The mat (or ecp) fimbrial operon is ubiquitous and conserved in Escherichia coli, but its functions remain poorly described. In routine growth media newborn meningitis isolates of E. coli express the meningitis-associated and temperature-regulated (Mat) fimbria, also termed E. coli common pilus (ECP), at 20 6C, and here we show that the six-gene (matABCDEF)-encoded Mat fimbria is needed for temperature-dependent biofilm formation on abiotic surfaces. The matBCDEF deletion mutant of meningitis E. coli IHE 3034 was defective in an early stage of biofilm development and consequently unable to establish a detectable biofilm, contrasting with IHE 3034 derivatives deleted for flagella, type 1 fimbriae or S-fimbriae, which retained the wild-type biofilm phenotype. Furthermore, induced production of Mat fimbriae from expression plasmids enabled biofilm-deficient E. coli K-12 cells to form biofilm at 20 6C. No biofilm was detected with IHE 3034 or MG1655 strains grown at 37 6C. The surface expression of Mat fimbriae and the frequency of Mat-positive cells in the IHE 3034 population from 20 6C were high and remained unaltered during the transition from planktonic to biofilm growth and within the matured biofilm community. Considering the prevalence of the highly conserved mat locus in E. coli genomes, we hypothesize that Mat fimbria-mediated biofilm formation is an ancestral characteristic of E. coli. INTRODUCTIONEscherichia coli is a multifaceted bacterium that colonizes the mammalian intestine as a harmless commensal but also causes a repertoire of intestinal and extraintestinal infectious diseases (Dobrindt, 2005) and survives in the environment (Savageau, 1983). E. coli from the mother and/or the surrounding environment are generally among the earliest colonizers in the oxygenous neonatal gut. During the first months of life a succession of bacterial populations progresses to a complex, more stable and adult-like microbial community dominated by strict anaerobes (Adlerberth & Wold, 2009). However, some E. coli strains are able to persist as a member of the normal microbiota and constitute a major portion of the facultative intestinal flora on adult mucosal surfaces. E. coli also frequently colonizes anatomical locations outside the gastrointestinal tract, e.g. vagina and urinary bladder (Obata-Yasuoka et al., 2002; Rosen et al., 2007). The population structure of E. coli is largely clonal (Ochman & Selander, 1984;Selander et al., 1986), and isolates are frequently categorized into distinct pathogroups according to specific combinations of phenotypic traits. Based on multilocus enzyme electrophoresis, E. coli strains fall into four main phylogenetic groups (designated A, B1, B2 and D), each containing varying proportions of different pathogroups and non-pathogens. E. coli strains resident in childhood and adult microbiota commonly include pathogenic variants (Sarff et al., 1975;Siitonen, 1992), predominantly belonging to the virulenceassociated phylogenetic group B2 (Nowrouzian et al., 2005; Obata-Yasuoka et al., 200...

SummaryHeterogeneity of cell population is a key component behind the evolutionary success of Escherichia coli. The heterogeneity supports species adaptation and mainly results from lateral gene transfer. Adaptation may also involve genomic alterations that affect regulation of conserved genes. Here we analysed regulation of the mat (or ecp) genes that encode a conserved fimbrial adhesin of E. coli. We found that the differential and temperature-sensitive expression control of the mat operon is dependent on mat promoter polymorphism and closely linked to phylogenetic grouping of E. coli. In the mat promoter lineage favouring fimbriae expression, the mat operonencoded regulator MatA forms a positive feedback loop that overcomes the repression by H-NS and stabilizes the fimbrillin mRNA under low growth temperature, acidic pH or elevated levels of acetate. The study exemplifies phylogenetic group-associated expression of a highly common surface organelle in E. coli.

The monoterpene indole alkaloids (MIAs) are a valuable family of chemicals that include the anticancer drugs vinblastine and vincristine. These compounds are of global significance-appearing on the World Health Organization's list of model essential medicines-but remain exorbitantly priced due to low in planta levels. Chemical synthesis and genetic manipulation of MIA producing plants such as Catharanthus roseus have so far failed to find a solution to this problem. Synthetic biology holds a potential answer, by building the pathway into more tractable organisms such as Saccharomyces cerevisiae. Recent work has taken the first steps in this direction by producing small amounts of the intermediate strictosidine in yeast. In order to help improve on these titers, we aimed to optimize the early biosynthetic steps of the MIA pathway to the metabolite nepetalactol. We combined a number of strategies to create a base strain producing 11.4 mg/L of the precursor geraniol. We also show production of the critical intermediate 10-hydroxygeraniol and demonstrate nepetalactol production in vitro. Lastly we demonstrate that activity of the iridoid synthase toward the intermediates geraniol and 10-hydroxygeraniol results in the synthesis of the nonproductive intermediates citronellol and 10-hydroxycitronellol. This discovery has serious implications for the reconstruction of the MIA in heterologous organisms.

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Paclitaxel is an important diterpenoid commonly used as an anticancer drug. Although the paclitaxel biosynthetic pathway has been mostly revealed, some steps remain to be elucidated. The difficulties in plant transformations and the scarcity of the precursor of paclitaxel, (+)-taxa-4(5), 11(12)-diene (taxadiene), have hindered the full comprehension of paclitaxel biochemistry and, therefore, its production by biotechnological approaches. One solution is to use the budding yeast, Saccharomyces cerevisiae, as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (GGPP), needs to be increased in yeast. In this study, we screened various GGPP synthases (GGPPS) to find the most suitable GGPPS for taxadiene production in yeast. We also optimized the taxadiene production by increasing the flux toward the terpenoid pathway. Finally, to remove selection markers, we integrated the required genes using a CRISPR/Cas9 system in the yeast genome. Our result showed that a titer of 2.02 ± 0.40 mg/L (plasmid) and 0.41 ± 0.06 mg/L (integrated) can be achieved using these strategies. This platform strain can be used to readily test the gene candidates for microbial paclitaxel biosynthesis in the future.