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J. Biol. Chem., Vol. 282, Issue 24, 17738-17748, June 15, 2007

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Comparative Analysis of Two UDP-glucose Dehydrogenases in Pseudomonas aeruginosa PAO1^*

Ruei-Jiun Hung, Han-Sheng Chien, Ruei-Zeng Lin, Ching-Ting Lin, Jaya Vatsyayan, Hwei-Ling Peng, and Hwan-You Chang¹

From the Institute of Molecular Medicine, National Tsing Hua University and the Department of Biological Science and Technology, National Chiao Tung University, Hsin Chu 300, Taiwan, Republic of China

Received for publication, March 1, 2007 , and in revised form, April 17, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD⁺-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K_m values of PA2022 and PA3559 for UDP-glucose are 0.1 and 0.4 mM, whereas the K_m values for NAD⁺ are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg²⁺, which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
UDP-glucose dehydrogenase (UGDH)² (EC 1.1.1.22 [EC] ) converts UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcUA) accompanied by the reduction of two molecules of NAD⁺. The importance of UDP-GlcUA is readily apparent considering the downstream polymers that utilize this compound or its descendents, such as UDP-xylose, UDP-arabinose, and UDP-galacturonic acid (1). The cellular functions of UGDH have been investigated in a number of organisms, and these studies demonstrate its importance in detoxification, polysaccharide biosynthesis, and embryonic development (2). In the liver, glucuronide conjugation of xeno- and endobiotic compounds by UDP-glucuronosyltransferases is an important way to aid in solubilizing these metabolites (3). In plants, both UDP-GlcUA and its derivative, UDP-xylose, are essential to synthesizing cell wall polysaccharides such as hemicellulose (4). Animals require UDP-GlcUA to synthesize major structural polysaccharides such as hyaluronic acid (HA), heparan sulfate, and chondroitin sulfate (5). An inability to synthesize these structural polysaccharides may partly explain why a UGDH deficiency disrupts the Wingless signal pathway in Drosophila melanogaster (6–8) and arrests mesoderm and endoderm migration during gastrulation in mice (9).

In many pathogenic bacteria, UDP-GlcUA is necessary for the synthesis of exopolysaccharides (EPS) and lipopolysaccharides (LPS). The formation of these polysaccharides is critical to bacterial virulence (10, 11) because it enables the bacteria to evade attacks by a host immune system, such as phagocytosis (12, 13). Some pathogens, including group A and C streptococci, can even synthesize nonimmunogenic EPS such as HA to prevent antibody-mediated killing (14). Recent studies demonstrate that UGDH mutation in the pathogenic fungus Cryptococcus neoformans alters the cell integrity and nucleotide sugar pool. The cells also become temperature-sensitive and fail to grow in an animal model (15).

Pseudomonas aeruginosa, a Gram-negative bacterium, is noted for its environmental versatility, resistance to antibiotics, and ability to cause life-threatening infections in cystic fibrosis, burn, and immunocompromised patients (16). Epidemiological data indicate that P. aeruginosa is the third leading cause of nosocomial infection (17). The significance of EPS in P. aeruginosa virulence is well documented in the cystic fibrosis strains, in which the synthesis of a large quantity of alginate has frequently been observed. However, many other pathogenic P. aeruginosa strains, such as PAO1, do not normally produce alginate. The precise structure and composition of the EPS and the genes required for EPS synthesis in these P. aeruginosa strains remain unclear. Because UDP-GlcUA is one of the most commonly found sugar donors for bacterial EPS synthesis, this study attempts to identify the UGDH encoding gene in P. aeruginosa PAO1 and to investigate the biochemistry and functional roles of this enzyme in this important pathogen. This study first demonstrates that at least two P. aeruginosa genes can encode a product with UGDH activity, each with a distinct expression pattern and enzyme kinetic parameters. The presence of two types of UGDH motivated the construction of deletion mutants for each of the genes, and this study analyzes and reports on the functional roles of these genes in several biological properties of P. aeruginosa PAO1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains and Growth Conditions, Plasmids, Oligonucleotide Primers, and Chemicals—Table 1 lists the bacterial strains and plasmids used in this study. Escherichia coli and P. aeruginosa were cultured in standard Luria-Bertani broth (LB: 10 g/liter tryptone, 5 g/liter yeast extract, 10 g/liter sodium chloride) or on LB agar. The basal medium employed in the determination of minimal inhibition concentration (MIC) of antibiotics and in RNA extraction for quantitative PCR contained 30 mM glucose, 40 mM K₂HPO₄, 22 mM KH₂PO₄, 7 mM (NH₄)₂SO₄, and either 2.0 or 0.02 mM MgSO₄ for Mg²⁺-sufficient or Mg²⁺-deficient conditions, respectively. The following antibiotic concentrations were used to select recombinant E. coli: tetracycline, 10 µg/ml; ampicillin, 100 µg/ml; kanamycin, 25 µg/ml. The concentration of tetracycline for selecting recombinant P. aeruginosa was 50 µg/ml. Unless indicated otherwise, all chemicals were obtained from Sigma and were of the highest purity available. Table 2 lists the oligonucleotide primers used in this study.

