Title: Dynamics of bacterial communities driven by antagonistic quorum sensing pathways Background: Cell-cell communication has the potential to transform a discrete group of cells into a thriving community. Communication between unicellular bacteria can convert them into multicellular communities that can withstand several stressors effectively. This is particularly important for microorganisms competing for a specific niche, to establish their dominance over other microbes occupying the same habitat. A bacterial community in general is composed of isogenic but functionally diverse bacterial population subsets which stem from cellular heterogeneity. Such heterogeneous populations ensures that few subsets of bacteria adopt phenotypes that accommodate for fluctuating conditions in the environment during colonisation or infection of a host. This phenotypic plasticity in an isogenic bacterial population is key for the genesis of division of labour, however, the molecular basis of such functional diversification is largely uncharacterized and unexplored. Quorum sensing is at the heart of bacterial communication and bacterial communities. Quorum sensing was first established in Streptococcus pneumoniae, a Gram-positive nasal dweller which causes a significant global health burden. Competence Stimulating Peptide (CSP) was the first quorum sensing molecule that was discovered following decoding the mechanism for development of competence. Response to CSP involves regulation of a plethora of gene expression programs in S. pneumoniae which ultimately results in the uptake of copious amounts of DNA, both self and non-self. This results in increased fitness as well as antimicrobial resistance in the population. In nasal passages, the primary environmental reservoir of S. pneumoniae, it primarily resides in biofilms which is mainly composed of its own DNA. This essentially hints towards an orchestrated self or kin-killing pathway. Indeed, a kin-killing pathway, known as fratricide, has been discovered in S. pneumoniae (Peterson et al., 2004) where competent cells kill non-competent cells. This facilitates release of DNA, which not only provide a scaffold for biofilm but also acts as a source of nutrition (Trappetti et al., 2011). Moreover, lysis of non-competent cells releases the pore-forming toxin pneumolysin that induces host inflammation, which is a prerequisite for pneumococcal transmission from one host to another (Martner et al., 2008). Interestingly, expression of this critical virulence factor in S. pneumoniae is regulated by autoinducer-2, an interspecies quorum sensing molecule. Collectively, these point towards an unknown mechanism that maintains the proportion of competent and non-competent cells in S. pneumoniae population, to ensure intra-host sustenance as well as inter-host transmission. However, how such population dynamics are maintained is unknown. Problems to be addressed: Diversification of bacterial populations by stochastic response to competing quorum sensing pathways can be a smart strategy for prolonged sustenance and efficient periodic transmission. We hypothesize that in an S. pneumoniae population, the interplay between the above-mentioned CSP and AI-2 quorum sensing pathways leads to functional diversification of singular cells that results in the development of competent vs non-competent phenotypes, respectively. Since AI-2 is internalized and subsequently sequestered for phenotypic effects, it may not be available to other cells which could respond to CSP and be committed to competence pathway. We also speculate that AI-2 pathway inhibits CSP signaling, thereby blocking the initiation of competence development in AI-2 committed cells and thus potentiating the development of competent and non-competent population dynamics. Approach: 1. Evaluation of temporal population dynamics in S. pneumoniae. We first aim to generate a double mutant SPN strain that lacks the capability to produce both CSP (produced by comC gene) and AI-2 (synthesized by luxS gene). In this double mutant (ΔcomC ΔluxS), we plan to translationally fuse ssbB (a CSP responsive gene) with rfp and ply (an AI-2 responsive gene) with gfp. Using this reporter strain, we aim to decipher population dynamicity following separate or simultaneous activation of both CSP and AI-2 quorum sensing pathways by exogenous addition of CSP and/or AI-2 by employing flow cytometry and time-lapse fluorescence microscopy. 2. Examining mechanistic basis of fratricide in S. pneumoniae population. Our preliminary data shows that AI-2 committed cells are killed in the presence of cells responsive to CSP signaling. Next, we aim to determine the genetic basis of this fratricide event by gene expression analysis of cells committed to either CSP or AI-2 pathways. We trust that such analysis would allow us to pinpoint the mechanism for antagonism between competing quorum sensing pathways. Effect on the broader field Exploring the mechanistic basis of the origin of heterogeneity as well as the maintenance of population dynamicity in bacterial communities would provide a deeper insight into the formation and maintenance of bacterial biofilms as a whole. Apart from its significance in demystifying biofilms, this area of research can shed light on the genesis of the persisting population, which ultimately results in the development of antibiotic resistance. References: SN Peterson et al. (2004). Molecular Microbiology, 51(4), 1051–1070. C Trappetti et al. (2011) Infection and Immunity, 79(11), 4550–4558. A Martner et al. (2008). Infection and Immunity, 76(9), 4079. J Paradisi et al. (2001). Clinical Microbiology and Infection : The Official Publication of the European Society of Clinical Microbiology and Infectious Diseases, 7 Suppl 4(SUPPL. 4), 34–42.