INTRODUCTION

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Pseudomonas aeruginosa is a leading cause of nosocomial infections (30) and one of the main causes of debilitating pulmonary infections in patients with cystic fibrosis (CF) (9, 30). It can express a number of cell surface and extracellular polysaccharides that are virulence factors and that contribute to its success as an opportunistic pathogen (3, 9). Specifically, this species produces two distinct types of lipopolysaccharide (LPS) (17, 26), and strains infecting CF patients often overproduce the extracellular polysaccharide alginate (9, 18). The protein encoded by the algC gene possesses both phosphomannomutase (PMM) and phosphoglucomutase (PGM) activities, which are required for the interconversion of mannose-6-phosphate (Man-6-P) or glucose-6-phosphate (Glc-6-P) to Man-1-P or Glc-1-P, respectively (2, 33). Man-1-P and Glc-1-P are required for the formation of the precursors GDP-D-mannuronic acid, GDP-D-mannose, UDP-D-glucose, and TDP-L-rhamnose, which are used for alginate, LPS, and rhamnolipid biosynthesis (2, 23, 25, 26, 33, 34). Several pieces of evidence indicate that the algC gene product is indeed common and essential to the biosynthetic pathways of both LPS and alginate in P. aeruginosa (2, 26, 33, 34). An algC homologue has been recently identified within the P. aeruginosa genome, but the enzymatic nature of the gene product has not been established (28). This open reading frame (ORF), provisionally called ORF540, theoretically coded for a protein of 445 amino acids. It was located in contig 54 (15 July 1999 Pseudomonas genome release), and its actual position on the single PAO1 contig is from position 5333450 to 5334784 (reverse complement) (15 December 1999 release) (Pseudomonas Genome Project [http://www.pseudomonas.com]). Interestingly, the corresponding amino acid sequence showed significant homology with proteins belonging to the phosphohexomutase family found in databases (11), particularly with gene products from Escherichia coli, Staphylococcus aureus, or Helicobacter pylori that were recently characterized as phosphoglucosamine mutases (5, 12, 21). These latter enzymes (GlmM) catalyze the formation of glucosamine-1-phosphate (GlcN-1-P) from GlcN-6-P, the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components such as peptidoglycan and LPS (11). We here report that the P. aeruginosa glmM gene can fully complement a well-characterized glmM deficiency in E. coli and that the protein purified to homogeneity showed the expected phosphoglucosamine mutase activity. It is also shown that GlmM enzymes from P. aeruginosa and E. coli exhibit additional PMM (relatively high) and PGM (low) activities.

	MATERIALS AND METHODS

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Bacterial strains, plasmid vectors, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. P. aeruginosa IST27N is a nonmucoid variant spontaneously derived from a mucoid strain isolated from a Portuguese CF patient at the Sta. Maria Hospital in Lisbon, Portugal (16, 28). This strain was used to obtain the genomic DNA used as a template to amplify ORF540 by PCR. P. aeruginosa strain 8858, carrying an algC mutation, is an alginate-negative mutant (34) and was used in complementation assays. E. coli strains JM83 (32) and GPM83 (21) were used as hosts for plasmids and for the preparation of the overproduced GlmM enzyme. Strain GPM83, which carries an inactivated copy of the glmM gene on the chromosome and a wild-type copy of glmM on a plasmid whose replication is thermosensitive, was used in complementation tests. The plasmid vector pTrc99A was purchased from Pharmacia (Uppsala, Sweden), and the pTrcHis60 vector was recently described (24). Recombinant plasmid pNZ49 carries the P. aeruginosa algC gene, encoding a protein with PMM and PGM activities, into the cloning vector pMMB66(HE) (34). This plasmid and the algC mutant 8858 were a kind gift from A. M. Chakrabarty (34). The broad-host-range controlled expression vector pMMB66(HE) was described earlier (7). Unless otherwise noted, 2YT (22) was used as a rich medium to grow E. coli strains, and Lennox L broth medium (Sigma Chemical Co., St. Louis, Mo.) was used to grow P. aeruginosa strains. Solid medium used to grow P. aeruginosa strains was Pseudomonas Isolation Agar (Difco Laboratories, Detroit, Mich.). Growth was monitored at 600 nm with a spectrophotometer (UV-1601; Shimadzu, Duisburg, Germany). For strains carrying drug resistance genes, antibiotics were used at the following concentrations, in micrograms per milliliter: ampicillin, 100; kanamycin, 30; chloramphenicol, 25 (for E. coli); and carbenicillin, 300 (for P. aeruginosa).

