This #find more randurls[1|1|,|CHEM1|]# approach of growth curve synchronization has several advantages over sampling a system at different times. Firstly, the endpoint measurements can all be performed at the same time, thereby decreasing experimental variability. Secondly, efficiency will be improved compared to processing multiple samples at different times. Thirdly, no invasive sampling is necessary and the method requires no constant vigilance or presence. Finally, as we discuss throughout the paper, it allows measuring the division rate of cells

directly from optical density with very high precision. We exemplify the growth curve synchronization method by analyzing rhamnolipid secretion by the bacterium Pseudomonas aeruginosa. P. aeruginosa is an opportunistic human pathogen found in long-term, often terminal, infections in cystic fibrosis patients and various nosocomial infections occurring in immunocompromized TPCA-1 ic50 patients [2–9]. Rhamnolipids are among the predominant virulence factors of P. aeruginosa [9, 10]. These glycolipid surfactants are involved in the formation and maintenance of biofilms, cytolysis of polymorphonuclear leukocytes (PMNs) and swarming motility ([8, 11]; reviewed in [12]). Their synthesis is regulated by quorum sensing, a mechanism for cell density-dependent

gene regulation. As such, rhamnolipid secretion in P. aeruginosa is a valuable model system to investigate how pathogenic bacteria coordinate population-wide traits at the molecular level [13]. The rhamnolipid quorum-sensing regulation consists of at least two hierarchical systems governed by two different autoinducers [14–23].

These two systems, called rhl and las, share a common motif. An autoinducer synthase (RhlI and LasI) synthesizes eltoprazine the autoinducer (N-butyryl-L-homoserine lactone or C4-HSL and N-(3-oxododecanoyl)-L-homoserine lactone or 3O-C12-HSL), which binds to its cognate transcription factor (RhlR and LasR) that, in turn, up-regulates the autoinducer synthase in a positive feedback. LasR controls expression of RhlR, and thereby the las system is hierarchically above rhl. The rhl system induces expression of rhlAB, resulting in rhamnolipid production [24]. In spite of this knowledge, the rhamnolipid system has puzzled microbiologists because it does not behave like the paradigm of quorum sensing [13, 25, 26]. In either rhlI – or lasI – bacteria, adding autoinducers to the growth media does not induce rhamnolipid secretion from the outset of the culture, indicating there is at least one other factor regulating rhlAB expression [13]. Here we illustrate our growth curve synchronization method by integrating high-resolution spectrophotometric measurements of cell density and gene expression with endpoint rhamnolipid quantification to produce multi-measurement time series of the latter.