The chitobiose transporter, chbC, is required for chitin utilization in Borrelia burgdorferi

Abstract

Background: The bacterium Borrelia burgdorferi, the causative agent of Lyme disease, is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine (GlcNAc), from its tick or vertebrate host. GlcNAc can be imported into the cell as a monomer or dimer (chitobiose), and the annotation for several B. burgdorferi genes suggests that this organism may be able to degrade and utilize chitin, a polymer of GlcNAc. We investigated the ability of B. burgdorferi to utilize chitin in the absence of free GlcNAc, and we attempted to identify genes involved in the process. We also examined the role of RpoS, one of two alternative sigma factors present in B. burgdorferi, in the regulation of chitin utilization.

Results: Using fluorescent chitinase substrates, we demonstrated an inherent chitinase activity in rabbit serum, a component of the B. burgdorferi growth medium (BSK-II). After inactivating this activity by boiling, we showed that wild-type cells can utilize chitotriose, chitohexose or coarse chitin flakes in the presence of boiled serum and in the absence of free GlcNAc. Further, we replaced the serum component of BSK-II with a lipid extract and still observed growth on chitin substrates without free GlcNAc. In an attempt to knockout B. burgdorferi chitinase activity, we generated mutations in two genes (bb0002 and bb0620) predicted to encode enzymes that could potentially cleave the beta-(1,4)-glycosidic linkages found in chitin. While these mutations had no effect on the ability to utilize chitin, a mutation in the gene encoding the chitobiose transporter (bbb04, chbC) did block utilization of chitin substrates by B. burgdorferi. Finally, we provide evidence that chitin utilization in an rpoS mutant is delayed compared to wild-type cells, indicating that RpoS may be involved in the regulation of chitin degradation by this organism.

Conclusions: The data collected in this study demonstrate that B. burgdorferi can utilize chitin as a source of GlcNAc in the absence of free GlcNAc, and suggest that chitin is cleaved into dimers before being imported across the cytoplasmic membrane via the chitobiose transporter. In addition, our data suggest that the enzyme(s) involved in chitin degradation are at least partially regulated by the alternative sigma factor RpoS.

Figure 1

Chitin utilization in medium supplemented with boiled rabbit serum. Wild-type cells (B31-A) were cultured in BSK-II without GlcNAc and supplemented with 7% boiled rabbit serum. Late-log phase cells were diluted to 1.0 × 10⁵ cells ml^-1 and the following substrates were added: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle), 50 μM chitotriose (open triangle), 25 μM chitohexose (closed square) or 0.4% chitin (open square). Cells were enumerated daily by darkfield microscopy. This is a representative experiment that was repeated five times.

Figure 2

Chitin utilization in boiled medium without BSA. BSK-II without GlcNAc and BSA was supplemented with 7% rabbit serum. Wild-type cells were cultured in unboiled medium (A) or medium that was boiled for 10 min (B). Late-log phase cells were diluted to 1.0 × 10⁵ cells ml^-1 and the following substrates were added: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle), 50 μM chitotriose (open triangle) or 25 μM chitohexose (closed square). Cells were enumerated daily by darkfield microscopy. This is a representative experiment that was repeated three times.

Figure 3

Chitin utilization in serum-free medium containing a lipid supplement. Serum-free BSK-II was supplemented with a lipid mixture. Wild-type cells in late-log phase were diluted to 1.0 × 10⁵ cells ml^-1 in the absence of free GlcNAc and supplemented with the following substrates: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle) or 25 μM chitohexose (closed square). Cells were enumerated daily by darkfield microscopy. This is a representative experiment that was repeated three times.

Figure 4

β-N-acetylhexosaminidase (bb0002) and β-glucosidase (bb0620) double mutant utilizes chitin. Growth of RR60 (double mutant) in the presence of chitobiose or chitohexose. Late-log phase cells were diluted to 1.0 × 10⁵ cells ml^-1 in BSK-II containing 7% boiled serum, lacking GlcNAc and supplemented with the following substrates: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle) or 25 μM chitohexose (open triangle). Cells were enumerated daily by darkfield microscopy. This is a representative experiment that was repeated twice.

Figure 5

Growth of a chbC mutant and complemented mutant on chitin. (A) Growth of RR34 (chbC mutant) in the presence of chitobiose, chitotriose and chitohexose. Late-log phase cells were diluted to 1.0 × 10⁵ cells ml^-1 in BSK-II containing 7% boiled serum, lacking GlcNAc and supplemented with the following substrates: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle), 50 μM chitotriose (open triangle) or 25 μM chitohexose (closed square). Cells were enumerated daily by darkfield microscopy. (B) Growth of JR14 (RR34 complemented with BBB04/pCE320) in the presence of chitobiose, chitotriose and chitohexose. Late-log phase cells were diluted to 1.0 × 10⁵ cells ml^-1 in BSK-II containing 7% boiled serum, lacking GlcNAc and supplemented with the following substrates: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle), 50 μM chitotriose (open triangle) or 25 μM chitohexose (closed square). Cells were enumerated daily by darkfield microscopy. These are representative growth experiments that were repeated four times.

Figure 6

RpoS regulates chitobiose and chitin utilization. Growth of A74 (rpoS mutant) in BSK-II without GlcNAc and supplemented with 7% unboiled (A) or boiled serum (B). Late-log phase cells were diluted to 1.0 × 10⁵ cells ml^-1 and cultures were supplemented with the following substrates: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle) or 25 μM chitohexose (open triangle). Cells were enumerated daily by darkfield microscopy. This is a representative experiment that was repeated three times.