Overexpression and Purification of His₆-tagged PA2022 and PA3559 Products—The production of PA2022 and PA3559 in E. coli was carried out by growing the recombinant strains in LB broth supplemented with 25 µg/ml kanamycin and 10 µg/ml tetracycline. The cells were first cultivated by shaking at 37 °C until the A₆₀₀ = 0.5. Isopropyl thio--D-galactopyranoside was subsequently added at a final concentration of 0.2 mM and growth continued for 5 h at 24 °C under shaking. Cells were harvested by centrifugation at 4,000 x g for 10 min, resuspended in binding buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5 mM imidazole), and lysed by sonication on ice with a macroprobe sonicator (model XL2020, Misonix) at a power setting of 4 for 15 min. After centrifugation at 5000 rpm at 4 °C for 20 min, the supernatant was applied to a nickel-Sepharose column (GE Healthcare), and the chromatography was performed as recommended by the manufacturer. The eluting buffer consisted of 500 mM imidazole. The purity of the protein was then evaluated by SDS-PAGE.

Ion Pair HPLC Analysis of Nucleotide Sugars—This study used a Waters HPLC system (Waters 2796 Bioseparations Module) composed of an autosampler and a UV detector (Waters 2996 photodiode array detector) to analyze the amount of nucleotide sugars in cells. Separation of nucleotide sugars, NAD⁺, and NADH was performed at 34 °C on a µBondapak C18 column (10-µm particle size, 3.9 x 300 mm) and a guard column cartridge (5-µm particle size, 4.6 x 20 mm, Atlantis®, Waters). The method was performed as described previously (19, 20). Two buffers were used for the HPLC analysis. Buffer A was 100 mM KH₂PO₄/K₂HPO₄ plus 8 mM tetrabutylammonium-bisulfate hydrogen sulfate used as the pairing reagent, pH 5.3. Buffer B was 70% buffer A plus 30% methanol, pH 5.9. Concentrate KOH or phosphoric acid was used for adjusting the buffer pH. All solutions were freshly prepared, filtered through a 0.2-µm filter (Millipore), and degassed before use. The sequence of chromatographic gradient was as follows: 100% buffer A for 2.5 min, 0–40% buffer B for 14 min, 40–100% buffer B for 1 min, 100% buffer B for 6 min, 100–100% buffer B for 1 min, followed by an equilibration phase of 100% buffer A for 8 min. The flow rate was 1.0 ml/min.

