Bacterial strains and growth conditions
H. somni 2336 is a pathogenic isolate from bovine pneumonia, 738 is an LOS phase variant of 2336 obtained following subculture and passage in a bovine, and 129Pt is a non-pathogenic commensal from the healthy bovine prepuce . The bacteria were grown on Columbia agar with 5% sheep blood (CBA) in 3-5% CO2, in Columbia broth, or Terrific broth (Difco, BD Diagnostic Systems, Sparks, MD); the latter two supplemented with 0.1% Trizma base (no pH adjustment), 0.01% thiamine monophosphate (TMP)  (CTT or TTT, respectively), and 1% glucose. The bacteria were grown at 37°C or 42°C with rapid shaking (~200 rpm) in flasks with a large headspace and harvested in early stationary phase (~5 × 109 colony forming units [CFU]/ml). Alternatively, the bacteria were grown under low oxygen tension in a bottle filled with medium to minimize the headspace and shaken slowly (75 rpm) to favor biofilm formation . Bacteria were also grown in a strict anaerobic environment on CBA in a BD GasPak system (BD Diagnostic Systems), or in CTT containing Oxyrase for Broth™ (Oxyrase, Mansfield, OH). For some experiments, the medium was supplemented with 2% NaCl, or the bacteria were harvested during mid- to late-stationary phase (48-72 h post-inoculation). For growth supplementation with Neu5Ac, 1 mg (50-μg/ml final concentration) of Neu5Ac (Sigma Chemical Co.) was added to CBA, TTT, or to a chemically defined medium .
H. somni was grown on CBA plates incubated in 5% CO2 or anaerobic conditions for 48-72 h at 37°C. The cells were scraped from the plates and suspended in phosphate buffered saline, pH 7.2, (PBS) to a turbidity of 150 Klett units (about 109 CFU/ml). After vigorous vortexing at room temperature, the cell suspension was incubated at 37°C for 1 h, vortexed again, and the cells removed by centrifugation (10,000 × g for 15 min). Cetavlon (hexadecyltrimethyl ammonium bromide) was added to a final concentration of 0.005 M. Any precipitate that formed was harvested and solubilized in distilled water. No further purification was done on this sample. Alternatively, the bacteria were grown to late stationary phase in CTT (48-72 h post-inoculation), the bacteria harvested as above, and Cetavlon added to the supernatant. Any precipitate that formed following addition of Cetavlon was further purified by enzyme digestion (RNase, DNase, and Proteinase K), phenol extraction, and ultracentrifugation to remove LOS, as described for purification of the capsular polysaccharide of Actinobacillus pleuropneumoniae .
The bacteria were also grown at 37°C in filled 1-L bottles containing TTT with shaking at 75 rpm for 4-5 days. The clear supernatant was carefully removed and the sediment was extracted with 45% aqueous phenol at room temperature, digested with DNase, RNase, and Proteinase K, and subjected to ultracentrifugation at 125,000 × g at 4°C, as described for purification of H. somni LOS , except that the supernatant from the ultracentrifugation step was retained. Polysaccharide in the supernatant was precipitated by the addition of 30 mM sodium acetate (final) and 5 volumes of cold (-20°C) 95% ethanol, and incubated at -20°C for at least 4 hours. The pellet obtained by centrifugation was suspended in distilled water, and eluted through a Sephacryl S-400 column (2.5 × 50 cm) with distilled water as eluent. The first fractions containing carbohydrate (determined by phenol-sulfuric acid assay)  were pooled and lyophilized.
For comparative analysis of polysaccharide production under various environmental conditions, the bacteria were cultured in CTT at 37°C either anaerobically (with Oxyrase) or with 2% NaCl aerobically, or at 42°C aerobically. All cultures were grown to 4 × 109 CFU/ml (early stationary phase). The bacteria were harvested and 0.005 M Cetavlon (final concentration) was added to the supernatants to precipitate large molecular mass, negatively charged components. The precipitate was then solubilized with 0.9 M NaCl, 5 volumes of cold ethanol were added, and the mixture incubated at -20°C overnight. The precipitate was resuspended in water, lyophilized, and weighed to determine the amount of polysaccharide in each sample. The cell pellets were washed with PBS and the concentration of protein in each sample was determined by BCA protein assay (Pierce, Rockford, IL).
Polyacrylamide gel electrophoresis and alcian blue silver staining
Polyacrylamide gel electrophoresis (PAGE) for polysaccharides was done as described by Pelkonen et al. , followed by alcian blue and silver staining by a modified method of Min and Cowman  using a Bio-Rad silver stain kit.
Rabbits were immunized subcutaneously in 4 different sites with a total of 50 μg of purified polysaccharide (in 1 ml of sterile water) mixed 1:1 with Freund's Complete Adjuvant, followed by a second immunization 3 weeks later with the same formulation of 50 μg of polysaccharide in Freund's Incomplete Adjuvant. The rabbits were then immunized intravenously with 50 μg of the polysaccharide until high-titer immune serum was obtained . The IgG fraction of the antiserum was isolated by Protein A/G affinity chromatography .
