Ryan C. Fink

Abstract Shotgun metagenomics and marker gene amplicon sequencing can be used to directly measure or predict the functional repertoire of the microbiota en masse , but current methods do not readily estimate the functional capability of individual microorganisms. Here we present BugBase, an algorithm that predicts organism-level coverage of functional pathways as well as biologically interpretable phenotypes such as oxygen tolerance, Gram staining and pathogenic potential, within complex microbiomes using either whole-genome shotgun or marker gene sequencing data. We find BugBase’s organism-level pathway coverage predictions to be statistically higher powered than current ‘bag-of-genes’ approaches for discerning functional changes in both host-associated and environmental microbiomes.

Salmonella enterica serovar Typhimurium must successfully transition the broad fluctuations in oxygen concentrations encountered in the host. In Escherichia coli, FNR is one of the main regulatory proteins involved in O2 sensing. To assess the role of FNR in serovar Typhimurium, we constructed an isogenic fnr mutant in the virulent wild-type strain (ATCC 14028s) and compared their transcriptional profiles and pathogenicities in mice. Here, we report that, under anaerobic conditions, 311 genes (6.80% of the genome) are regulated directly or indirectly by FNR; of these, 87 genes (28%) are poorly characterized. Regulation by FNR in serovar Typhimurium is similar to, but distinct from, that in E. coli. Thus, genes/operons involved in aerobic metabolism, NO. detoxification, flagellar biosynthesis, motility, chemotaxis, and anaerobic carbon utilization are regulated by FNR in a fashion similar to that in E. coli. However, genes/operons existing in E. coli but regulated by FNR only in serovar Typhimurium include those coding for ethanolamine utilization, a universal stress protein, a ferritin-like protein, and a phosphotransacetylase. Interestingly, Salmonella-specific genes/operons regulated by FNR include numerous virulence genes within Salmonella pathogenicity island 1 (SPI-1), newly identified flagellar genes (mcpAC, cheV), and the virulence operon (srfABC). Furthermore, the role of FNR as a positive regulator of motility, flagellar biosynthesis, and pathogenesis was confirmed by showing that the mutant is nonmotile, lacks flagella, is attenuated in mice, and does not survive inside macrophages. The inability of the mutant to survive inside macrophages is likely due to its sensitivity to the reactive oxygen species generated by NADPH phagocyte oxidase.

An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.