A bistable prokaryotic differentiation system underlying development of conjugative transfer competence

Abstract

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7–4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements. Horizontal gene transfer processes among prokaryotes have raised wide interest, which is attested by broad public health concern of rapid spread of antibiotic resistances. However, we typically take for granted that horizontal transfer is the result of some underlying spontaneous low frequency event, but this is not necessarily the case. As we show here, mobile genetic elements from the class of integrative and conjugative elements (ICEs) impose a coordinated program on the host cell in order to transfer, leading to an exclusive differentiated set of transfer competent cells. We base our conclusions on single cell microscopy studies to compare the rare activation of ICE promoters in individual cells in bacterial populations, and on mutant and RNA-seq analysis to show their dependency on ICE factors. This is an important finding because it implies that conjugation itself is subject to natural selection, which would lead to selection of fitter elements that transfer better or become more widespread.