General DNA techniques. DNA restriction and modification enzymes were obtained from New England Biolabs (Beverly, Mass.), Appligene Oncor (Illkirch, France), and Gibco BRL (Gaithersburg, Md.). DNA fragments were purified with the Wizard purification system (Promega Corporation, Madison, Wis.). Total DNA was extracted from cells of P. aeruginosa IST27N grown overnight in Lennox L broth liquid medium at 30°C with orbital agitation by the method of Goldberg and Ohman (8). Small- and large-scale plasmid isolations from E. coli cells were carried out by the alkaline lysis method, and standard procedures for endonuclease digestions, ligation, and agarose electrophoresis were used (27). E. coli cells were made competent for transformation with plasmid DNA by treatment with CaCl₂ (4) or by electroporation. The ability of plasmids to complement the thermosensitive glmM mutant strain GPM83 was tested as described earlier (21). The ability of plasmids to complement the P. aeruginosa algC mutant was tested by introducing plasmid pNZ49, plasmid pIT352 (prepared as described below), or the cloning vector pMMB66(HE) into P. aeruginosa strains by triparental filter mating using the helper plasmid pRK2013 (6) as previously described (15). P. aeruginosa transconjugants were selected in Pseudomonas Isolation Agar plates with carbenicillin and incubated at 37 or 30°C (14) for up to 5 days to compare mucoidy.

Construction of plasmids. PCR primers used to amplify ORF540 from the P. aeruginosa IST27N chromosome were designed from the P. aeruginosa PAO1 genomic database sequence found to flank this ORF, according to the results of the Pseudomonas Genome Project (15 July 1999 release) (http://www .pseudomonas.com). These primer oligonucleotides were purchased from Pharmacia, and BamHI and HindIII restriction sites (shown in boldface below) were incorporated at the 5' ends of the sense and the antisense primers, respectively. The sense primer was specific for a 81-bp region upstream from the ORF540 initiation codon (5'-GGATCCGGCCAAGGGCGCACGGATAAT-3' [primer A]). The antisense primer used corresponds to oligonucleotides complementary to the 53-bp sequence downstream from the TGA stop codon (5'-AAGCTTGGGGCAAAGTGGGCGCAGATGTT-3' [primer B]). Amplification reactions were carried out in a final volume of 50 µl containing 0.3 nmol of each primer, 10 nmol of each deoxynucleoside triphosphate, 125 nmol of MgCl₂, 2.5 U of Taq polymerase (Appligene), and 250 ng of genomic DNA. Thermocycling reactions were performed in a PTC-100 thermal cycler (MJ Research, Inc., Watertown, Mass.) and consisted of an initial denaturation at 95°C (120 s) and 30 cycles of primer annealing at 61.8°C (30 s), extension at 72°C (60 s), and denaturation at 95°C (120 s). The 1,504-bp amplification product was recovered, purified from the gel using the Wizard PCR Preps DNA purification kit from Promega, and cloned into the vector plasmid pCR2.1 (Invitrogen, San Diego, Calif.), giving rise to plasmid pIT351. Plasmid pIT352 was constructed by ligating the HindIII 1,562-bp fragment of pIT351, encompassing the ORF540 sequence, under the control of the tac promoter into the broad-host-range controlled expression vector pMMB66(HE).

Preparation of crude enzyme. JM83, PAE831, and PAE832 cells were grown at 37°C in 2YT-ampicillin medium (0.5-liter cultures). When the optical density of the culture reached 0.1, IPTG (Eurogentec Seraing, Belgium) was added at a final concentration of 1 mM, and growth was continued for 3 h. Cells of thermosensitive mutant strain GPM83 were grown at 30°C, or first at 30°C and then for 5 h at the restrictive temperature of 42°C, as previously described (21). Cells were harvested and washed with 40 ml of cold 20 mM potassium phosphate buffer (pH 7.4) containing 0.5 mM MgCl₂ and 0.1% -mercaptoethanol (buffer A). The cell pellet was then resuspended in 5 ml of the same buffer and disrupted by sonication (VibraCell sonicator; Bioblock, Illkirch, France) for 3 min in the cold. The resulting suspension was centrifuged at 4°C for 30 min at 200,000 × g, and the supernatant was dialyzed overnight at 4°C against 100 volumes of buffer A. The resulting solution (5 ml; 10 to 12 mg of protein · ml¹), designated crude enzyme, was stored at 20°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins was performed as previously described using 12% polyacrylamide gels (13), and protein concentrations were determined by the method of Bradford (1), using bovine serum albumin as standard.

