It is now well established that bacterial populations utilize cell-to-cell signaling (quorum-sensing, QS) to control the production of public goods and other co-operative behaviours. Evolutionary theory predicts that both the cost of signal production and the response to signals should incur fitness costs for producing cells. Although costs imposed by the downstream consequences of QS have been shown, the cost of QS signal molecule (QSSM) production and its impact on fitness has not been examined. We measured the fitness cost to cells of synthesising QSSMs by quantifying metabolite levels in the presence of QSSM synthases. We found that: (i) bacteria making certain QSSMs have a growth defect that exerts an evolutionary cost, (ii) production of QSSMs negatively correlates with intracellular concentrations of QSSM precursors, (iii) the production of heterologous QSSMs negatively impacts the production of a native QSSM that shares common substrates, and (iv) supplementation with exogenously added metabolites partially rescued growth defects imposed by QSSM synthesis. These data identify the sources of the fitness costs incurred by QSSM producer cells, and indicate that there may be metabolic trade-offs associated with QS signaling that could exert selection on how signaling evolves.

Bacterial cells within populations communicate with each other to control social behaviors by producing diffusible quorum sensing (QS) signal molecules.Evolutionary theory predicts that both the cost of signal production and the response to signals should incur fitness costs for producing cells. Here we provide the first empirical evidence that the production of QS signals incurs fitness costs to producing cells.Since QS plays a major role in bacterial pathogenicity, this finding will underpin novel antimicrobial strategies that are urgently needed to replace currently available antimicrobials that are becoming obsolete through the ever-rising incidence of resistance. SummaryIt is now well established that bacterial populations utilize cell-to-cell signaling (quorum-sensing, QS) to control the production of public goods and other cooperative behaviours. Evolutionary theory predicts that both the cost of signal production and the response to signals should incur fitness costs for producing cells. Although costs imposed by the downstream consequences of QS have been shown, it has not been demonstrated that the production of QS signal molecules (QSSMs) results in a decrease in fitness. We measured the fitness cost to cells of synthesising QSSMs by quantifying metabolite levels in the presence of QSSM synthases. We found that: (i) bacteria making QSSMs have a growth defect that exerts an evolutionary cost, (ii) production of QSSMs correlates with reduced intracellular concentrations of QSSM precursors, (iii) the production of heterologous QSSMs negatively impacts the production of a native QSSM that shares common substrates, and (iv). CC-BY-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/050229 doi: bioRxiv preprint first posted online Apr. 26, 2016; Ruparell et al.Page 3 of 36 supplementation with exogenously added metabolites partially rescued growth defects imposed by QSSM synthesis. These data provide the first direct experimental evidence that the production of QS signals carries fitness costs to producer cells.

Bacterial infection of humans, animals and plants relies heavily on secreted proteases that degrade host defences or activate bacterial toxins. The largest family of proteins secreted by Gram-negative pathogenic bacteria, the Autotransporters (ATs), includes key proteolytic virulence factors. There remains uncertainty about the mechanistic steps of the pathway ATs share to exit bacteria, and how it is energetically driven. This study set out to shed light on the AT secretion pathway with the ultimate aim of uncovering novel antimicrobial targets that would be unlikely to trigger the development of resistance mechanisms in bacteria. To do this, two AT virulence factors with distinct proteolytic functions, EspC (secreted from Enteropathogenic Escherichia coli) and AaaA (tethered to the extracellular surface of Pseudomonas aeruginosa) were chosen. EspC and AaaA were fluorescently labelled using two separate methods to establish the localization patterns of ATs as they are secreted from a bacterial cell. Super resolution microscopy revealed that localization of ATs occurs via a helical route along the bacterial cytoskeleton. In addition to requiring the conserved C-terminal β-barrel translocator domain of the AT, we present the first evidence that secretion is dependent on a dynamic interaction with a structure reliant upon the actin homologue MreB and the Sec translocon. These findings provide a step forward in the mechanistic understanding of the secretion of this widely distributed 2 family of proteins that have pivotal roles in bacterial pathogenesis and conserved structural properties that could serve as novel broad-range antimicrobial targets. SignificanceSecreted bacterial proteases facilitate the infection of human, animal and plant hosts by degrading host defences or activating bacterial toxins. The autotransporter family is the largest family of proteins secreted from Gram-negative bacteria, and includes proteolytic virulence factors crucial to bacterial infection. Precisely how autotransporters migrate from the inside to the outside of the cell, and how this movement is energetically driven is a mystery. We demonstrate a spiral pathway of autotransporter secretion, presenting evidence that it involves a dynamic interaction with the actin homologue MreB that comprises the bacterial cytoskeleton. Our findings open the way to unravelling the mechanism of autotransporter secretion and offer the possibility to identify novel antimicrobial targets unlikely to trigger the development of antimicrobial resistance.