Enzymatic Activity and Kinetic Characterization—UGDH activity was measured by monitoring the change in absorbance at 340 nm, which accompanies the reduction of NAD⁺ to NADH. The assay was performed at room temperature in 0.1 M Tris-HCl buffer, pH 7.5, containing 5 mM UDP-Glc, 1 mM dithiothreitol (DTT), 1 mM MgCl₂, and 5 mM NAD⁺. One unit of UGDH activity is defined as the amount of enzyme required to produce 2 µmol of NADH per min at room temperature. Prior to HPLC analysis, the reaction mixture was mixed thoroughly with an equal volume of methanol, and the protein precipitate was removed by centrifugation at 20,000 x g for 30 min. The K_m and V_max values for UDP-Glc and NAD⁺ were determined independently using standard assay conditions. Kinetic parameters for UDP-Glc as the substrate were measured by holding NAD⁺ constant (5 mM) and varying UDP-Glc from 0.02 to 5 mM. Similarly, NAD⁺ kinetic measurements were made by holding UDP-Glc constant (5 mM) and varying NAD⁺ from 0.02 to 5 mM. GraphPad Prism4 software plotted the data, and K_m and V_max were calculated by fitting the data to the equation (V = V_max [S]^h/([S]^h + K_m^h)) where h is the Hill coefficient. Substrate specificity was measured by substituting UDP-Glc with the same concentration of ADP-Glc, TDP-Glc, GDP-Man, UDP-Gal, or UDP-GlcNAc in the standard reaction. To determine the K_i value of UDP-GlcUA, the K_m value of UDP-Glc at a variety of concentrations of UDP-GlcUA was first measured. The results were then fitted onto a curve where X is the concentration of UDP-GlcUA, and Y is the observed K_m value. Linear regression was used to determine the x axis intercept, which equals the negative K_i value. The measurement of K_i for NADH was carried out similarly in the presence of variable concentrations of the compound.

Molecular Mass Determination—The molecular masses of PA2022 and PA3559 in aqueous solution were determined using a Superdex-200 10/300 GL column (GE Healthcare) on a fast protein liquid chromatograph in 100 mM Tris-HCl, pH 7.5, containing 300 mM NaCl, at a flow rate of 0.5 ml/min. Size determination was made by comparing molecular mass standards (GE Healthcare) chromatographed under the same conditions. The molecular mass standards used were as follows: ferritin, 440 kDa; aldolase, 158 kDa; albumin, 67 kDa; RNase A, 15 kDa. A calibration curve was plotted as the log (molecular mass) versus K_av, where K_av = (V_e - V₀)/(V_t - V₀), giving correlation values above 0.99. The term V_e is the elution volume for the protein; V₀ is the column void volume, and V_t is the column total bed volume. The molecular masses of PA2022 and PA3559 were subsequently determined from the calibration curve.

Construction of PA2022 and PA3559 Deletion Strains—Constructing the PA3559 gene-specific deletion mutants in P. aeruginosa involved obtaining DNA fragments of 1 kb in size both upstream and downstream of the targeted sequences to be deleted by PCR amplification of genomic DNA of P. aeruginosa PAO1. Primers pmrE05 and pmrE03 were used for amplifying upstream fragments, and primers pmrE04 and pmrE06 (Table 2) were used for the downstream region. The amplified fragments were then cloned in tandem into the suicide vector pEX18Tc. For PA2022 mutant construction, a 4.1-kb DNA segment comprising the PA2022 gene was first cloned into the PstI and EcoRI sites of the suicide vector pSUP202. The next step was to insert a kanamycin resistance gene in the unique BamHI site of the PA2022 gene. In either case, the resulting gene replacement plasmids were transformed individually into E. coli SM10 and subsequently mobilized into P. aeruginosa PAO1 or its derivatives via conjugation. The transconjugants were selected on LB agar containing tetracycline (50 µg/ml) and ampicillin (100 µg/ml), which was used to counterselect donor E. coli. The surviving colonies were then propagated in LB broth containing 5% sucrose, which selected against cells containing the sacB gene harbored on pEX18Tc. These colonies were also tested for tetracycline sensitivity to ensure plasmid loss. The resultant mutants were confirmed by PCR amplification and Southern hybridization with a probe specific to each of the genes.

