Bacteria, medium and growth conditions
All strains of Komagataeibacter xylinus (ATCC 53582) were cultured in liquid or on solid Schramm-Hestrin (SH)/agar medium at 30°C. For preparations requiring cells free of cellulose, 0.3% (v/v) cellulase from Trichoderma reesei ATCC 26921 (Sigma-Aldrich) was added to the culture medium 24 h before harvesting to digest the cellulose and obtain a uniform suspension. E. coli strains used in this study (DH5α, 5-alpha F’ Iq, and Mach1 pir116+), were grown in Luria-Bertani (LB) broth at 37°C under static conditions or on a rotatory shaker (250 rpm). For selection of resistant markers, antibiotics were used at the following concentrations: kanamycin, 50 µg/mL; ampicillin, 100 µg/mL; and streptomycin, 50 µg/mL. The E. coli strain Mach1 pir116+ was kindly provided by Dr. Ayush Kumar (University of Manitoba), whereas DH5α, 5-α F’ Iq, and dam−/dcm− strains were from New England Biolabs.
EMS mutagenesis and mutant isolation
Wild type cells were mutagenized by treating 3.2 x 107 cells/ml with 140 mM ethyl methane sulfonate (EMS) for 50 min at 30°C. This resulted in survival of ~1.6 x 107 cells/ml. Approximately 4 million mutagenized cells were used to seed four 96-well plates with 0.2 mL SH containing 10 µM pellicin. The wells were monitored for the presence of a pellicle after 5 days incubation at 30°C under static conditions. Pellicles that formed were collected and digested with cellulase, diluted and used to inoculate another four sets of 96-well plates. This was repeated one more time and pellicle forming cells were diluted and spread-plated on solid medium containing 10 µM pellicin to isolate individual colonies that were resistant to pellicin. In total, 18 isolates that showed consistent pellicin resistance were retained for further analysis.
Preparation of cultures to determine the degree of pellicin resistance
Cells from a single colony were first cultivated in SH liquid medium on a rotary shaker at 30°C for 7 days. A cellulose-free uniform suspension cells was obtained by adding cellulase to the growth medium at a final concentration of 0.3% (v/v). The initial inoculum was prepared as described above. The presence or absence of a pellicle was assessed after 7 days under static conditions at 30°C. Each well of a 96-well plate was loaded with a total volume of 200 µL of bacterial culture containing either dimethylsulfoxide (DMSO) or pellicin dissolved in DMSO; pellicin concentrations ranged from 5 to 200 µM. The results are representative of nine technical replicates obtained from three biological replicates.
Growth kinetics of wild-type and plr cultures
In order to determine the effect of pellicin on the growth of K. xylinus wild
type and the plr15 mutant, cultures were examined in SH medium containing or lacking 10 µM pellicin. Growth kinetic data was collected from cultures grown in 96-well microtitre plates inoculated with a total volume of 200 µL SH medium containing 0.3% (v/v) cellulase and either DMSO or pellicin dissolved in DMSO. The inoculum was prepared by harvesting 5-day old, cellulase-digested cultures by centrifugation at 17,000 x g for 5 minutes at room temperature, followed by three washes with fresh SH medium. The starting inoculum was adjusted to an optical density at 600 nm (OD600) of 0.02 in SH broth. The bacterial culture plates were incubated at 30°C with shaking at 150 rpm. The optical density was measured using Bio-Rad xMarkTM Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories Ltd., Mississauga, ON). Bacterial growth was monitored for 154 hours. The data from eight technical replicates obtained from two biological replicates were averaged and used for statistical analysis.
Determination of crystalline cellulose content
The crystalline cellulose from pellicles formed by plr mutants and wild type was determined as previously described (30). Briefly, inocula from plr15 and wild type were prepared as described above. Pellicles from 7 day-old cultures were harvested, treated with 0.1 N NaOH at 80°C for 20 min to lyse cells, neutralized by shaking in ultra-pure water for 24 h with two water changes, washed with acetone five times and air-dried at room temperature for 10 days. The dried pellicles were weighed and 15 mg of each pellicle transferred to a glass test tube. Each pellicle fraction was soaked in 3 mL of Updegraff solution (1:2:8 acetic acid:water:nitric acid) in a boiling water bath for 1 h. The acid resistant pellicles were transferred to pre-weighed Whatman GF/A glass fibre filters and washed several times with 70% ethanol by vacuum. These insoluble cellulose fractions were then allowed to dry overnight at room temperature. The pellicles and glass fibre filters were placed in 6-well plates and 5 mL of 67% (v/v) sulfuric acid was added to each well on a rotatory shaker at room temperature to completely hydrolyze the cellulose. The total crystalline cellulose content in each pellicle fraction was estimated as glucose equivalents using an anthrone assay (30).
