2.1 Plasmids, primers, bacterial strains and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1 and Table 2, and the primers used are listed in Table 3. K. pneumoniae CG43, a clinical isolate of serotype K2, is high virulent to mice [120]. E.coli and K. pneumoniae strains were generally propagated at 37°C in Luria-Bertani (LB) broth. M9 minimal medium was also used in some specific assay. Bacterial growth was assessed by measuring the absorbance of optical density at 600 nm (OD600). The antibiotics used include ampicillin (100 µg/ml), chloramphenicol (35 µg/ml), kanamycin (25 µg/ml), tetracycline (5 µg/ml) and streptomycin (500 µg/ml). Microaerobic culture for the MrkA expression was added with mineral oil (M5310) which was purchased from Sigma.
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2.2 Construction of the site-directed mutants RcsB
pHY121 that carrying rcsB and its approximately 600 bp adjacent regions was used as the template. The plasmids with site-directed mutations were constructed by quick change method. pHY121 was amplified with the complementary primer sets rcsB-D56E(＋)/rcsB-D56E(－) , wc30/wc31 and wc32/wc33 encompassing the mutation site (167th bp for D56A and 168th bp for D56E of pHY121insert) by using PfuUltra II Fusion HS DNA polymerase (Agilent Technologies) to generate mutant alleles of rcsB with the D56E or D56A mutations. The PCR product was resolved on an agarose gel, recovered, treated with DpnI for 2 hr to remove the template plasmid and transformed into E. coli JM109. The plasmid, pHY121*, carrying the mutation allele encoding RcsB (RcsB*, D56E and D56A mutations) was then prepared from the transformant colony and confirmed by sequence analysis. The mutated fragment RcsB-D56E and RcsB-D56A were subcloned into pRK415 to yield pYX001 and pYX002, respectively.
pYX001 and pYX002 were then individually mobilized from E. coli S17-1 λpir to the K. pneumoniae CG43S3 ∆rcsB strain by conjugation, and the subsequent selection was performed as described above. Each site-directed mutation in K. pneumoniae was confirmed by DNA sequencing.
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2.3 Measurement of bacterial growth
Cultures of the parental strain K. pneumoniae CG43S3, along with rcsB deletion mutant strains, ∆rcsB[pRK415-RcsB], ∆rcsB[pRK415-RcsB-D56E], ∆rcsB[pRK415-RcsB-D56A] complement strains were grown overnight in LB medium. Twenty microliters of ovenight LB cultures of K.
pneumoniae strains was used to inoculate 4 ml of LB broth. The cultures were incubated at 37°C with shaking, and the optical density was recorded as the absorbance at 600 nm at the indicated time points. Values were the average and standard deviation from triplicate samples from one of the three independent trials.
2.4 Extractions and Quantification of CPS
Bacterial CPS was extracted using the method described [121]. Briefly, 500 µl of overnight grown bacterial was mixed with 100 µl of 1%
Zwittergent 3-14 (Sigma-Aldrich, Milwaukee, WI, USA) in 100mM citric acid (pH 2.0) and incubated at 50°C for 20 min. After centrifugation, 250 µl of the supernatant was transferred to a new tube, and the CPS was precipitated with 1 ml of absolute ethanol. The pellet was dried, dissolved
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in 200 µl de-ionized water, and then 1200 µl of 12.5 mM borax in H2SO4
was added. The mixture was vigorously mixed, boiled for 5 min, cooled down and then 20 µl of 0.15% 3-hydroxydiphenol (Sigma-Aldrich, Milwaukee, WI, USA) was added. The absorbance at 520 nm was measured, and the glucuronic acid content was determined from a standard curve of glucuronic acid and expressed as µg per 109 CFU or 1010CFU.
2.5 Western Blot Analysis
Samples for Western blot analysis were prepared by obtaining whole bacterial lysates, cells were grown in LB medium at 37°C for overnight.
Each culture was harvested for 400 µl, after centrifugation, cells were resuspended in 100 µl of de-ionized water, and then were boiled at 100°C for 10 min. Quantification was taken to equalized the concentration of all cultures, and added with 2 × protein dye that containing 20% of DTT (Dithiothreitol) to boil again at 100°C for 10 min. Proteins were separated on SDS-polyacrylamide gels (13.5% to 5%). For Western blotting, proteins were transferred onto PVDF (Polyvinylidene fluoride) membranes by electroblotting. When blotting was completed, add blocking buffer which
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contains 5% milk in TBS buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.5) and blocked for 16 hr at 4°C. Probing was carried out with diluted antibodies: anti-GAPDH, anti-RcsB N-terminal, anti-MrkA, anti-FimA or anti-YfdX at a ratio of 1: 10000 for 2 hr at room temperature. Before incubating the blot in second antibody, wash the membrane twice by TBST buffer (TBS buffer with 0.1% tween-20) for 10 min per time, and then wash by TBS buffer for 10 min to remove the non-specific binding of first antibody. Secondary hybridization was incubated with diluted anti-rabbit IgG conjugated with alkaline phosphatase at a ratio of 1: 5000 for 1 hr at room temperature. Bound ligands were detected by using the alkaline phosphatase staining method, which contain alkaline phosphate buffer (150 mM Tris-HCl, 5 mM MgCl2 6H2O, pH 9.5), BCIP (50 mg/ml of 5-bromo-4-chloro-3-indolyl phosphate in 95% dimethylformamide) and NBT (50 mg/ml of p-nitro-blue tetrazolium chloride in 70% dimethylformamide).
