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<p>Abstract</p> <p>Background</p> <p><it>Borrelia burgdorferi </it>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 <it>B. burgdorferi</it>, 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.</p> <p>Results</p> <p>By comparing the growth of wild-type cells to insertional mutants for <it>rpoS </it>and <it>rpoN </it>we determined that RpoS, but not RpoN, partially regulates both biphasic growth and chitobiose utilization. The <it>rpoS </it>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 <it>rpoS </it>complemented mutant. Expression analysis of <it>chbC</it>, which encodes the membrane-spanning protein of the chitobiose phosphotransferase system, suggests the delay is due to the inability of the <it>rpoS </it>mutant to up regulate <it>chbC</it>. Furthermore, supplementing GlcNAc starved cultures with high concentrations (75 or 150 μM) of chitobiose resulted in biphasic growth of the <it>rpoS </it>mutant compared to a single exponential phase for the wild type and <it>rpoS </it>complemented mutant. In contrast, growth of the <it>rpoN </it>mutant under all conditions was similar to the wild type. 5' Rapid amplification of cDNA ends (5' RACE) revealed the transcriptional start site for <it>chbC </it>to be 42 bp upstream of the translational start site. Analysis of the <it>chbC </it>promoter region revealed homology to previously described RpoD and RpoS <it>B. burgdorferi </it>promoters. We also determined that yeastolate, a component of the growth medium (BSK-II), is not essential for second exponential phase growth.</p> <p>Conclusion</p> <p>Together these results suggest that RpoD and RpoS, but not RpoN, regulate biphasic growth and chitobiose utilization in <it>B. burgdorferi </it>by regulating the expression of the chitobiose transporter (<it>chbC</it>). 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.</p>