DNA isolation from K. xylinus cultures was carried out using a modified protocol based on Murray and Thompson (37). Briefly cells from a 50 mL bacterial culture were harvested by centrifugation and lysed with 500 µL of 2X cetyl trimethyl ammonium bromide (CTAB) buffer (2% (w/v), 1.4 M NaCl, 100 mM Tris-HCl pH 8.0, and 20 mM EDTA) containing 50 µg (w/v) RNaseA for 30 min at 65°C. The lysed cells were cooled to room temperature then an equal volume of chloroform was added and the contents were mixed by inversion. The solution was centrifuged for 10 minutes at 17,000 x g to separate the aqueous and organic phases. The upper aqueous phase was transferred to a fresh tube and mixed with an equal volume of 2-propanol, then centrifuged for 20 minutes at 7,000 x g to pellet the DNA. The pellet was washed with 70% ethanol by centrifugation at 7,000 x g for 5 minutes. The ethanol layer was removed and the pellet was air dried for 15 minutes. Finally, the pellet was re-suspended in 100 µL of TE buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA).
The bcs operon genes bcsA, bcsB, bcsC, and bcsD from wild type and plr15 cultures were amplified using a touchdown PCR protocol using oligonucleotide primers designed according to the published cellulose synthase operon sequence of K. xylinus ATCC 53582 (GenBank accession number X54676.1). The parameters for touchdown PCR were: 98°C for 2 minutes, followed by 10 cycles of 98°C for 30s, 60°C (decreasing by 1°C/cycle) for 30s, and 72°C for 3 minutes; followed by 30 cycles of 98°C for 10s, 56°C for 30s, and 72°C for 3 minutes with a final extension at 72°C for 5 minutes. Primer pairs used for amplification and DNA sequencing are listed in Additional file 3.
Site-directed mutagenesis and allele replacement
The bcsA gene was PCR-amplified with high fidelity Phusion polymerase (New England Biolabs) using wild type genomic DNA as template. The PCR primers used for this amplification were BcsA BamHI Frwd (5’- TACGGATCCAACGAAGAAGAATCCTAAGGC-3’) and BcsA SpeI Rev (5’-ATCACTAGTGACGGGTTGTTCGTATCGT-3’). These primers introduced a 5’ BamHI site and a 3’ SpeI site on the ends of the PCR product. PCR conditions for this amplification were: 98°C for 2 minutes, followed by 10 cycles of 98°C for 30 s, 60°C (decreasing by 1°C/cycle) for 45 s, 72°C for 2 minutes and 30 s, followed by 30 cycles 98°C for 10 s, 56°C for 45 s, and 72°C for 2 minutes and 30 s with a final extension at 72°C for 5 minutes.
The bcsA PCR fragment was subsequently blunt-end cloned into the EcoRV site of pZErO-2 (Invitrogen). The resulting plasmid, named pZErO-2 bcsA, was sequenced to confirm the correct sequence of the bcsA gene was present. The bcsA fragment was then subcloned using the engineered BamHI and SpeI sites into the suicide plasmid vector pKNG101, which was later used for allele replacement. The new plasmid, pKNG101 bcsA ColE1 ori, contained the ColE1 origin of replication from pZErO-2 in addition to the endogenous R6Kg ori origins of replication; neither origin of replication allows for plasmid replication in K. xylinus. The plasmid sequence was confirmed by sequencing (Additional file 4).
Site directed mutagenesis of the bcsA gene was performed by substituting the GCC codon at position 1345 of the coding sequence corresponding to A449 with ACC, which codes for threonine. This was done using the forward primer BcsA Ala1345Thr Fwrd (5’ CATGTTCCACACCGTCGGCACG-3’) and reverse primer BcsA Ala1345Thr Rev (5’-TGCGGGATGGCATAGGCC-3’). The PCR reaction conditions were: 98°C for 2 minutes, followed by 25 cycles of 98°C for 30s, 60°C for 45s, and 72°C for 2.5 minutes and a final extension at 72°C for 5 minutes. A 10X Kinase-Ligase-DpnI Mix (10X KLD) was then used for phosphorylation, intramolecular circularization of the PCR product, and removal of the template as per manufacturer's directions (New England Biolabs). The construct was transformed using high-efficiency DH5α competent cells. The presence of the mutation was confirmed by DNA sequencing.
