species set the rate of vesicle-mediated horizontal gene transfer

Most bacteria release extracellular vesicles (EVs). Recent studies have found these vesicles are capable of gene delivery, however the consequences of vesicle-mediated transfer on the patterns and rates of gene flow within microbial communities remains unclear. Previous studies have not determined the impact of both the genetic cargo and the donor and recipient species on the rate of vesicle-mediated gene exchange. This report examines the potential for EVs as a mechanism of gene transfer within heterogeneous microbial populations. EVs were harvested from three species of Gram-negative microbes carrying different plasmids. The dynamics of gene transfer into recipient species was measured. This study demonstrates that vesicles enable gene exchange between five species of Gram-negative bacteria, and that the identity of the genetic cargo, donor strain, and recipient strain all influence gene transfer rates. Each species released and acquired vesicles containing genetic material to a variable degree, and the transfer rate did not correlate with the relatedness of the donor and recipient species. The results suggest that EVs may be a general mechanism to exchange non-specialized genetic cargo between bacterial species.


Genetic cargo and bacterial species set the rate of vesicle-mediated horizontal gene transfer
Frances Tran and James Q. Boedicker

Supplementary Figure 1. E. coli
EVs load different plasmids, pUC19, pLC291, and pZS2501. PCR targeting each plasmid is performed on EVs isolated from E. coli liquid culture transformed with one of three plasmids. DNA gel shows PCR products with expected lengths: pUC19 product ~380bp, pLC291 product ~160bp, and pZS2501 product ~120bp. Calculating the vesicle number:

Supplementary
The density of vesicles harvested from donor cell cultures was estimated from the measurements of outer membrane protein concentrations and light scattering measurements of vesicle size. Omp, Omp, and Omp are the major protein component of a bacterial membrane, and are used as a proxy for the total amount of lipid harvested from donor cultures 6-8 .
To calculate the area of lipid (A lipid ), we use: A lipid = m omp 6 x 10 -9 µg OmpA/µm 2 , (Equation S1) where m omp is the mass of Omp A protein from the protein gel in µg. Reference 92 in the main text reports that E. coli has 6 x 10 -9 µg OmpA/µm 2 .
To convert the area of lipid to the number of vesicles, we use: where d ev is the average diameter of an extracellular vesicle. Here we use 0.2 µm as the average diameter of all vesicles in our study, in agreement with dynamic light scattering measurements reported in Figure 1D. From these calculations we find on the order of 6 x 10 11 vesicles were harvested from 400 mL of stationary phase culture. For transfer experiments, we calculate 1.3 x 10 10 EVs were added to 4 mL of culture. This would give: 1.3 x 10 10 vesicles / (4 mL * 4 x 10 9 cells/mL) = 0.8 vesicles/cell. (Equation S4) Therefore, vesicle uptake by recipient cells was measured at a ratio of vesicles to cells only a factor of 2 higher than estimated for stationary phase cultures.
From Figure 2C, we found that a factor of 10 reduction in the number of vesicles resulted in about a 1.5 hour increase in the transfer time. Although not tested, if we assume a 10 fold change in either vesicle number or recipient cell number result in similar delays in transfer time, we could estimate how long it would take to observe transfer between two stationary phase cultures with a volume of 1 mL. 1 mL reduces the cell number by a factor of 4 as compared to the 4 mL cultures used in the transfer experiments. The number of vesicles produced by 1 mL of stationary phase culture is 1.6 x 10 9 vesicles (based on 0.4 vesicles/cell calculated above), or an 8 fold reduction in vesicle number. Together, that would be equivalent to a 32 fold reduction in vesicles. Based on Figure 2C, this suggests a single vesicle transfer would occur within 1 mL of stationary phase culture every 7.3 hours. These are very rough calculations, but does suggest that vesicle mediated gene transfer would be occurring within populations of a billion cells over time scales less than a day. As shown in Figure 2B and 4B-D, the plasmid transferred and the donor and recipient strains also modulate the vesicle transfer times by multiple hours.
Additionally it is known that culturing conditions and cell stress both influence vesicle production. It is likely that exchange times under a specific set of circumstances could be anywhere from 1 hour to multiple days. More experimental work is needed to further quantify vesicle exchange times per cell under a variety of conditions.