Preparation of the Cell-free Extracts for UGDH Activity Determination—The bacteria grown overnight were refreshed in 50 ml of LB broth for 2 h and collected by centrifugation. The pellet was resuspended in 30 ml of buffer containing 100 mM Tris-HCl, 20 mM NaCl, 1 mM MgCl₂, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, pH 7.5, disrupted by a brief ultrasonication, and centrifuged for 20 min at 14,000 x g to remove the debris. The clarified cell lysates were dialyzed overnight against 100 mM Tris-HCl, 1 mM MgCl₂, 1 mM DTT, pH 7.5, prior to determination of the UGDH activity. The cell lysate (15 µg of protein) in this specific assay was first incubated with NAD⁺ to determine the endogenous NAD⁺ reduction rate. UDP-Glc was then added to the reaction mixture, and the difference in the NAD⁺ reduction rate was measured. Here the definition of 1 unit is the activity that produces 1 µmol of NADH per min.

Extraction of Intracellular Nucleotide Sugars—Bacterial cells from the 0.5-ml overnight culture were collected by centrifugation. The pellet was briefly washed with phosphate-buffered saline, and 185 µl of a cold 0.5 M perchloric acid solution was added to the pellet and mixed vigorously. After incubation on ice for 2 min, the samples were centrifuged at 10,000 x g at 4 °C, for 5 min. The supernatants containing the soluble molecules were neutralized with 42 µl of ice-cold 2.5 M KOH in 1.5 M K₂HPO₄ and incubated on ice for 2 min. The neutralized samples were centrifuged again and filtered with 0.2-µm filters (Millipore, Billerica, MA) to remove potassium perchlorate precipitate. The samples were stored at -70 °C until analysis.

Susceptibility to Antibiotics—Antibiotic-impregnated disks (BBL^TM, Oxoid or Difco) were used to measure the sensitivity of the bacterial strains to antibiotics. For the disk diffusion assay, overnight aerobically grown cultures in LB were diluted to an A₆₀₀ of 0.1, and 100-µl aliquots were spread uniformly on an LB plate. The disk loads were as follows: ampicillin, 10 µg; polymyxin B, 300 IU; ceftazidime, 30 µg; cefotaxime, 30 µg; chloramphenicol, 30 µg; gentamicin, 10 µg; imipenem, 10 µg; kanamycin, 30 µg; lincomycin, 2 µg; and tetracycline, 30 µg. Comparing the zone of inhibition to a standard sensitivity chart (BD Biosciences) revealed the resistance to an antibiotic. The MIC of each antibiotic was determined by the microdilution method in multiple-well plates. The MIC was defined as the lowest concentration of antibiotic that prevented growth of the testing bacterial strains after 18 h of incubation at 37 °C.

Bacterial Motility Assays—For swimming motility assays (21), tryptone swim plates (1% tryptone, 0.5% NaCl, 0.3% agar) were inoculated with a sterile toothpick and incubated for 12–14 h at 25 °C. Motility was then assessed qualitatively by examining the circular turbid zone formed by the bacterial cells migrating away from the point of inoculation. For the twitching motility assay, cells were stab-inoculated with a toothpick through a thin (3 mm) LB agar layer (1% agar) to the bottom of the Petri dish. After incubation from 24 to 48 h at 37 °C, a hazy zone of growth at the interface between the agar and the polystyrene surface was revealed by crystal violet staining (1% (w/v) solution) (22).

LPS Analysis—The hot phenol-water method (23) and the proteinase K digestion method (24) were utilized to extract LPS. The LPS preparations were separated on a 12.5% SDS-polyacrylamide gel and silver-stained according to a previously published method (25).

Reverse Transcription and Real Time-PCR—Total RNA was isolated by using the High Pure RNA isolation kit (Roche Applied Science). Two micrograms of total RNA was reverse-transcribed in a 20-µl reaction mixture containing 50 units of Moloney murine leukemia virus reverse transcriptase, 5 µM DTT (both from Invitrogen), 40 units of RNaseOUT recombinant ribonuclease inhibitor (Promega) 0.5 µM of random hexanucleotide primers (GE Healthcare), and 500 µM of dNTP mixture. The reverse transcription reaction was carried out at 50 °C for 60 min. Heating the reaction mixture at 70 °C for 15 min was subsequently performed to terminate the reaction, and the cDNA was stored at -20 °C.