The pKNG101 bcsA ColE1 ori variants were used for allele replacement of the endogenous bcsA gene. The sacB gene on the pKNG101 plasmid encodes a levansucrase enzyme which in several genera of gram-negative bacteria has been shown to be lethal in the presence of 5% sucrose (31). This property facilitated selection for the integration of the mutated bcsA gene into the chromosome and for the excision of the vector. Transformation of K. xylinus with pKNG101 bcsA ColE1 ori resulted in the appearance of streptomycin-resistant transformants (SmR) from the first homologous recombination. A total of 30 independent streptomycin-resistant transformants obtained from electroporation of K. xylinus wild type cells with the pKNG101 bcsA ColE1 ori construct were grown in SH medium with shaking (150 rpm) at 30°C overnight. A volume of 150 µL of diluted cultures was plated on SH medium supplemented with 5% sucrose. A total of 400 sucrose-tolerant colonies were then grown in 200 µL SH medium containing 30 µM pellicin. A total of 8 pellicle-positive clones were obtained using this procedure. Genomic DNA from pellicle-positive and pellicle-negative colonies was extracted using the CTAB method described above. DNA sequencing of PCR products generated from both positive and negative clone genomic DNA was used to confirm the presence or absence of the site-directed mutation in the bcsA gene.
Scanning electron microscopy
Pellicles were prepared for electron microscopy as previously described (29). Observations were made using a Hitachi FlexSEM 1000 scanning electron microscope.
In vitro cellulose synthase assays and 14[C]-glucose incorporation into cellulose
Cellulose synthase assays using crude membrane preparations were performed as previously described (29). Radioactive 14[C]-glucose incorporation into cellulose of K. xylinus cultures were carried out by inoculating 1 mL of SH medium containing 1% glucose with cells to an OD600 = 0.5. Cultures were incubated for 2 h at 30°C under static conditions, followed by addition of 5 µCi of 14[C]-glucose and incubated for an additional 5 h under the same conditions. Cells were harvested by centrifugation and washed 3 times with 1 mL fresh SH medium. To each pellet, 1 mL Updegraff solution (30) and incubated in a boiling water bath for 1 h. Insoluble material was collected onto Whatman GF/A glass fibre filters and the flow-through collected in fresh tubes. Filters were then washed 3 times with 4 mL water and once with 4 mL methanol. Filters were allowed to dry overnight at room temperature. The total activity of soluble and insoluble material was determined using a liquid scintillation counter.
All statistics were performed using SigmaPlot 12.5 software. A student’s t-test or a one-way ANOVA test were performed for statistical analysis, and values were
determined to be significant at a P value of < 0.05. A Saphiro-Wilk test was run in
parallel to the one-way ANOVA test with P values for normality and equal variance set up at 0.050 in order to determine homogeneity of variance.
Structural analysis of K. xylinus BcsA was performed using the Phyre2 Protein Fold Recognition Server (38) based on the Rhodobacter sphaeroides cellulose synthase template PDB 4hg6A, to model a cellulose translocation intermediate of cellulose synthase subunit A. The pdb files created from these models were visualized using PyMOL software (39).
Powder X-ray Diffraction
Sample preparation for X-ray diffraction (XRD) to assess the crystallinity of cellulose was as previously described (29). Briefly, samples were prepared by collecting pellicles of K. xylinus cultures grown in SH medium containing DMSO or by centrifugation (in the case of wild type) or pellicles (in the case of plr mutants) of cultures grown in the presence of 10 mM pellicin. Lyophilized material was finely ground, passed through a #60-gauge mesh and examined by Powder X-ray diffraction. X-ray diffractograms were recorded with a Philips PW3710 with Cu Ka radiation (1.54060 Å) with the generator working at 10kV and 10 mA. Angular scanning was at a 2 q step size of 0.020, a step time of 2.5 s. The relative crystallinity index (RCI) was calculated according to Segal et al. (40) using the formula RCI = (It-Ia/It) x 100, where It is the total intensity of the (110) peak for cellulose Ia at 22.73° 2q 22° and, for wild type culture with pellicin, of the (020) peak for cellulose II at 21.73° 2q. The Ia is the amorphous intensity at 18° 2q for cellulose Ia and 16° 2q for cellulose II (Additional file 5a). Peak identity was confirmed by comparison to diffraction patterns of cellulose Ia (41) using Match! Software (Crystal Impact, Germany).