2.6 Biofilm Formation Assay
Overnight grown bacteria were diluted 1: 100 in LB broth supplemented with appropriate antibiotic and then inoculated into each well
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of a 96-well microtiter dish (Orange Scientific) for statically incubation at 37°C for 12 hr and 48 hr After removal of the bacteria, the plate was washed by de-ionized water once, and 150 µl of 1% (w/v) crystal violet was added to each well. The plate was incubated at room temperature on an orbital shaker for 30 min, and then washed three times. The dye was solubilized in 1% (w/v) SDS, and absorbance at 595 nm was determined.
2.7 Yeast-cell Agglutination
Agglutination of yeast Saccharomyces cerevisiae AH109 was carried out as described [122]. Briefly, bacteria (~108 CFU/ml) were suspended in saline (0.85% of NaCl) with or without 5% mannose and then mixed with 1% of yeast (Sigma-Aldrich) on a glass slide. After 5 min incubation at room temperature on an orbital shaker, agglutination of yeast caused by bacteria could be assessed.
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2.8 Disc Diffusion Assay
Bacteria were overnight grown with 1: 20 dilution in LB broth supplemented with appropriate antibiotic and then incubated at 37°C until OD600 reached 0.3- 0.4. Spread 100 µl of each strain of culture on M9 agar plate, and then place a nitrate disc on the center of the plate. Add 5 µl of 10 mM paraquat on the nitrate disc and place the agar plate by staying the agar at the bottom in 37°C incubator for overnight.
2.9 Acid Stress Response
Bacterial resistance to acid challenge was determined essentially as previously described [123]. Overnight grown bacteria were diluted 1: 20 in LB broth supplemented with an appropriate antibiotic and then incubated at 37°C until OD600 reached 0.4 - 0.6. All strains were further divided into two groups of tubes, while each tube containing 1 ml of bacteria culture. Both groups of culture was centrifuged and resuspended in 1 ml of pH 4.4 LB medium for a 1 hr adaption period before the acid challenge. After adaptation, each culture from one of the group was centrifuged and resuspended in 1 ml of pH 3.0 M9 medium for 45 min. After the acid
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challenge, 100 µl of each culture in the second group was immediately removed for serial tenfold dilution in saline, and 100 µl of the 10-6 diluted sample was plated onto LB agar plates at what was considered to be the initial time point (t0). The group with pH 3.0 acid challenge was removed 100 µl immediately and serially diluted, 100 µl of each culture to the 10-6 dilution was plated onto LB agar plates and incubated at 37°C overnight.
The survival rates of each strain were then calculated by dividing the number of colonies with acid challenge by the number of colonies at t0.
2.10 Measurement of Promoter Activity
A laboratory stocked plasmid, placZ15-PyfdX, which is the promoter selection plasmid placZ15 that inserted with the yfdX promoter region in front of a promoter-less lacZ gene [124]. placZ15-PyfdX was then mobilized from E. coli S17-1 λpir to K. pneumoniae wild type and
∆rcsB strains by conjugation. β-galactosidase activity was determined as previously described [124]. In brief, overnight cultures were diluted 1: 20 in LB broth supplemented with appropriate antibiotic and then incubated at 37°C until OD600 reached 0.8 - 1.0. Harvest 1 ml of each
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culture in a tube, and wash with 1 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol) for twice. All cultures were resuspended after washed, 100µl of each culture was taken to mix with 900 µl of Z buffer, 17 µl of 0.1% SDS and 35 µl of chloroform, mixture was shaken vigorously. After incubation in 30°C water bath for 10 min, the reaction was initiated by adding 200 µl of 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) (Sigma-Aldrich). Upon the yellow-colored appearance, the reaction was terminated by adding 500 µl of 1 M Na2CO3. OD420 of each strain was recorded and the β-galactosidase activity was expressed as Miller units
(1 Miller unit = 1000 ∗(𝑡∗𝜐∗OD600)OD420 ; OD420 is the absorbance of the yellow o-nitrophenol; t = reaction time in minutes; v = volume of culture assayed in milliliters; OD600 = reflects optical density of bacteria culture) [125]. Each sample was assayed in triplicate, at least three independent experiments were carried out. The data shown were calculated from one representative experiment and shown as the means and standard deviation from triplicate samples.
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2.11 RcsB N-terminal region antiserum preparation
The N-terminal domain of rcsB was PCR amplified using primers Yen001 and Yen002 and cloned into the PCR cloning vector yT&A (Yeastern Biotech, Taiwan). The NdeI/SalI fragments form the resulting plasmid (yT&A-RcsB N-terminal) were then cloned individually into pET30a (Novagen, Madison, Wis, USA) to yield pET30a-RcsB N-terminal allowing the in-frame fusion to the N-terminus His-tag. Plasmid pET30a-RcsB-Nterminal was then introduced into E. coli BL21 (DE3) (Invitrogen, USA) for overexpression of the His6::RcsB N-terminal recombinant protein.
The recombinant protein was over-produced from the mid-log phased culture (OD600 around 0.4 - 0.6) by induction with 1 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) for 4 hr at 37°C. The proteins were then purified from total cell lysate by affinity chromatography using His-Bind resin (Novagen, Madison, Wis). After purification, the eluent was dialyzed against 1× protein storage buffer (10 mM Tris-HCl pH 7.5, 138 mM NaCl, 2.7 mM KCl, and 10% glycerol) at 4°C overnight. RcsB N-terminal region antiserum was prepared by immunizing a New Zealand white rabbit with 2.5 mg of the purified His6::RcsB N-terminal recombinant protein and the immunized rabbit was exsanguinated on day 60.
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2.12 Statistical Methods
The results of the biofilm-forming activity and β-galactosidase activity assays were derived from a single experiment that was representative of three independent experiments. Each sample was assayed in triplicate and the data were presented as the mean ± standard deviation (SD). Differences between groups were evaluated by a two-tailed Student’s t-test. P-values less than 0.05 were considered statistically significant difference.
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