Abstract
BackgroundBorrelia burgdorferi has limited biosynthetic capabilities and must scavenge N-acetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment. Spirochetes cultured in the absence of free GlcNAc exhibit biphasic growth; however, addition of chitobiose (a dimer of GlcNAc) substitutes for free GlcNAc resulting in a single exponential phase. We evaluated the effect of RpoS and RpoN, the only alternative sigma factors in B. burgdorferi, on biphasic growth and chitobiose utilization in the absence of free GlcNAc. In addition, we investigated the source of GlcNAc in the second exponential phase.ResultsBy comparing the growth of wild-type cells to insertional mutants for rpoS and rpoN we determined that RpoS, but not RpoN, partially regulates both biphasic growth and chitobiose utilization. The rpoS mutant, cultured in the absence of free GlcNAc, exhibited a significant delay in the ability to initiate a second exponential phase compared to the wild type and rpoS complemented mutant. Expression analysis of chbC, which encodes the membrane-spanning protein of the chitobiose phosphotransferase system, suggests the delay is due to the inability of the rpoS mutant to up regulate chbC. Furthermore, supplementing GlcNAc starved cultures with high concentrations (75 or 150 μM) of chitobiose resulted in biphasic growth of the rpoS mutant compared to a single exponential phase for the wild type and rpoS complemented mutant. In contrast, growth of the rpoN mutant under all conditions was similar to the wild type. 5' Rapid amplification of cDNA ends (5' RACE) revealed the transcriptional start site for chbC to be 42 bp upstream of the translational start site. Analysis of the chbC promoter region revealed homology to previously described RpoD and RpoS B. burgdorferi promoters. We also determined that yeastolate, a component of the growth medium (BSK-II), is not essential for second exponential phase growth.ConclusionTogether these results suggest that RpoD and RpoS, but not RpoN, regulate biphasic growth and chitobiose utilization in B. burgdorferi by regulating the expression of the chitobiose transporter (chbC). The data also demonstrate that the second exponential phase observed in wild-type cells in the absence of free GlcNAc is not due to free chitobiose or GlcNAc oligomers present in the medium.
Highlights
Borrelia burgdorferi has limited biosynthetic capabilities and must scavenge Nacetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment
Effect of rpoS mutation on growth in BSK-II without GlcNAc To determine if the alternative sigma factor, RpoS, is involved in the regulation of biphasic growth, we compared the growth of an rpoS mutant strain (A74) to a wildtype strain (B31-A) in the presence and absence of free GlcNAc (Fig. 1)
When cells were cultured in the absence of free GlcNAc there was a considerable difference in the ability of the two strains to initiate a second exponential phase
Summary
Borrelia burgdorferi has limited biosynthetic capabilities and must scavenge Nacetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment. Spirochetes cultured in the absence of free GlcNAc exhibit biphasic growth; addition of chitobiose (a dimer of GlcNAc) substitutes for free GlcNAc resulting in a single exponential phase. We evaluated the effect of RpoS and RpoN, the only alternative sigma factors in B. burgdorferi, on biphasic growth and chitobiose utilization in the absence of free GlcNAc. In addition, we investigated the source of GlcNAc in the second exponential phase. The spirochete is maintained in an enzootic cycle, alternating between a tick vector (Ixodes scapularis) and vertebrate host. Uninfected larval ticks acquire B. burgdorferi after feeding on a vector-competent host, and spirochetes colonize and persist within the tick midgut for months as the tick molts to the nymphal stage [1]. In the infected-unfed tick, B. burgdorferi is associated with the midgut epithelium, existing in a non-replicative state in a nutrient poor environment. The spirochetes move from the midgut of the feeding tick to the hemolymph and to the salivary glands where they can be transferred to a naïve host, a process that occurs no earlier than 24 hours after tick attachment [3]
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