We show that H. volcanii biofilms contain a biologically
formed layer of pleomorphic adherent cells at high density
and in close association (Figure 3B,C,D and Additional
file 1: Figure S2). The observed spatial arrangement
and diversity of cellular structure is consistent with the
model of cytoplasmic bridge formation and membrane
fusion during mating [95], previous electron micrographs
showing elongated cells with intercellular structures [39],
and/or the involvement of additional contact-dependent
cargo-transfer mechanisms not described in H. volcanii,
such as lipoprotein transfer [96], vesicular trafficking
[97] and cell-to-cell secretion systems [98]. Based
on these observations, we suggest biofilms are the microenvironments
where mating likely occurs in nature,
and propose this mode of growth as an excellent way
for studying and visualizing HGT mechanisms in live
cells and in real time