All real time PCR analyses were performed on an ABI PRIZM 7000 instrument. Each reaction included a 20-µl reaction mixture containing 0.1 µM of each primer, 10 µl of 2x SYBR Green PCR master mix (Applied Biosystem, including AmpliTaq Gold DNA polymerase with buffer, dNTPs mix, SYBR Green I dye, Rox dye, and 10 mM MgCl₂), and 1 µl of the template cDNA. The typical amplification program included activation of the enzyme at 94 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 15 s, and annealing and extension at 60 °C for 1 min. The C_T (cycle threshold) value for each gene was determined by automated threshold analysis function of the ABI instrument and normalized to C_T(G3PDH) to obtain dC_T (= C_T(G3PDH) - C_T(test)). The difference in gene expression at different Mg²⁺ concentrations was indicated with the ddC_T values, which were calculated as dC_{T(low Mg²⁺)} - dC_{T(high Mg²⁺)}. The difference of n between two C_T or dC_T values indicates a 2ⁿ-fold difference in amount of the target sequence between the two cDNA samples being compared.

Biofilm Assay—The kinetics of biofilm formation in a static system was measured as described by O'Toole and Kolter (26). Briefly, LB was inoculated with an overnight culture of each strain at a 1/100 dilution, and 100-µl aliquots of the diluted bacterial suspension were added to a 96-well polystyrene microtiter dish. The microtiter dish was incubated at 37 °C in a humidified chamber. The liquid culture was removed by aspiration, and the wells were rinsed thoroughly two times with phosphate-buffered saline at each time point. Samples were stained by adding 100 µl of 1% crystal violet solution to each well and allowing them to sit for 15 min. After staining, the wells were washed three times with water, and any remaining crystal violet in the wells was dissolved in 100 µl of 95% ethanol, and the absorbance at 595 nm was determined. Alternatively, the biofilm-forming capability of these strains was evaluated by using a polydimethylsiloxane flow cell connected to a bacterial culture system, which allowed the culture media flowing through the flow cells at uni-direction. Before perfusion, 10 ml of mid-log phase (A₆₀₀ = 0.5) P. aeruginosa was injected into the microchannels by a polypropylene syringe. The flow cells were incubated for 1 h at 25 °C to allow initial bacteria attachment. A peristaltic pump then passed a 1:10 dilution of LB broth through the flow cell at a flow rate of 10 ml/min. After 24 h, the biofilms formed in the flow cell were labeled with SYBR Green solution for 5 min and then examined under a Zeiss LSM 510 laser-scanning confocal microscope. Optical sections with a step size of 3 µm in the z axis were collected and reconstructed into three-dimensional cross-sections.

View larger version (88K): [in this window] [in a new window]   FIGURE 1. Sequence comparison of UGDH homologs. Multiple sequence alignment of S. pyogenes SpUGDH (GenBankTM accession number NP_270108), P. aeruginosa PA2022 (NP_250712), PA3559 (NP_252249), PA3540 (NP_252230), and PA3159 (NP_251849) is shown. The asterisks indicate the position of the Rossmann fold, which contains a consensus sequence GXGXXG. The arrow points to a residue that determines the substrate specificity of the enzymes. The arrowhead indicates the catalytic Cys-266 of PA2022, with a flanking sequence of GGXCXXXD. Residues identical in at least three of the comparison sequences are highlighted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oligomeric Structure of PA2022 and PA3559 Gene Products—The oligomeric structure of nucleotide sugar dehydrogenases varies considerably in different organisms. The P. aeruginosa UDP-GlcNAc dehydrogenase (WbpA) is a trimer (29), whereas SpUGDH is a monomer (27) in solutions. On the other hand, E. coli UGDH (32) and P. aeruginosa GMDH display a dimeric and a tetrameric form, respectively (33). Gel filtration chromatography reveals that the native conformation of PA2022 and PA3559 is 104 and 117 kDa, respectively (Fig. 2). Because the predicted mass of PA2022 and PA3559 is 54.5 and 57.4 kDa, respectively, both proteins are probably present in a dimeric form in native conditions, as also found for the E. coli UGDH.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Determination of the oligomeric states of PA2022 and PA3559. Purified PA2022 and PA3559 were fractionated individually by gel filtration fast protein liquid chromatography on a Superdex 200 HR 10/30 column. The size was determined by comparing molecular weight standards resolved under the same conditions. The average retention time in the column was plotted versus the log of the molecular weight for each standard. The sizes of PA2022 and PA3559 are indicated with and , respectively.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Characterization of the reaction catalyzed by PA2022 and PA3559. The standard UGDH assay was performed in the absence (A) or presence of the gene product of either PA2022 (B) or PA3559 (C). Peak 1, UDP-Glc; peak 2, NAD; peak 3, UDP-GlcUA; peak4, NADH. mAU is milliabsorption units at 260 nm.

