SummaryPhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens. Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF s factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF s factor s EcfG are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and s EcfG control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein-protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/s EcfG regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium-legume interaction via yet undisclosed mechanisms.

Conjugative transfer of the integrative and conjugative element ICE clc in the bacterium Pseudomonas knackmussii is the consequence of a bistable decision taken in some 3% of cells in a population during stationary phase. Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICE clc transfer rates and activation of two key ICE clc promoters (P int and P inR ) decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on P inR , whereas one of the gene products from the P inR -controlled operon (InrR) transmits activation to P int and other ICE clc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the P int and P inR promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated P int and P inR , whereas a double-copy rpoS-mcherry –containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate P inR and thus ICE clc transfer. Double promoter–reporter fusions confirmed that expression of P inR is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from P int is dominated by intrinsic noise, indicating it is specific to the ICE clc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.

The integrative and conjugative element ICEclc is a mobile genetic element in Pseudomonas knackmussii B13, and an experimental model for a widely distributed group of elements in Proteobacteria. ICEclc is transferred from specialized transfer competent cells, which arise at a frequency of 3-5% in a population at stationary phase. Very little is known about the different factors that control the transfer frequency of this ICE family. Here we report the discovery of a three-gene operon encoded by ICEclc, which exerts global control on transfer initiation. The operon consists of three consecutive regulatory genes, encoding a TetR-type repressor MfsR, a MarR-type regulator and a LysR-type activator TciR. We show that MfsR autoregulates expression of the operon, whereas TciR is a global activator of ICEclc gene expression, but no clear role was yet found for MarR. Deletion of mfsR increases expression of tciR and marR, causing the proportion of transfer competent cells to reach almost 100% and transfer frequencies to approach 1 per donor. mfsR deletion also caused a two orders of magnitude loss in population viability, individual cell growth arrest and loss of ICEclc. This indicates that autoregulation is an important feature maintaining ICE transfer but avoiding fitness loss. Bioinformatic analysis showed that the mfsR-marR-tciR operon is unique for ICEclc and a few highly related ICE, whereas tciR orthologues occur more widely in a large variety of suspected ICE among Proteobacteria.

Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, which arises only in 3-5% of individual cells. The mechanisms controlling this bistable switch between non-active and transfer competent cells have long remained enigmatic. Using a variety of genetic tools and epistasis experiments in P. putida, we uncovered an 'upstream' cascade of three consecutive transcription factor-nodes, which controls transfer competence initiation. One of the uncovered transcription factors (named BisR) is representative for a new regulator family. Initiation activates a feedback loop, controlled by a second hitherto unrecognized heteromeric transcription factor named BisDC. Stochastic modeling and experimental data demonstrated the feedback loop to act as a scalable converter of unimodal (population-wide or 'analog') input to bistable (subpopulation-specific or ‘digital’) output. The feedback loop further enables prolonged production of BisDC, which ensures expression of the 'downstream' functions mediating ICE transfer competence in activated cells. Phylogenetic analyses showed that the ICEclc regulatory constellation with BisR and BisDC is widespread among Gamma- and Beta-proteobacteria, including various pathogenic strains, highlighting its evolutionary conservation and prime importance to control the behaviour of this wide family of conjugative elements.

15 Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas 16 requires development of a transfer competence state in stationary phase, which arises only 17 in 3-5% of individual cells. The mechanisms controlling this bistable switch between non-18 active and transfer competent cells have long remained enigmatic. Using a variety of 19 genetic tools and epistasis experiments in P. putida, we uncovered an 'upstream' cascade 20 of three consecutive transcription factor-nodes, which controls transfer competence 21 initiation. Initiation activates a feedback loop, which stochastic modeling and 22 experimental data demonstrated acts as a scalable converter of unimodal input to bistable 23 output. The feedback loop further enables prolonged production of a transcription factor 24 that ensures 'downstream' transfer competence formation in activated cells. Phylogenetic 25 analyses showed that the ICEclc regulatory factors are widespread among Gammaand 26 Beta-proteobacteria, highlighting its evolutionary conservation and prime importance to 27 control the behaviour of this wide family of conjugative elements. 28 Keywords 29 bistability, regulation, ICEclc, stochastic modeling, adaptation, feedback control, Pseudomonas 30 putida, horizontal gene transfer 31 32 Introduction 33 34Biological bistability refers to the existence of two mutually exclusive stable states within a 35 population of genetically identical individuals, leading to two distinct phenotypes or 36 developmental programs 1 . The basis for bistability lies in a stochastic regulatory decision 37 resulting in cells following one of two possible specific genetic programs that determine their 38 phenotypic differentiation 2 . Bistability has been considered as a bet-hedging strategy leading 39to an increased fitness of the genotype by ensuring survival of one of both phenotypes 40 depending on environmental conditions 3 . A number of bistable differentiation programs is well 41 known in microbiology, notably competence formation and sporulation in Bacillus subtilis 4,5 , 42 colicin production and persistence in Escherichia coli 6 , virulence development of 43 Acinetobacter baumannii 7 , or the lysogenic/lytic switch of phage lambda 8,9 . 44 The dual lifestyle of the Pseudomonas integrative and conjugative element (ICE) ICEclc has 45 also been described as a bistable phenotype (Fig. 1A) 10 . In the majority of cells ICEclc is 46 maintained in the integrated state, but a small proportion of cells (3-5%) in stationary phase 47 activates the ICE transfer competence program 10,11 . Upon resuming growth, transfer competent 48 (tc) donor cells excise and replicate the ICE 12 , which can conjugate to a recipient cell, where 49 the ICE can integrate 11 . ICEclc transfer competence comprises a differentiated stable state, 50 because initiated tc cells do not transform back to the ICE-quiescent state. Although tc cells 51 divide a few times, their division is compromised by the ICE and eventually arrests 52 completely 13,14 . 53ICEs have ...