Immuno-transmission electron microscopy (ITEM) for analysis of polysaccharide on cells and in the biofilm
To determine if the polysaccharide formed a well-associated structure around cells of H. somni, the bacteria were grown anaerobically or in CO2, and gently scraped off plates to a turbidity of 150 Klett units (~109 cells/ml). Immunofixation was done as previously described  using 1.5 ml of bacterial suspension incubated for 1 h at 37°C with 1 ml of a rabbit IgG (0.3 mg/ml) to the polysaccharide. Thin sections were examined with a JEOL 100 CX-II transmission electron microscope.
Biofilms were grown on coverslips in TTT to stationary phase , and fixed overnight in a 1-ml mixture of 4% paraformaldehyde and 5% dimethyl sulfoxide. Samples were then embedded in situ in OCT (Sakura Finetek USA, Inc., Torrance, Calif.) on the coverslip surface upon which they were formed. For cryo-ITEM the coverslip was removed by freezing the sample in liquid nitrogen and shattering the glass, leaving the biofilm within the OCT. The OCT block was cut into 10 μm thick sections using a Cryostat (MICROM HM 505E) .
OCT sections were washed with PBS, blocked with 5% NGS (normal goat serum) (Electron Microscopy Sciences, Hatfield, PA) for 15 min, and washed with PBS. The sections were incubated with a 1:25 dilution of polyclonal rabbit antiserum to the polysaccharide at 4°C overnight, washed with PBS, and incubated with the secondary antibody (goat anti-rabbit IgG) conjugated to 10-nm colloidal gold (Electron Microscopy Sciences) for 2 hours. The samples were subsequently washed in PBS, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, treated with 1% osmium tetroxide, dehydrated through a graded series of ethanol (30%, 50%, 70%), and flat-embedded in Spurr's resin using a Chien embedding mold (Polysciences, Niles, IL). Thin sections (70 nm) were cut with a Leica EM UC-6E ultramicrotome, collected on Formvar-coated nickel grids, and stained with uranyl acetate and lead citrate. The grids were dried and observed using a JEOL 1230 TEM.
Scanning electron microscopy (SEM)
H. somni 2336 and 129pt were grown as a biofilm in chemically defined medium  with and without Neu5Ac (50 μg/ml) on glass coverslips in a 12-well plate (Falcon 3911, Microtest), and incubated for 5 days at 37°C without shaking. The coverslips were washed gently with PBS and fixed in 2.5% glutaraldehyde. The samples were processed as described , and examined using a Philips 505 scanning electron microscope.
Lectin binding to biofilms
The OCT resin sections were incubated with the fluorescein-conjugated Moringa M lectin (EY Laboratories, San Mateo, Calif.), which is specific for mannose, and counter-stained with the nucleic acid stain To-Pro3 (Molecular Probes, Invitrogen) as described . The sections were washed in PBS three times, mounted with a coverslip, and examined by confocal laser scanning microscopy with red and green channels.
Analytical and structural methods
To determine if supplementation of cultures with Neu5Ac modified LOS under different culture conditions, LOS was extracted from bacteria grown as a biofilm, as planktonic cells, or on blood agar plates supplemented with and without Neu5Ac as previously described, and then O-deacylated (OdA LOS) (12). The OdA LOS samples were analyzed by negative ion electrospray mass spectrometry (ES-MS) on a VG Quattro triple quadrupole mass spectrometer (Fisons Instruments) with selective ion scanning at m/z 290, specific for Neu5Ac, as described previously .
To determine the presence of Neu5Ac on the polysaccharide from cells grown as a biofilm, polysaccharide purified from the biofilm (1 mg) was dried over P2O5 for 1 h under diminished pressure and treated with methanol/2 M HCl at 80°C for 18 h. The solution was extracted twice with equal volumes of n-hexane to remove contaminant fatty acid methyl esters, the methanolic phase was dried, and the O-methyl glycosides were acetylated with dry pyridine (200 μl) and Ac2O (100 μl) at 80°C for 30 min. The reactants were removed by evaporation, and the mixture of peracetylated O-methyl glycosides was analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS).
Sugar residues and their absolute configuration were determined by GLC and GLC-MS, which were carried out as described . Monosaccharides were identified as acetylated O-methyl glycoside derivatives. After methanolysis (2 M HCl/MeOH, 85°C, 24 h) and acetylation with acetic anhydride in pyridine (85°C, 30 min) the polysaccharide sample was analyzed by GLC-MS. Linkage analysis was carried out by methylation, as described . The sample was hydrolyzed with 4 M trifluoroacetic acid (100°C, 4 h), carbonyl-reduced with NaBD4, acetylated, and analyzed by GLC-MS.
For enzymatic hydrolysis of the polysaccharide, 10 mg was dissolved in 50 mM Na+CH3COO- (2 ml) and treated with α-mannosidase (200 μl, Sigma) at 30°C for 7 days. After lyophilization the sample was fractionated through a 1.5 × 100 cm column of Sephadex G-15 (Pharmacia), and eluted with 10 mM NH4HCO3 at a flow rate of 45 mL/h. Fraction volumes of 2 ml were collected.