Nucleotide Sugar Profiling of PAO1 and UGDH Mutant Strains—Whether the cellular nucleotide sugar pool is affected in the UGDH mutants was unclear. Presumably, the accumulation of UDP-Glc and the absence of UDP-GlcUA would be observed in the UGDH mutants, in particular the double deletion mutant RJ103. Nevertheless, reverse-phase ion pair HPLC analysis of the cell extract indicates that UDP-Glc (retention time = 8.63 min) seems to remain at a constant level in all the strains. On the other hand, UDP-GlcUA could not be detected in any of the extracts, suggesting that the compound turnover rate is rapid in the bacterial cells. The metabolite profile of RJ101 is similar to that of RJ103, showing an additional peak compared with wild type PAO1. The extra peak eluted at a time point very close to UDP-Gal, suggesting that any extra UDP-Glc in RJ101 and RJ103 was partially converted to UDP-Gal (supplemental Fig. S3). Finally, the total uronic acid content amount in the EPS in each strain was examined. Contrary to expectations that the uronic acid content would be reduced because of the UGDH deficiency, all UGDH mutant strains synthesized a higher quantity of uronic acid than the wild type PAO1 (Table 6). A possible explanation for this finding is that GlcUA accounts for only a minor fraction of the uronic acid pool in P. aeruginosa cells, and the UDP-Glc that accumulated because of UGDH deficiency is re-directed to pathways for the synthesis of other uronic acids such as ManUA or GlcNAcUA.

Susceptibility of the UGDH Mutants to Antibiotics—P. aeruginosa is notorious for its natural resistance to many antibiotics partly because of the permeability barrier afforded by its cell surface components. As UGDH normally plays an important role in LPS and EPS biosynthesis, it would be interesting to know if there is any difference in antibiotic susceptibility between the UGDH-deficient mutants and the wild type PAO1. An antibiotic sensitivity assay was performed by the standard disk diffusion assay. As Table 7 demonstrates, the susceptibility of PA2022 and PA3559 mutants to polymyxin B, lincomycin, ampicillin, and kanamycin showed no significant differences from the wild type PAO1 when tested on LB plates. Interestingly, the mutants RJ101 and RJ103 were more susceptible to chloramphenicol and cefotaxime but not RJ102. Testing the MIC of each of the antibiotics to these bacterial strains further verified the findings. Two different Mg²⁺ concentrations were also tested to differentiate the possible roles of the two UGDH in antibiotic resistance. Our results indicate that RJ101 and RJ103 are indeed more susceptible than wild type PAO1 and RJ102 to ampicillin, chloramphenicol, and cefotaxime in either high (2.0 mM) or low concentrations of Mg²⁺ (0.02 mM) medium, although high concentrations produce a more pronounced effect (Table 8). On the other hand, RJ102 and RJ103 were more sensitive to polymyxin B when grown in low Mg²⁺ conditions, but not RJ101, implying that PA3559 is responsible for the polymyxin B resistance.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Analysis of PA2022 and PA3559 gene expression by reverse transcription-quantitative PCR. Wild type PAO1 was cultured in basal medium containing either 0.02 or 2.0 mM Mg2+. The PA2022 and PA3559 gene expressions were determined as folds of G3PDH expression. The results are the average of three independent experiments.

