Numerous bacteria, including Yersinia pestis, express the poly-N-acetylglucosamine (PNAG) surface carbohydrate, a major component of biofilms often associated with a specific appearance of colonies on Congo red agar. Biofilm formation and PNAG synthesis by Y. pestis have been reported to be maximal at 21 to 28°C or "flea temperatures," facilitating the regurgitation of Y. pestis into a mammalian host during feeding, but production is diminished at 37°C and thus presumed to be decreased during mammalian infection. Most studies of PNAG expression and biofilm formation by Y. pestis have used a low-virulence derivative of strain KIM, designated KIM6+, that lacks the pCD1 virulence plasmid, and an isogenic mutant without the pigmentation locus, which contains the hemin storage genes that encode PNAG biosynthetic proteins. Using confocal microscopy, fluorescence-activated cell sorter analysis and growth on Congo red agar, we confirmed prior findings regarding PNAG production with the KIM6+ strain. However, we found that fully virulent wild-type (WT) strains KIM and CO92 had maximal PNAG expression at 37°C, with lower PNAG production at 28°C both in broth medium and on Congo red agar plates. Notably, the typical dark colony morphology appearing on Congo red agar was maintained at 28°C, indicating that this phenotype is not associated with PNAG expression in WT Y. pestis. Extracts of WT sylvatic Y. pestis strains from the Russian Federation confirmed the maximal expression of PNAG at 37°C. PNAG production by WT Y. pestis is maximal at mammalian and not insect vector temperatures, suggesting that this factor may have a role during mammalian infection.
Importance: Yersinia pestis transitions from low-temperature residence and replication in insect vectors to higher-temperature replication in mammalian hosts. Prior findings based primarily on an avirulent derivative of WT (wild-type) KIM, named KIM6+, showed that biofilm formation associated with synthesis of poly-N-acetylglucosamine (PNAG) is maximal at 21 to 28°C and decreased at 37°C. Biofilm formation was purported to facilitate the transmission of Y. pestis from fleas to mammals while having little importance in mammalian infection. Here we found that for WT strains KIM and CO92, maximal PNAG production occurs at 37°C, indicating that temperature regulation of PNAG production in WT Y. pestis is not mimicked by strain KIM6+. Additionally, we found that Congo red binding does not always correlate with PNAG production, despite its widespread use as an indicator of biofilm production. Taken together, the findings show that a role for PNAG in WT Y. pestis infection should not be disregarded and warrants further study.