The chbG gene of the chitobiose (chb) operon of Escherichia coli encodes a chitooligosaccharide deacetylase

Abstract

The chb operon of Escherichia coli is involved in the utilization of the β-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.

Fig 1

The chbG gene is transcribed as a part of the chb operon. (A) Schematics of the chb operon showing the binding sites of regulators and the proteins encoded by the operon. (B) Agarose gel electrophoresis of the PCR samples corresponding to different cycles of semiquantitative RT-PCR of the chb mRNA using primers (represented as inverted arrows in panel A) that span the chbF-chbG interval.

Fig 2

MALDI-TOF analysis of the deacetylation of chitobiose by purified ChbG. Spectra were obtained from CHCA matrix alone (A), 2 mM chitobiose (GlcNAc)₂ dissolved in water (B), purified ChbG with 10 mM chitobiose in the presence of 2 mM MgCl₂ (C), purified ChbG with 10 mM chitobiose in the absence of MgCl₂ (D), purified ChbG without chitobiose in the presence of 2 mM MgCl₂ (E), and chitobiose without ChbG in the presence of 2 mM MgCl₂ (F).

Fig 3

ESI-MS analysis of deacetylation of chitobiose by purified ChbG. Spectra were recorded from reactions containing 10 mM chitobiose without ChbG (A) and 10 mM chitobiose with purified ChbG (B).

Fig 4

Transcription from the chb promoter measured from a chb-lacZ transcriptional fusion in different genetic backgrounds in the presence of chitobiose, chitosan dimer, and chitotriose. (A) β-Galactosidase activity in the presence of 2 mM chitobiose and chitotriose in JM-chb1 (wild type), JM-chb1ΔchbG (ΔchbG), JM-chb1-1 (ΔybfM), and JM-chb1ΔchbG (ΔchbG) transformed with the plasmids pTRC99A and p99A-wt-chbG. The expression of wild-type chbG in plasmid p99A-wt-chbG was induced by adding 0.01 mM IPTG. (B) β-Galactosidase activity in the presence of chitosan dimer, chitobiose, and chitotriose in JM-chb1 (WT), JM-chb1ΔchbG (ΔchbG), JM-Rji84 (ΔnagC), JM-Rji88 (ΔnagC ΔchbG), and JM-Rji88 (ΔnagC ΔchbG) transformed with pACDH-wt-chbG. Values shown are means ± the standard deviations (SD; n > 3).

Fig 5

Effect of chbG deletion on transcriptional activation of the chb promoter in Cel⁺ mutants carrying a deletion of nagC and different activating mutations in chbR. (A) β-Galactosidase activity measured from chb-lacZ fusion in JM-Rji84(ΔnagC) and Cel⁺ mutants, JM-Rji89 (ΔnagC chbR1), JM-Rji90 (ΔnagC chbR2), JM-Rji91 (ΔnagC chbR3), JM-Rji92 (ΔnagC chbR4), and JM-Rji94(ΔnagC chbR5) carrying either wild-type chbG or deletion of chbG in the presence of 10 mM cellobiose and 2 mM chitobiose. (B) The different chbR alleles used. Values shown are means ± the SD (n > 3).

Fig 6

Role of chbG and chbF in ChbR-mediated activation of the chb promoter. The β-galactosidase activity from a chromosomal chb-lacZ fusion was measured in JM-chb1 (WT), JM-Rji84 (ΔnagC), JM-Rji84ΔchbF (ΔnagC ΔchbF), JM-Rji84ΔchbA (ΔnagC ΔchbA), JM-Rji101 (ΔnagC ΔchbF ΔchbG)/pACDH, and JM-Rji101 (ΔnagC ΔchbF ΔchbG)/pACDH-wt-chbG. Values shown are means ± the SD (n = 3).

Fig 7

Effect of substitution of conserved residues on chbG function. Transcription from the chb promoter measured as β-galactosidase activity from a chb-lacZ fusion in JM-chb1 (wild type)/pACDH, JM-chb1ΔchbG/pACDH, and JM-chb1ΔchbG transformed with plasmids carrying different mutants of E. coli chbG, and the chbG homologs from S. sonnei (SschbG) and V. cholerae (A0904). Values shown are means ± the SD from (n = 3).

Fig 8

Model for chitooligosaccharide utilization in E. coli. See the text for details.