The Pseudomonas aeruginosa algC Gene Encodes ... - NCBI

Vol. 176, No. 12

JOURNAL OF BACTERIOLOGY, June 1994, p. 3500-3507

0021-9193/94/$04.00+0

Copyright © 1994, American Society for Microbiology

The Pseudomonas aeruginosa algC Gene Encodes Phosphoglucomutase, Required for the Synthesis of a Complete Lipopolysaccharide Core MICHAEL J. COYNE, JR., KRISTIN S. RUSSELL, CARYN L. COYLE, AND JOANNA B. GOLDBERG* Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts Received 10 December 1993/Accepted 8 April 1994 We have constructed strains of Pseudomonas aeruginosa with mutations in the algC gene, previously shown to encode the enzyme phosphomannomutase. The algC mutants of a serotype 05 strain (PAO1) and a serotype 03 strain (PAC1R) did not express lipopolysaccharide (LPS) 0 side chains or the A-band (common antigen) polysaccharide. The migration of LPS from the algC mutant strains in Tricine-sodium dodecyl sulfatepolyacrylamide gels was similar to that of LPS from a PAO1 LPS-rough mutant, strain AK1012, and from a PAClR LPS-rough mutant, PAC605, each previously shown to be deficient in the incorporation of glucose onto the LPS core (K. F. Jarrell and A. M. Kropinski, J. Virol. 40:411420, 1981, and P. S. N. Rowe and P. M. Meadow, Eur. J. Biochem. 132:329-337, 1983). We show that, as expected, the algC mutant strains had no detectable phosphomannomutase activity and that neither algC strain had detectable phosphoglucomutase (PGM) activity. To confirm that the PGM activity was encoded by the algC gene, we transferred the cloned, intact P. aeruginosa algC gene to a pgm mutant of Escherichia coli and observed complementation of the pgm phenotype. Our finding that the algC gene product has PGM activity and that strains with mutations in this gene produce a truncated LPS core suggests that the synthesis of glucose 1-phosphate is necessary in the biosynthesis of the P. aeruginosa LPS core. The data presented here thus demonstrate that the algC gene is required for the synthesis of a complete LPS core in two strains with different LPS core and 0 side chain structures.

algC gene) might interrupt 0 side chain synthesis in strains with mannuronic acid residues in their 0 side chains. However, the structural alteration in the LPS core of the algC mutant AK1012 had previously been shown to involve the loss of glucose residues (21). To further investigate the involvement of algC in the synthesis of the P. aeruginosa LPS, we constructed algC mutants of strains PAO1 (serotype 05) and PAClR (serotype 03) and determined the effects of these defined mutations on the core LPS and on expression of both 0 antigen and A-band (or common antigen) LPS. The antigenically conserved A-band LPS, composed mostly of D-rhamnose (3), is expressed by P. aeruginosa strains on the same core as 0-antigenic LPS, independent of serotype (17). The 0 side chain antigen of strain PAClR does not contain mannuronic acid residues but rather contains tetrasaccharide repeating units consisting of L-rhamnose, D-glucosamine, D-bacillosamine, and L-galactosaminuronic acid (23). The LPS core structures of strains PAO1 and PAClR are known, and defined LPS core mutants of these strains are available for comparison (21, 39). Further, neither of these core structures contains mannuronic acid residues. Here, we show that algC mutants of PA01 and PAClR do not express LPS 0 side chain antigen or A-band LPS and synthesize an incomplete LPS core. In addition, we show that, compared with the wild-type strains, the algC mutants lack not only PMM activity but also phosphoglucomutase (PGM) activity, which suggests that the algC gene is required not only for the synthesis of mannose 1-phosphate but also for the formation of glucose 1-phosphate. This form of glucose is presumably a necessary intermediate in the pathway leading to the addition of glucose residues to the LPS core. In addition, we demonstrate that the P. aeruginosa algC gene is

We previously determined that DNA cloned from Pseudomonas aeruginosa PAO1 which complemented the rough lipopolysaccharide (LPS), serum-sensitive phenotype of mutant strain AK1012 was equivalent to the algC gene (12). Zielinski et al. have characterized the algC gene and determined that it encodes the enzyme phosphomannomutase (PMM) (49). This enzyme interconverts mannose 6-phosphate and mannose 1-phosphate and is an obligate enzyme of the pathway leading to the biosynthesis of the exopolysaccharide alginate. Zielinski et al. (49) detected an increase in PMM activity in P. aeruginosa attributable to the algC gene in recombinant plasmid pNZ49 and purified the enzyme from P. aeruginosa containing this plasmid. We found that recombinant plasmid pNZ49 complemented the LPS-rough phenotype of strain AK1012 and, conversely, that our cloned gene complemented the alginate mutation in a P. aeruginosa algC mutant, strain 8858

