A Simple Biosensor-Based Assay for Quantitative Autoinducer-2 Analysis.

Bacteria produce and react to interspecies signaling molecules in order to control the expression of genes that are particularly beneficial when they are expressed by a bacterial community. In addition to intraspecies communication, the signaling molecule autoinducer-2 (AI-2) can also serve for interspecies communication between Gram-positive and Gram-negative bacteria and is therefore of particular interest. The analysis and quantification of AI-2 are essential for understanding population density-dependent changes in bacterial behavior and pathogenicity. However, currently available bioassays for AI-2 quantification are rather complex, have narrow detection ranges, and are very sensitive to trace components of, for example, growth media. To facilitate and improve the detection of AI-2, we have developed an Escherichia coli biosensor-based assay that is sensitive, cheap, fast, robust, and reliable in the quantification of biologically active AI-2. The bioassay is based on an lsr promoter-fluorescent reporter gene fusion cassette that we chromosomally integrated in a biosensor strain, but the cassette can also be used in a low-copy number plasmid for the application in other Gram-negative bacterial species. We show here that AI-2 quantification was possible in a concentration range from 400 nM to 100 µM and that a critical interpretation of the kinetics of the measurements can reveal sugar interference. With the help of our biosensor strain, coculture experiments were done to test the capability and kinetics of AI-2 secretion by various Gram-negative bacteria in real time. Finally, calibration curves were used to calculate the absolute AI-2 concentration in cell-free bacterial samples.

Two Polyketides Intertwined in Complex Regulation: Posttranscriptional CsrA-Mediated Control of Colibactin and Yersiniabactin Synthesis in Escherichia coli.

Bacteria have to process several levels of gene regulation and coordination of interconnected regulatory networks to ensure the most adequate cellular response to specific growth conditions. Especially, expression of complex and costly fitness and pathogenicity-associated traits is coordinated and tightly regulated at multiple levels. We studied the interconnected regulation of the expression of the colibactin and yersiniabactin polyketide biosynthesis machineries, which are encoded by two pathogenicity islands found in many phylogroup B2 Escherichia coli isolates. Comparative phenotypic and genotypic analyses identified the BarA-UvrY two-component system as an important regulatory element involved in colibactin and yersiniabactin expression. The carbon storage regulator (Csr) system controls the expression of a wide range of central metabolic and virulence-associated traits. The availability of CsrA, the key translational regulator of the Csr system, depends on BarA-UvrY activity. We employed reporter gene fusions to demonstrate UvrY- and CsrA-dependent expression of the colibactin and yersiniabactin determinants and confirmed a direct interaction of CsrA with the 5' untranslated leader transcripts of representative genes of the colibactin and yersiniabactin operons by RNA electrophoretic mobility shift assays. This posttranscriptional regulation adds an additional level of complexity to control mechanisms of polyketide expression, which is also orchestrated at the level of ferric uptake regulator (Fur)-dependent regulation of transcription and phosphopantetheinyl transferase-dependent activation of polyketide biosynthesis. Our results emphasize the interconnection of iron- and primary metabolism-responsive regulation of colibactin and yersiniabactin expression by the fine-tuned action of different regulatory mechanisms in response to variable environmental signals as a prerequisite for bacterial adaptability, fitness, and pathogenicity in different habitats. IMPORTANCE Secondary metabolite expression is a widespread strategy among bacteria to improve their fitness in habitats where they constantly compete for resources with other bacteria. The production of secondary metabolites is associated with a metabolic and energetic burden. Colibactin and yersiniabactin are two polyketides, which are expressed in concert and promote the virulence of different enterobacterial pathogens. To maximize fitness, they should be expressed only in microenvironments in which they are required. Accordingly, precise regulation of colibactin and yersiniabactin expression is crucial. We show that the expression of these two polyketides is also interconnected via primary metabolism-responsive regulation at the posttranscriptional level by the CsrA RNA-binding protein. Our findings may help to optimize (over-)expression and further functional characterization of the polyketide colibactin. Additionally, this new aspect of concerted colibactin and yersiniabactin expression extends our knowledge of conditions that favor the expression of these virulence- and fitness-associated factors in different Enterobacterales members.