Characterizing 5-oxoproline sensing pathways of Salmonella enterica serovar typhimurium.

5-Oxoproline (5OP) is a poorly researched ubiquitous natural amino acid found in all life forms. We have previously shown that Salmonella enterica serovar Typhimurium (Salmonella) responds to 5OP exposure by reducing cyclic-di-GMP levels, and resultant cellulose dependent cellular aggregation in a YfeA and BcsA dependent manner. To understand if 5OP was specifically sensed by Salmonella we compared the interaction of Salmonella with 5OP to that of the chemically similar and biologically relevant molecule, L-proline. We show that L-proline but not 5OP can be utilized by Salmonella as a nutrient source. We also show that 5OP but not L-proline regulates cellulose dependent cellular aggregation. These results imply that 5OP is utilized by Salmonella as a specific signal. However, L-proline is a 5OP aggregation inhibitor implying that while it cannot activate the aggregation pathway by itself, it can inhibit 5OP dependent activation. We then show that in a L-proline transporter knockout mutant L-proline competition remain unaffected, implying sensing of 5OP is extracellular. Last, we identify a transcriptional effect of 5OP exposure, upregulation of the mgtCBR operon, known to be activated during host invasion. While mgtCBR is known to be regulated by both low pH and L-proline starvation, we show that 5OP regulation of mgtCBR is indirect through changes in pH and is not dependent on the 5OP chemical structure similarity to L-proline. We also show this response to be PhoPQ dependent. We further show that the aggregation response is independent of pH modulation, PhoPQ and MgtC and that the mgtCBR transcriptional response is independent of YfeA and BcsA. Thus, the two responses are mediated through two independent signaling pathways. To conclude, we show Salmonella responds to 5OP specifically to regulate aggregation and not specifically to regulate gene expression. When and where in the Salmonella life cycle does 5OP sensing takes place remains an open question. Furthermore, because 5OP inhibits c-di-GMP through the activation of an external sensor, and does not require an internalization step like many studied biofilm inhibitors, 5OP or derivatives might be developed into useful biofilm inhibitors.

Large Overlap Between the Intestinal and Reproductive Tract Microbiomes of Chickens.

Recent work characterized the chicken reproductive tract (oviduct) microbiome composition and its similarity to the egg and chick microbiomes. However, the origin of the oviduct microbiome has not been addressed yet. Here, we characterized the microbiome composition along the oviduct (infundibulum, magnum, and shell gland) as well as in the gut (jejunum and cecum) of broiler breeders at 37 weeks of age of the Cobb industrial breed. We found that while the microbiome composition along the oviduct is similar, the three sites, jejunum, cecum, and oviduct hold distinct microbiomes. However, there was also a large overlap in the composition of the gut and oviduct microbiomes, with 55 and 53% of amplicon sequence variants (ASVs) representing 96 and 90% of the total abundance in the jejunum and cecum, respectively, shared with the magnum. Furthermore, we identified a strong correlation between the relative abundance of ASVs in the gut and their probability to be found in the oviduct. These results suggest that material from the gut travels the full length of the oviduct. This is possibly the result of chicken physiology which includes the cloaca, a cavity to which both the intestinal and reproductive tracts open into. As the cloaca is common to birds, reptiles, amphibians, most fish, and monotremes, our finding may be relevant to many vertebrates. Importantly, these results indicate that mere presence in, and ascending of the oviduct are not virulence characteristics specific to pathogens, as commonly thought, but are the result of chicken physiology and characterize all gut bacteria. Furthermore, whereas a vertical transmission route from the hen to the chick has been suggested, our work starts laying a mechanistic foundation to this route, by describing the movement of gut bacteria to the oviduct, where they may be enclosed in the developing egg. Last, as our results show that gut material travels the full length of the oviduct, fertilization in poultry occurs in the presence of at least bacterial products if not live bacteria, and therefore food additives, probiotics, and diet possibly have a much more direct effect on reproduction and egg formation than previously considered.