Person:

Dutton, Rachel Janelle

The Escherichia coli membrane protein DsbD functions as an electron hub that dispatches electrons received from the cytoplasmic thioredoxin system to periplasmic oxidoreductases involved in protein disulfide isomerization, cytochrome c biogenesis, and sulfenic acid reduction. Here, we describe a new class of DsbD proteins, named ScsB, whose members are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs significantly. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, we searched for the substrates of ScsB. We discovered that ScsB provides electrons to the first peroxide reduction pathway identified in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. We show that PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Moreover, we identified two additional proteins that depend on ScsB for reduction, a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC. Altogether, our results reveal that the array of proteins involved in reductive pathways in the oxidative cell envelope is significantly broader than was previously thought. Moreover, the identification of a new periplasmic peroxiredoxin indicates that in some bacteria, it is important to directly scavenge peroxides in the cell envelope even before they reach the cytoplasm.

Background: The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. The physical interaction of these two proteins represents an alternative mechanism for regulating the activity of (\sigma^{54}) transcription factors. This study is to characterize the interaction of the two proteins and the consequences of the interaction on their regulatory activity. Results: FliX and FlbD form stable complexes, which can stand the interference of 2.65 M NaCl. The stability of FliX and FlbD was affected by the co-existence of each other. Five FliX mutants (R71A, L85K, (\Delta)117-118, T130L, and L136K) were created by site-directed mutagenesis in conserved regions of the protein. All mutants were successfully expressed in both wild-type and (\Delta fliX) Caulobacter strains. All but (FliX_{L85K}) could rescue the motility and cell division defects of a (\Delta fliX) mutant strain. The ability of FliX to regulate the transcription of class II and class III/IV flagellar promoters was fully diminished due to the L85K mutation. Co-immunoprecipitation experiment revealed that (FliX_{L85K}) was unable to physically interact with FlbD. Conclusions: FliX interacts with FlbD and thereby directly regulates the activity of FlbD in response to flagellar assembly. Mutations in highly conserved regions of FliX could severely affect the recognition between FliX and FlbD and hence interrupt the normal progression of flagellar synthesis and other developmental events in Caulobacter.

Long-term diet influences the structure and activity of the trillions of microorganisms residing in the human gut1–5, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here, we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila, and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale, and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals2, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi, and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids, and the outgrowth of microorganisms capable of triggering inflammatory bowel disease6. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.