(12). In this previous study (12), we suggested that the algC gene might be required for production of mannuronic acid residues, similar to those found in alginate, which are present in the 0 antigen side chain of P. aeruginosa PAO1 (serotype 05). Since the serotype 05 0 side chain trisaccharide repeating units consist of two molecules of 2-acetamido-3-acetamidino-2,3dideoxy-mannuronic acid and one molecule of N-acetyl-Dfucosamine (23), we hypothesized that the loss of an enzyme involved in the catabolism of mannose (PMM encoded by the * Corresponding author. Mailing address: Channing Laboratory, Harvard Medical School, 180 Longwood Ave., Boston, MA 021155899. Phone: (617) 432-2193. Fax: (617) 731-1541. Electronic mail address: [email protected].

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algC MUTANTS OF PSEUDOMONAS AERUGINOSA

TABLE 1. Bacterial strains, phage, and plasmids Strain, phage, or plasmid

Revelant characteristic(s)

Strains P. aeruginosa PAOI AK1012 PAClR PAC605 PAC612 PAC556 PAC557 PAC610 PAC608 PAO1 algC::tet PAClR algC::tet E. coli

DH5ox W1485 W1485pgmA::tet Bacteriophage E79 Plasmids pUCP18 pLPS188 pKI11 pNOT19 pMOB3 pKR3 pRK2013 pMMB66HE pNZ49 pML14

Source or reference

Serotype 05 Rough LPS derivative of PAO1 Serotype 03 Rough LPS derivative of PACIR Rough LPS derivative of PAClR Rough LPS derivative of PAC1 Rough LPS derivative of PACI Rough LPS derivative of PAClR Rough LPS derivative of PAClR algC mutant of PAO1 algC mutant of PACIR

25 20 39 33, 39 39 39 39 39 39 This study This study

Plasmid host Wild-type strain pgm mutant of W1485 0 side chain specific

Gibco-BRLU 30 30 25

Ap', broad-host-range plasmid algC gene in pUCP18 Contains Tcr cartridge pUC19 + NotI site sacB Cmr oriT algC::Tc' in pNOT19 + pMOB3 Kmr helper plasmid Apr broad-host-range plasmid algC gene in pMMB66HE Sp', contains E. coli pgm geneb

43 12 19 44 44 This study 9 10 49 30

aGibco-BRL Corporation, Gaithersburg, Md. Spr, spectinomycin resistance.

b

able to complement an Escherichia coli pgm strain, thereby restoring this strain with the ability to ferment galactose.

MATERIALS AND METHODS Bacterial strains, plasmids, plasmid mobilization, and media. Bacterial strains, plasmids, and bacteriophage are described in Table 1. Plasmids were transformed into E. coli with CaCl2-treated (5) or RbCl2-treated (15) competent cells as recipients and mobilized into P. aeruginosa by triparental mating as previously described (13) or introduced by electroporation (46). P. aeruginosa was selected on cetrimide agar (Difco Laboratories, Detroit, Mich.). L agar or tryptic soy agar was used otherwise. MacConkey agar base (Difco Laboratories) containing 1% galactose was used to distinguish E. coli pgm+ strains (red) from pgm mutant strains (pink) (1, 32). Isopropyl-p3-D-thiogalactopyranoside (IPTG, 40 ,ug/ml) was added to induce the tac promoter of pNZ49. Antibiotics used for selection were tetracycline (10 ,ug/ml for E. coli and 100

[ig/ml for P. aeruginosa), chloramphenicol (25 ,ug/ml for E. coli), ampicillin (50 pug/ml for E. coli), carbenicillin (500 p.g/ml for P. aeruginosa), and spectinomycin (80 ,ug/ml for E. coli). IPTG and 5-bromo-4-chloro-3-indolyl-,3-D-galactopyranoside (X-Gal, 40 pg/ml) were added to solid media to detect the loss of lacZoc complementation in cloning experiments utilizing appropriate vector-host combinations. DNA manipulations. Restriction endonuclease, alkaline phosphatase, and T4 DNA ligase reactions were carried out as recommended by the supplier (Boehringer Mannheim Biochemicals Corporation, Indianapolis, Ind.). Plasmid DNA was isolated from strains of E. coli by the method of Birnboim and