Effects of UGDH on Bacterial Motility—Bacterial motility has been shown to closely associate with biofilm formation. For instance, flagella participate in the initial stages of P. aeruginosa biofilm development. Type IV pilus-mediated twitching motility is also involved in biofilm formation (19), particularly when microcolonies grow to mature mushroom-like structures on a glass surface (14). Determining whether the UGDH mutant strains had a defect in either swimming or twitching motility might explain the abnormal biofilm development. None of the strains exhibited significant differences in swimming or twitching motility.

Multiple UGDH Homologs in Pseudomonas sp.—The presence of two UGDH genes in P. aeruginosa PAO1 is most likely advantageous to the bacterium when confronting environmental changes. This finding prompted investigation of whether other Pseudomonas species also possess multiple copies of UGDH genes. Using PA2022 and PA3559 as templates to search homologous genes in the genome of 11 Pseudomonas species revealed that all of the tested genomes, including P. aeruginosa, P. fluorescens, P. putida, and P. syringae, harbored multiple copies of UGDH homologous gene (Table 9). Phylogenetic tree analysis indicates that these UGDH-like genes can be divided into four clades as follows: one is very similar to PA2022, another group resembles PA3559, one group contains UDP-GlcNAc dehydrogenase encoding genes, and the rest are more closely related to GMDH. We would like to note that PA2022-like genes only occur in P. aeruginosa, whereas PA3559 orthologs are widely distributed in all Pseudomonas sp. (Fig. 7). In addition, PA2022 is closely linked to the galU gene that encodes UDP-Glc pyrophosphorylase, an enzyme that synthesizes UDP-Glc. In comparison, no UGDH-like gene can be found near the galU and gor, which encodes a glutathione reductase, in other Pseudomonas species. On the other hand, all PA3559-like genes in Pseudomonas species, except for P. putida, are adjacent to the 7-gene polymyxin resistance (pmr) gene cluster. These genes are designated PA3552–3558 in PAO1 and have been annotated to encode enzymes for polysaccharide synthesis (34). Phylogenetic analysis suggests that PA2022, a unique member of UGDH gene family only found in P. aeruginosa, may be evolved from PA3559 by gene duplication to adapt to environmental changes. The distinct biochemical properties and gene expression profiles of PA2022 and PA3559 support this notion.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Kinetics of biofilm formation in P. aeruginosa PAO1 and UGDH mutants. The parental and mutant strains were grown in static culture in microtiter dishes. Biofilm formation kinetics was analyzed at different time points. The data are the means from two independent experiments, with each sample assayed in triplicate.

View larger version (73K): [in this window] [in a new window]   FIGURE 6. Micrographs of the biofilm formed by the wild type PAO1 and the double mutant RJ103. The tested bacteria were grown in a flow cell culture system and labeled by SYBR Green. Photographs were taken after 24 h of incubation using a laser-scanning confocal microscope at x100 magnification with scans taken through the z axis to obtain 3-µm optical sections. The panels above and to the right of each top-down micrograph show side view micrographs of axis reconstructions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The three-dimensional molecular structure of SpUGDH was solved and reported by Campbell et al. (27). Subsequently Snook et al. (33) determined the structure of a related enzyme, GMDH of P. aeruginosa PAO1. Using these structures as the template, the PA2022 and PA3559 structure predictions were performed at the Swiss-Model, and Fig. 8 shows the results. The structure model did not include C-terminal 100 amino acids because the high diversity of this region failed to fit proper templates. Overall, the structures of the two gene products resemble P. aeruginosa GMDH more than SpUGDH. The NAD⁺ and UDP-Glc binding sites and catalytic sites of PA2022 and PA3559 were identified by comparing highly conserved SpUGDH domains and are indicated in Fig. 8, A and B. Both proteins contain a NAD⁺ dinucleotide-binding domain in the N terminus and a nucleotide sugar-binding domain in the C-terminal regions, which are interconnected with a long (33 residues) -helix. Superimposing PA2022 and PA3559 reveals that both main structures are highly similar except for some flexible loops. Cys-266 in PA2022 and PA3559 is at a position equivalent to the catalytic nucleophile Cys-260 in SpUGDH. The distance between the UDP-Glc-binding site and the catalytic site is too great for the reaction occurring in a single protein molecule. Therefore, this study proposes that the enzymes become active only when they form domain-swapped dimers that place the N-terminal domain of one monomer in close association with the C-terminal domain of the adjacent monomer. However, this speculation must be tested experimentally.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Phylogenetic analysis of UGDH-like genes from several Pseudomonas species. The genes were grouped into four families as follows: PA2022-like UGDH (I), GMDH (II), UDP-GlcNAc DHase (III), and PA3559-like UGDH (IV). The following prefixes indicate the species: PA 14, P. aeruginosa PA14; Pfl_, P. fluorescens PfO-1; PFL, P. fluorescens Pf-5; PP, P. putida KT2440; Psyr, P. syringae pv. syringae B728a; PSPPH, P. syringae pv. phaseolicola 1448A; PSPTO, P. syringae pv. tomato str. DC3000.

