Figure 1.
Genetic organization of the Burkholderia cenocepacia J2315 bce-II gene cluster, and alignment of amino acid sequences of the B. cenocepacia J2315 BceN protein (CAR54861) with GMDs from B. cepacia IST408 (JN987864), P. aeruginosa (AAG08838), A. thermoaerophilus (AAS55711) and E. coli (AAC77842).
Upper part: Schematic representation of the bceII gene cluster evidencing the bceN gene in black. Lower part: Sequence alignment of the indicated GMDs, evidencing the Wierenga motif and the catalytic triad typical of the short-chain dehydrogenases/reductases (SDR) family of proteins (highlighted in light grey). The specific GMD motifs are highlighted in dark grey. Asterisks indicate identical amino acid residues. One or two dots indicate semi-conserved or conserved substitutions, respectively. The predicted secondary structure of B. cenocepacia J2315 and B. cepacia IST408 BceN proteins is shown above the alignment segments, where cylinders represent α-helices and arrows represent β-sheets. Alignments and the secondary structure predictions were performed with CLUSTAL X2 and the PSIPRED software, respectively. Genes bceM to bceU putatively encode GDP-D-rhamnose reductase (bceM), GDP-D-mannose dehydratase (bceN), acyltransferase (bceO), unknown (bceP), repeat unit flippase (bceQ), glycosyltransferase (bceR), acyltransferase (bceS), UDP-glucose pyrophosphorylase (bceT), acyltransferase (bceU).
Figure 2.
(A) SDS-PAGE analysis of the 6×His-tagged BceN from B. cenocepacia J2315 by E. coli BL21 (DE3). Lanes from left panel: t0, total soluble proteins from E. coli BL21 (DE3) with plasmid pJRF4 before induction with IPTG; t2, total soluble proteins from E. coli BL21 (DE3) with plasmid pJRF4 after 2 hours of induction with IPTG; A–D, 6×His-tagged BceN protein eluted from Ni2+-NTA affinity chromatography column with increasing concentrations of imidazole. Lanes from right panel (Western-blot): 1, BSA used as negative control; 2, purified 6×His-tagged BceN protein from B. cenocepacia J2315. The monoclonal anti-polyhistidine antibody conjugated with peroxidase (Sigma-Aldrich) was used in the Western-blot. (B) Discontinuous native PAGE analysis of 6×His-tagged BceN. Lanes: M1, BSA; M2, ovalbumin; M3, aldolase; M4, catalase; A–B, purified 6×His-tagged BceN from B. cenocepacia J2315 with no GMD activity; C, purified 6×His-tagged BceN from B. cenocepacia J2315 with GMD activity. The analysis of the discontinuous native PAGE showed a predominant band with approximately 162 kDa compatible with a tetrameric form of the protein. Trimeric, dimeric and monomeric forms are also visible. (C) Structural models of the BceN of B. cenocepacia J2315 and GMD from P. aeruginosa PAO1 (PDB ID: 1RPN). Graphics were generated using the RasWin Molecular Graphics (Windows Version 2.7.5).
Figure 3.
NMR spectroscopy studies of the 6×His-tagged BceN-catalysed reaction.
(A) Conversion of GDP-D-mannose (compound A) into GDP-4-keto-6-deoxy-D-mannose keto form (compound B) and the gem-diol form of compound B (compound C). The anomeric protons in each molecule are marked as Ha. (B) HSQC spectra of the reaction mixture containing 5 mM of GDP-D-mannose, after 14 hours of incubation at 25°C. The anomeric protons and corresponding signals in HSQC are showed. (C) Time-course spectra of the 6×His-tagged BceN reaction mixture with 2 mM of GDP-D-mannose at 25°C, showing the peaks corresponding to GDP-D-mannose (A), GDP-4-keto-6-deoxy-D-mannose keto form (B), and the gem-diol form of compound B (C). (D) Michaelis-Menten plot of the initial rate of GDP-D-mannose consumption as determined by NMR.
Table 1.
Kinetics parameters for different bacterial GMD enzymes using GDP-D-mannose as substrate.
Figure 4.
Involvement of BceN in cepacian biosynthesis.
Comparison of the amount of cepacian produced by B. cepacia IST408 (black columns), B. cepacia JRF1 (white columns), B. multivorans ATCC17616 (dark grey columns), and B. multivorans (pJFR6) (light grey columns) in SARA liquid medium, after 24, 48 and 72 hours of incubation at 30°C and 37°C with orbital agitation. The P-value was determined with the Unpaired t test with Welch correction and are represented by * when P<0.01, ** when P<0.0001, *** when P<0.001.