Global transcriptomic profiling and mutant analysis suggest linked 3-chlorobenzoate metabolism and activation of an integrative and conjugative element in Pseudomonas putida.

UNLABELLED: Conjugation, the exchange of genetic material between different bacterial species, is an important process underlying adaptation. The reason for this is that conjugative elements carry additional genes potentially providing adaptive benefit to the host, such as antibiotic resistance or pathways for degradation of environmental contaminants. However, there is increasing evidence that the conjugative process itself is also responsive to cellular or environmental cues and may, thus, be under rate-dependent selection. Here, we investigate the integrative and conjugative element ICEclc as a model for both metabolic pathway "engrafting" (i.e., how a Pseudomonas putida-encoded benzoate pathway is expanded to 3-chlorobenzoate; 3-CBA) and understanding how metabolic cues or states may lead to ICE activation. We compared differences in gene expression of P. putida with or without ICEclc grown on 3-CBA, benzoate, and succinate, both in exponential and stationary phase conditions. Mutants were constructed in the 3-CBA metabolic pathway, and effects on ICE-transfer were examined. We further deleted parts of a seemingly redundant methyltetrahydrofolate (mTHF) biosynthesis operon that was highly expressed during 3-CBA growth and studied its effects on ICE-activation. Our results demonstrate that 3-CBA metabolism is necessary for ICE activation and reveal an unexpected connection between mTHF pathway induction and ICEclc activation and transfer. These findings show how metabolic states may arise that are perceived by the ICE and lead to an increase in its transfer frequency, thereby mobilizing the very genes that partly caused this state to appear.

IMPORTANCE: Bacterial conjugation is widely appreciated for its diversity, its molecular details, and evolutionary consequences. However, the regulation of the onset of the conjugative process in or by the host cell is frequently taken for granted, whereas any influence of environmental or cellular cues on the rates of conjugation can have drastic consequences on both positive and negative outcomes of conjugation-dependent adaptation (e.g., the distribution of genes for antibiotic resistance). We study here a particular case of an integrative and conjugative element (ICEclc), representative of a widely distributed family of elements pervasive in pseudomonads, which has a modulable conjugation rate in donor populations that is dependent on the proportion of transfer-competent cells arising in stationary phase. Transfer-competent cell appearance permits to quantify and understand conditions favoring ICE activation and transfer, and we show here how the ICE reacts to differences in the physiological states of the host cell under influence of its metabolism of aromatic compounds.