Genetics and Regulation of Chitobiose Utilization in Borrelia burgdorferi

Abstract

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.

Adaptation to Glucosamine Starvation in Borrelia burgdorferi is Mediated by recA

“…This cell density is equivalent to that reached by cells in the second exponential phase when cultured in BSK-II lacking free GlcNAc, though about one-tenth the cell densities reached by cells cultured in complete BSK-II. In some experiments cells diluted from the second exponential phase were followed for extended periods of time after reaching their peak cell density (~2.5  10 7 cells ml -1 ) in a single exponential phase, and while there was a decline in cell density a second exponential phase was not observed as previously described (Tilly et al 2001). …”

“…1A), with growth in the second exponential phase dependent on the expression of the chitobiose transporter chbC (Tilly et al 2001;Rhodes et al 2010). To determine if cells in the second exponential phase were adapted to grow in the absence of free GlcNAc we followed cell growth in BSK-II lacking GlcNAc.…”

“…second exponential phase growth) is physiologic or genetic in nature. A previous report by Tilly et al (Tilly et al 2001) suggested that second exponential phase growth was a physiologic response, as cells from the second exponential phase exhibited biphasic growth after being transferred to fresh medium lacking GlcNAc. However, in that experiment starvation-adapted cells grew almost 10-fold higher in the first exponential phase as compared to cells that were not previously exposed to the starvation condition, leaving open the possibility that second exponential phase growth is due to the outgrowth of a mutant population.…”

“…In the absence of free GlcNAc B. burgdorferi can degrade chitin (a polymer of GlcNAc) to chitobiose and import this dimer through a dedicated chitobiose phosphotransferase system to satisfy its requirement for exogenous GlcNAc (Rhodes et al 2010). However, in the absence of both free GlcNAc and chitin this spirochete exhibits a unique biphasic growth pattern in which the second exponential phase is dependent on the expression of the chitobiose transporter (Tilly et al 2001;Rhodes et al 2009;Rhodes et al 2010). In the first exponential phase cells grow to approximately 2.0  10 6 cells ml -1 within three days by exhausting the small amount of free GlcNAc and/or GlcNAc oligomers from complex medium components such as rabbit serum and yeastolate.…”

“…burgdorferi is a limited genome organism and must scavenge most essential nutrients from its tick vector or vertebrate host (Das 2000;Saier MH 2000). The organism requires a complex medium for propagation in the laboratory, and specific addition of free N-acetylglucosamine (GlcNAc), a component of the bacterial cell wall, is necessary for cells to reach optimal cell densities in a single exponential phase (Barbour 1984;Tilly et al 2001;Rhodes et al 2009;Rhodes et al 2010). In the absence of free GlcNAc B. burgdorferi can degrade chitin (a polymer of GlcNAc) to chitobiose and import this dimer through a dedicated chitobiose phosphotransferase system to satisfy its requirement for exogenous GlcNAc (Rhodes et al 2010).…”