Purification of the His₆-tagged GlmM enzyme. The one-step purification procedure was carried out under native conditions, basically following the recommendations of the manufacturer (Qiagen, Santa Clarita, Calif.): binding of His₆-GlmM on Ni²⁺-nitrilotriacetate agarose and washing with buffer A containing 200 mM KCl and 20 mM imidazole to remove impurities, elution of the protein with increasing concentrations of imidazole added to buffer A (from 50 to 300 mM; the His₆-GlmM protein was eluted between 200 and 300 mM), and dialysis of eluted His₆-GlmM against buffer A containing 10% glycerol. The His₆-tagged enzyme prepared in this manner was more than 90% pure, as estimated by SDS-PAGE (Fig. 1).

View larger version (95K): [in this window] [in a new window]   FIG. 1.   Overproduction and purification of P. aeruginosa phosphoglucosamine mutase. Lane A, crude extract from parental JM83 cells; lanes B and C, crude extracts from PAE831 cells grown in the absence or presence of IPTG, respectively; lanes D and E, crude extracts from PAE832 cells grown in the absence or presence of IPTG, respectively. A high-level overproduction of the wild-type (lane C) and His6-tagged (lane E) forms of GlmM enzyme (arrowhead) is observed following induction of gene expression with IPTG. Lane F, purified His6-tagged GlmM enzyme. Molecular mass standards, indicated on the left, are as follows: phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; and ovalbumin, 43 kDa.

Enzymatic assays. (i) Biochemicals. ¹⁴C-labeled acetyl coenzyme A (acetyl-CoA) (1.9 GBq mmol¹) was from ICN (Irvine, Calif.). GlcN-6-P, acetyl-CoA, UTP, Glc-1-P, glucose-1,6-diphosphate (Glc-1,6-diP), Man-1-P, NAD, Glc-6-P dehydrogenase, and phosphomannose isomerase were bought from Sigma. Pure GlmU enzyme was prepared as previously described (24).

(ii) GlmM assay. The coupled assay in which the GlcN-1-P synthesized from GlcN-6-P by the mutase was quantitatively converted to UDP-N-acetylglucosamine in the presence of bifunctional GlmU enzyme (20, 24) was used. The standard assay mixture (100 µl) contained 50 mM Tris-HCl buffer (pH 8), 3 mM MgCl₂, 1 mM GlcN-6-P, 0.4 mM [¹⁴C]acetyl-CoA (700 Bq), 10 mM UTP, 0.7 mM Glc-1,6-diP, pure GlmU enzyme (1 µg), and GlmM enzyme (0.1 to 10 µg of protein, depending on overexpression or purification factors). Appropriate dilutions of the enzyme prior to assays were performed in buffer A supplemented with 100 µg of bovine serum albumin per ml. Reaction mixtures were incubated at 37°C for 30 min, and the reaction was stopped by the addition of 10 µl of glacial acetic acid. Reaction products were separated with high-voltage electrophoresis 3469 filter paper (Schleicher & Schuell, Dassel, Germany) in 2% formic acid (pH 1.9) for 90 min at 40 V · cm¹ using an LT36 apparatus (Savant Instruments, Hicksville, N.Y.). The radioactive spots were located and quantified with a radioactivity scanner (model Multi-Tracermaster LB285; EG&G Wallac/Berthold). One unit of enzyme activity was defined as the amount which catalyzed the synthesis of 1 µmol of product in 1 min.

(iii) PGM and PMM assays. The PGM activity of the GlmM enzyme was assayed at 37°C in a reaction mixture (100 µl) containing 50 mM Tris-HCl (pH 8), 5 mM MgCl₂, 1 mM Glc-1-P, 0.7 mM Glc-1,6-diP, 1 mM NAD, Glc-6-P dehydrogenase (2 µg), and pure GlmM enzyme (10 µg). The PMM activity of the GlmM enzyme was assayed at 37°C in a reaction mixture (100 µl) containing 50 mM Tris-HCl (pH 8), 5 mM MgCl₂, 1 mM Man-1-P, 0.7 mM Glc-1,6-diP, 1 mM NAD, Glc-6-P dehydrogenase (2 µg), phosphomannose isomerase (1 µg), and pure GlmM enzyme (1 µg). In both cases, the formation of NADH was monitored at 340 nm during a 2-h period with a Shimadzu UV-1601 spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that reduced 1 µmol of NAD per min under the assay conditions.