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Doly (4) and from P. aeruginosa as previously described (13) or from E. coli and P. aeruginosa by using the Magic Mini-Prep kit as suggested by the manufacturer (Promega Corporation, Madison, Wis.). Chromosomal DNA was isolated from P. aeruginosa strains either as previously described (13) or as described by Woo et al. (48). Southern blot hybridization was performed as previously described (13). Restriction fragments of plasmid DNA were recovered from agarose gels with the GeneClean II kit as recommended (BIO 101, Inc., La Jolla, Calif.) and subsequently labeled with [32P]dCTP by using a nick translation kit (Boehringer Mannheim Biochemicals Corporation). Construction of a vector for insertional inactivation of the algC gene. The strategy for construction of a gene replacement vector for inactivating the algC gene was based on the system developed by Schweizer (44). The wild-type algC gene, contained within recombinant plasmid pLPS188 (12), was insertionally inactivated by cloning the 1.4-kb tetracycline resistance (Tcr) cartridge from pKI11 (19) into the unique PstI site within the algC gene (49), thereby producing plasmid pKR1. The 3.6-kb SacI-NotI fragment from pKR1, which includes 2.2 kb of the 2.6-kb algC-containing insert from pLPS188 and all of the 1.4-kb Tcr cartridge, was cloned into the NotI-SacI sites of the ampicillin-resistant (Apr) vector, pNOT19 (44), thereby producing plasmid pKR2. The chloramphenicol resistance (Cmr), sacB, and oriT genes from pMOB3 (44) were recovered on a 5.8-kb NotI fragment and cloned into the unique Notl site of pKR2. The resultant plasmid, pKR3, contains a portion of the P. aeruginosa algC gene that was insertionally inactivated by the Tcr cartridge, as well as genes encoding Apr, Cmr, sucrose sensitivity (encoded by the sacB gene), and an origin of transfer, oriT. Plasmid pKR3 does not, however, contain an origin of replication usable by P. aeruginosa and is thus designated a nonreplicative integration vector. Serum resistance. Serum resistance was assessed by use of a rapid screening method. Bacterial cells were grown on media with appropriate selection overnight at 37°C and resuspended to an optical density at 600 nm of 0.2 in 0.9% saline; 100-,ul samples of this suspension were spread onto tryptic soy agar plates, and 2.5-,ul samples of normal human serum, either undiluted or serially diluted in saline, were spotted on the plates. The plates were incubated at 37°C overnight and were visually inspected. The serum resistance level of the strain being tested was assessed as the highest level of serum concentration at which growth of the bacterial lawn was unaffected. E79 sensitivity testing. Bacterial lawns of strains to be tested were spotted with 2.5 RI of the 0 side chain-specific bacteriophage E79 (25) at approximately 1010 PFU/ml. Sensitivity was indicated by a zone of clearing surrounded by growth of the bacteria after overnight incubation. LPS isolation. LPS was isolated by a modification of the method of Hitchcock and Brown (18). Strains were harvested in sterile phosphate-buffered saline (PBS) after overnight growth on appropriately supplemented L agar plates. The suspensions were autoclaved and vigorously vortexed, and the cells were pelleted by centrifugation. The supernatants were combined with four volumes of 95% ethanol and precipitated at -20°C overnight. The precipitates were collected by centrifugation and redissolved in sterile PBS. Aliquots of these preparations were digested with RNase and DNase (each at 100 ,ug/ml) at 37°C overnight and then with pronase (100 pg/ml) for 2 h at 56°C (enzymes were purchased from Boehringer Mannheim Biochemicals Corporation) and stored at -20°C until analysis. SDS-PAGE. Gels for sodium dodecyl sulfate-polyacrylamide

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COYNE ET AL.