Acetolysis of mannan (30 mg) was performed as reported . The acetylated products were applied to a column (1 × 150 cm) of TSK-40, and eluted with distilled water at a flow rate of 14 ml/h at room temperature; 2.5 ml fractions were collected. The fractionation yielded four fractions, as described in results.
Nuclear magnetic resonance (NMR) spectroscopy was used to obtain structural details of the polysaccharide. For structural assignments, 1D and 2D 1H-NMR spectra were recorded from a solution of 2 mg of polysaccharide in 0.5 ml of D2O, at 300 K, at pD 7, using a Bruker 600 DRX equipped with a cryo probe. The spectra were calibrated with internal acetone [δH 2.225, δC 31.45]. 31P NMR experiments were carried out using a Bruker DRX-400 spectrometer, with aqueous 85% phosphoric acid used as an external reference (0.00 ppm). Rotating frame Overhauser enhancement spectroscopy (ROESY) data sets (t1 × t2) were measured using 4096 × 256 points with a mixing time of 200 ms. Double quantum-filtered phase-sensitive correlation spectroscopy (COSY) experiments were performed with 0.258 s acquisition time, using data sets of 4096 × 256 points. Total correlation spectroscopy experiments (TOCSY) were performed with a spinlock time of 100 ms, using data sets (t1 × t2) of 4096 × 256 points. In all homonuclear experiments the data matrix was zero-filled in the F1 dimension to give a matrix of 4096 × 2048 points, and was resolution-enhanced in both dimensions by a sine-bell function before Fourier transformation. Coupling constants were determined on a first order basis from 2D phase-sensitive double quantum filtered correlation spectroscopy (DQF-COSY) . Heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were measured in the 1H-detected mode via single quantum coherence with proton decoupling in the 13C domain, using data sets of 2048 × 256 points. Experiments were carried out in the phase-sensitive mode. A 60 ms delay was used for the evolution of long-range connectivities in the HMBC experiment. In all heteronuclear experiments the data matrix was extended to 2048 × 1024 points using forward linear prediction extrapolation.
Real-time quantitative reverse transcription PCR (qRT-PCR)
Extraction of total RNA was performed from 3, 5, and 7 day-old biofilms using Total RNA Isolation (TRI) reagent (Molecular Research Centre, Inc., Cincinnati, OH) . Biofilms were grown in 1 L of broth as described above. The clear supernatant was carefully removed and the biofilm at the bottom of the flask was treated directly with TRI reagent following the manufacturer's protocol. To remove contaminating genomic DNA, approximately 10 μg of RNA was treated using Qiagen's RNeasy on-column DNase I (Q, 2.7 U DNase I/10 μg RNA), followed by Qiagen RNeasy MinElute (for DNase I removal) according to the manufacturer's protocol. The RNA concentration was determined spectrophotometrically using a Nanodrop ND-1000 instrument (Nanodrop Technologies, Wilmington, DE), and the integrity of the RNA was assessed by agarose gel electrophoresis. Planktonic cells were collected after centrifugation (6000 × g at 4°C) and resuspended in TRI reagent for extraction of RNA. Cell pellets were stored at -80°C until needed for RNA isolation.
Amplification, detection, and analysis of mRNA was performed using the ABI-Prism 7000 sequence detection system with a SYBR Green PCR master mix (Applied Biosystems, Carsbad, CA). The corresponding oligonucleotide primers were designed using Primer Express software (Applied Biosystems) and optimized for uniform size (90-100 bp) and consistent melting temperature (55°C). For each set of primers, a standard amplification curve was plotted [critical threshold cycle (Ct) against log of concentration] and only those with a slope of approximately -3 were considered reliable primers. SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen; C The qRT-PCR reaction mixture contained 1× SYBR Green PCR master mix (Applied Biosystems), 1 μl cDNA, and 0.5 μM of the forward and reverse PCR primers. Initial denaturation was at 95°C for 10 min, followed by a 40-cycle amplification of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. The critical threshold cycle, Ct, was defined as the cycle in which fluorescence becomes detectable above the background fluorescence, and is inversely proportional to the logarithm of the initial number of template molecules. A standard curve was plotted for each primer set with Ct values obtained from amplification of known quantities of H. somni cDNA. The standard curves were used for converting the Ct values into the relative number of cDNA molecules. Control reactions were used to determine any contamination by genomic DNA. The levels of expression of all genes tested by qRT-PCR were normalized using the housekeeping gene tryptophanyl-tRNA synthetase (trpS) of H. somni as an internal standard. There was no significant difference in the expression of the trpS under the different conditions or in the various samples tested (data not shown). Each assay was performed with three independent RNA samples in triplicate.
The Student's t test was used to calculate the statistical differences between the mean levels of polysaccharide expression of experimental samples (biofilm grown cells) and control samples (planktonic cells). A P value < 0.05 was considered significant. All statistical analyses were done using InStat software (InStat, San Diego, CA).