Isolation of sacculi and quantification of peptidoglycan. Exponential-phase cells (0.5-liter cultures) were grown at 37°C in 2YT medium supplemented with ampicillin. When the optical density of the culture reached 0.7 (about 3 × 10⁸ cells · ml¹), cells were harvested in the cold, washed with a cold 0.9% NaCl solution, and rapidly suspended by vigorous stirring in 20 ml of a hot (95 to 100°C) aqueous 4% SDS solution for 30 min. After standing overnight at room temperature, the suspensions were centrifuged for 30 min at 200,000 × g in a Beckman TL100 centrifuge, and the pellets were washed several times with water. Final suspensions made in 2 ml of water were homogenized by brief sonication. Aliquots were hydrolyzed and analyzed as previously described, and the peptidoglycan content of the sacculi was expressed in terms of its muramic acid content (19).

	RESULTS AND DISCUSSION

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The P. aeruginosa glmM gene complements a E. coli glmM mutation. A recent search for a potential algC homologue within the P. aeruginosa PAO1 genome database (http://www.pseudomonas.com) revealed a putative gene whose product showed 26% sequence identity with AlgC (28). A search within GenBank database showed significant homology of the AlgC homologue with enzymes belonging to the hexosephosphate mutase family. The highest degree of similarity was observed with recently characterized phosphoglucosamine mutases (GlmM) from E. coli, S. aureus, and H. pylori (40 to 60% identity for 445 amino acids). These enzymes catalyze the formation of GlcN-1-P from GlcN-6-P, an essential step in the pathway for UDP-N-acetylglucosamine synthesis (21, 29).

Overproduction and purification of P. aeruginosa phosphoglucosamine mutase in E. coli. When PAE831 and PAE832 cells were induced with 1 mM IPTG for 3 h, the accumulation of a protein species was observed (Fig. 1), whose molecular mass, about 50 kDa, was consistent with that calculated from the gene sequence (47.8 kDa). The overproduced protein represented about 10 to 15% of total cell proteins, and most of it (about 90%) was recovered in the soluble fraction after a typical cell fractionation. As shown in Table 2, the overproduction of this protein was correlated with an increase of phosphoglucosamine mutase activity in cell extracts. As the two E. coli strains PAE831 and PAE832 carried an inactivated copy of the glmM gene on the chromosome, they consequently expressed the P. aeruginosa gene present on plasmids as the sole source of phosphoglucosamine mutase. In the absence of IPTG, the GlmM activity that could be detected in these cells represented approximately 20% of the activity normally detected in a wild-type E. coli strain. It was sufficient, however, to complement a thermosensitive glmM mutant strain, suggesting that the E. coli GlmM enzyme is normally in great excess in wild-type cells with respect to specific cell requirements (21). When induced with IPTG, PAE831 and PAE832 cells contained 40-fold more phosphoglucosamine mutase activity than noninduced cells (Table 2). It should be mentioned that the levels of GlmM activity detected in these two strains were quite similar, suggesting that the presence of the His₆ tag extension at the C terminus of the protein had no significant effect on its enzymatic activity.

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Additional PGM and PMM activities of the P. aeruginosa phosphoglucosamine mutase. The ability of the P. aeruginosa GlmM enzyme to catalyze the interconversion of other sugar phosphates was investigated. The pure enzyme was shown to exhibit both PGM and PMM activities, which were 50- and 5-fold lower than its GlmM activity, respectively (Table 3). As reported recently (11), the E. coli GlmM enzyme also exhibits a PGM activity, which is 1,400-fold lower than its GlmM activity. The PMM activity of the E. coli enzyme was now tested and also appeared to be relatively high (1.75 U · mg of protein¹), representing almost 20% of its GlmM activity. The PMM activities of the two GlmM enzymes were thus much higher than their PGM activities and only slightly lower than their GlmM activities (Table 3). These results suggest that possibly all members of the phosphoglucosamine mutase family can use both mannose and glucose phosphates as substrates, besides glucosamine phosphate, although they can exhibit a preference for one substrate over another.