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Alysis uncovers three previously described loci (A to C) and three new loci (G to I) with interesting changes in expression.The downregulated locus D contains a three-gene operon involved in the synthesis of curli. Curli are common in Enterobacteriaceae and are typically involved in attachment to surfaces and biofilm formation (46). Although curli have not previously been described in Yersinia, it is known that Yersinia do not form biofilms at 37 . Therefore, this result makes sense if these curli are involved in biofilm formation rather than host-cell binding. The highly downregulated genes of locus E are all involved in synthesis of the bacterial flagella. It is well established that Y. enterocolitica flagella are downregulated at 37 , likely as an adaptation to avoid being recognized by Toll-like receptor 5 (TLR5) (18). This expected result confirms the utility of our technique in discovering systems that are differentially expressed upon changing environmental conditions. A more unexpected finding was seen in locus F with the upregulation of the glg operon, encoding five genes involved in the synthesis and metabolism of glycogen. Although the role of glycogen has not been studied in the context of a Y. enterocolitica infection, it has been demonstrated that E. coli O157:H7 glg operon mutants have a decreased ability to colonize the intestines of mice (47). It has been hypothesized that during colonization of the intestines, E. coli relies on internal stores of carbon during periods of low carbon availability (47). This is likely the case in Y. enterocolitica as well, with bacteria storing available carbon from the RPMI media as glycogen at 37 in preparation for colonization of the mammalian host. Y. enterocolitica transcriptional changes in response to extracellular infection. Bacteria infecting macrophages would be expected to upregulate virulence factors that aid in colonizationand evasion of the host immune response. We identified 180 genes with a 4-fold change in expression when comparing the transcriptomes of the extracellular bacteria to those in the conditioned RPMI after 120 min of infection (Fig. 3B), and we observed several loci of genes (A to C, G to I) that encode known and putative virulence factors (Fig. 5 and see Table S3 in the supplemental material). Interestingly, we see that all three of the downregulated loci are the same as upregulated loci in the previous comparison (A to C). The fimbria-encoding operons that were upregulated in response to the conditioned RPMI (A and C) are downregulated by bacteria attached to the macrophages (extracellular). This intriguing observation may indicate that the fimbria proteins are very stable once produced so that after the initial upregulation of the genes there is no need to create more fimbriae. Alternatively, they may be facilitating adherence to other cell types or noncellular host surfaces such as the soluble host factors present in the conditioned RPMI. The genes in locus B encode the sucrose uptake system, which was upregulated in the conditioned RPMI, an observation consistent with the hypothesis that the bacteria are storing carbon while in RPMI in preparation for the infection. Once the bacteria come into contact with the macrophages, they likely rely Tipifarnib In stock primarily on the stored glycogen as a carbon source and no longer actively import sugar. The highly upregulated locus G encodes the pH 6 antigen (Psa [Myf in Y.