View larger version (52K): [in this window] [in a new window]   FIGURE 8. The PA2022 and PA3559 structure models. The N terminus, NAD-binding site (NBS, yellow region), UDP-Glc-binding site (magenta loops), and the catalytic site (green regions) containing Arg-250 and Cys-266 in PA2022 (A) and PA3559 (B) are indicated.

This study provides direct evidence showing that UGDH is associated with antibiotic resistance in P. aeruginosa. In addition, PA2022 and PA3559 appear to play a distinct role in the phenomenon. The strains RJ101 and RJ103, in which PA2022 is deleted, are more susceptible to chloramphenicol and cefotaxime (Table 8) in both high and low Mg²⁺ conditions. This susceptibility reflects on the housekeeping role of PA2022. Because these antibiotics are distinct in their chemical structure and mode of action, changes in susceptibility to these antibiotics are most likely because of a nonspecific mechanism such as alterations in bacterial membrane permeability that modulate antibiotic entry into the cell. On the other hand, deleting PA3559 in P. aeruginosa caused a decrease in resistance levels to polymyxin B when propagated in low Mg²⁺ medium. Presumably, this effect is because of the higher expression levels of PA3559 in the growth conditions. Thus, PA3559 is likely to behave like its orthologous gene in the S. enterica serovar Typhimurium. The Salmonella UGDH gene participates in the pathway for synthesizing 4-amino-4-deoxy-L-arabinose, which affects lipid-A modification and produces polymyxin resistance (38). Nevertheless, other possibilities cannot be ruled out. For instance, the enzyme may modify or metabolize antibiotics directly, although we are not aware of any related finding being reported before.

Because of its critical role in EPS synthesis, UGDH has been considered as a crucial drug target (27). The presence of multiple UGDHs in Pseudomonas species makes this idea seem less attractive, however. Nevertheless, as a deficiency in UGDH could produce higher drug susceptibility in P. aeruginosa, a UGDH inhibitor may be used in combination with a clinically available antibiotic, as exemplified in the -lactam drugs/-lactamase inhibitor. Further investigation on how UGDH affects drug susceptibility in bacteria may reveal new implications for future drug development.

	FOOTNOTES

 
^* This work was supported by the National Science Council and the National Research Program of Genome Medicine, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Institute of Molecular Medicine, National Tsing Hua University, 101 Kuang Fu Rd., 2nd Sec., Hsin Chu 300, Taiwan. Tel.: 886-35742909; Fax: 886-35742910; E-mail: hychang{at}life.nthu.edu.tw.

² The abbreviations used are: UGDH, UDP-glucose dehydrogenase; UDP-GlcUA, UDP-glucuronic acid; DTT, dithiothreitol; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hyaluronic acid; HPLC, high pressure liquid chromatography; MIC, minimal inhibition concentration; LPS, lipopolysaccharide; EPS, of exo-polysaccharide; GMDH, GDP-Man dehydrogenase.

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