One possible explanation for the biphasic growth observed in the absence of free GlcNAc or limiting amounts of chitobiose is that
a mutation has occurred allowing for the outgrowth of a mutant population. A previous report from Tilly et al  suggested this was not the case as cells back-diluted from the second exponential phase into a medium without GlcNAc still exhibited biphasic growth. However, in that experiment cells that were back-diluted grew almost 10-fold higher in the first exponential phase compared to cells in the first exponential phase from the original culture. This suggests the back-diluted cells were now able to utilize a GlcNAc-containing medium component Volasertib mw that they were not previously able to use. In fact, unpublished data from our laboratory supports the hypothesis (Rhodes and
Selleck CBL-0137 Nelson, manuscript in preparation) that neopeptone see more (an enzymatic digest of protein) and rabbit serum supply GlcNAc sequestered in the form of glycoproteins or proteoglycans that B. burgdorferi can acquire and utilize for growth in the second exponential phase. Numerous reports have demonstrated adhesion of B. burgdorferi to mammalian cells through the binding of glycoproteins such as fibronectin , glycosaminoglycans such as heparin sulfate , and proteoglycans such as decorin . The ability to bind these substrates brings the spirochetes into close proximity with bound GlcNAc, and may represent a valuable source of this sugar when free GlcNAc or GlcNAc oligomers are not available. A deglycosylation mechanism has recently been described in Streptococcus pneumoniae, in which exoglycosidases sequentially remove sugar residues from host glycoproteins . We suggest that B. burgdorferi
may employ similar mechanisms by which they can release and utilize bound GlcNAc from host-derived glycoproteins, glycosaminoglycans and/or proteoglycans. Results described above suggest that some, if not all, of the GlcNAc imported into the cell in the second exponential phase comes in the form of chitobiose. The proposed mechanism for obtaining GlcNAc from glycoproteins would be consistent with this as the oligosaccharide portion of N-linked glycoproteins is attached to the amino acid asparagine through chitobiose . This core chitobiose residue as well as others present throughout Amino acid the oligosaccharide moiety may be sources of GlcNAc for B. burgdorferi during growth in the second exponential phase. A second possible explanation for biphasic growth is that it is the result of scavenging of GlcNAc released from dead B. burgdorferi cells. While it cannot be ruled out that some growth in the second exponential phase may be due to scavenging of GlcNAc from dead cells, it is unlikely that all of the growth is due to scavenging as the peak cell density in the second exponential phase is > 5-fold higher than the cell density reached in the first exponential phase.