gel electrophoresis (SDS-PAGE) were run essentially as described by Laemmli (26), using the Mighty Small II apparatus (Hoefer Scientific Instruments, San Francisco, Calif.). SDSPAGE analyses were performed with precast SDS-12% polyacrylamide gels (purchased from Jule Biotechnologies, Inc., New Haven, Conn.). In order to analyze LPS core samples, a two-buffer Tricine-based SDS-PAGE system was employed (28, 41). Tricine gels were cast as 18% separating gels. Before analysis by SDS-PAGE, an aliquot of the LPS sample was combined with an equal volume of 2x sample buffer (125 mM Tris, 4.1% SDS, 20% dithiothreitol, 0.001% bromophenol blue) and heated to 65°C for 15 to 30 min. Gels were silver stained by using a silver stain kit (Bio-Rad Laboratories, Hercules, Calif.) or electroblotted onto membranes for immunologic analysis (see below). Electroblotting and immunological detection. LPS samples separated by SDS-PAGE were transferred to Duralon-UV uncharged nylon membranes (Stratagene, La Jolla, Calif.) by semidry electrotransfer with the SemiPhor transfer unit (Hoefer Scientific Instruments) according to the manufacturer's instructions. The membranes were air dried and blocked by overnight incubation in blocking solution (5% skim milk, 10% PBS, 0.005% thimerosal). The blocked membranes were rinsed three times with PBS containing 0.05% Tween 20. To detect 0 side chain antigens, the membranes were incubated in polyclonal rabbit antisera (raised against purified P. aeruginosa LPS of the appropriate serotype; kindly provided by K. Hatano and G. B. Pier, Harvard Medical School, Boston, Mass. [36]) diluted 1:400 in PBS containing 10% blocking solution or, to detect A-band LPS, in a solution of mouse monoclonal antibody (MAb) NlFlO (kindly provided by J. Lam, University of Guelph, Guelph, Canada) (27). After being rinsed as described above, the membranes were further incubated for 2 h in alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G heavy- and light-chain antibodies diluted 1:2,000 (when polyclonal rabbit antiserum was the primary antibody) or (when MAb NlFlO was used as the primary antibody) in alkaline phosphatase-conjugated goat anti-mouse immunoglobulin M diluted 1:500 (both alkaline phosphatase-conjugated antibody preparations were purchased from Tago, Inc., Burlingame, Calif.) Secondary antibody dilutions were made in the blocking solution supplemented with 0.05% Tween 20. The membranes were rinsed again three times as described above and developed with 5-bromo-4-chloro-3-indolyl-phosphate-4nitroblue tetrazolium chloride phosphatase substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.). Enzyme assays. Cultures of the P. aeruginosa parental strains and the algC::tet mutants were grown overnight at 37°C in L broth with shaking at 250 rpm, diluted 1:100 into fresh L broth, and grown for an additional 6 h at 37°C. Cells were harvested by centrifugation at 8,000 x g for 20 min and washed twice in 0.9% saline. Pellets were stored at -70°C until use. The pelleted cells were resuspended in 100 mM MOPS (morpholinepropanesulfonic acid [pH 7.0])-i mM dithiothreitol-10% glycerol and broken in a French press (American Instrument Company, Urbana, Ill.) at a cell pressure of 12,000 lb/in2. The samples were centrifuged at 40,000 x g for 30 min, and the supernatants were used as a crude extract. Assays for the enzyme PMM were performed essentially as described previously (12, 40, 49). PMM activity was determined by quantitatively measuring the increase in optical density at 340 nm, caused by the reduction of NADP to NADPH after the addition of 1 pLmol of mannose 1-phosphate to the extracts. For quantitative assessment of PGM activity, 1 pumol of glucose 1-phosphate was added instead of mannose 1-phosphate, and spectrophotometric measurements were recorded

J. BACTERIOL.

as described above. One unit of PMM or PGM enzyme activity was defined as the amount leading to the reduction of 1 pLmol of NADP to NADPH per min. The total protein concentrations of the extracts were determined with a bicinchoninic acid protein assay reagent (Pierce, Co., Rockford, Ill.) with bovine serum albumin as a standard. Specific activity is expressed as the milliunits of activity per milligram of protein and is the average of two assays each for PMM and PGM enzyme activities.

RESULTS Construction of algC mutants of P. aeruginosa. Plasmid pKR3 contains a portion of the algC gene that was insertionally inactivated with a Tcr cartridge. In addition, the plasmid contains the structural gene for the Bacillus subtilis exoenzyme levansucrase, sacB, which confers sensitivity to sucrose (11). Plasmid pKR3 also contains an origin of transfer (oriT) and is thus conjugatively transferable from E. coli to P. aeruginosa when transfer functions are provided in trans by the helper plasmid pRK2013. This nonreplicative integration vector can be transferred to but cannot be maintained as a plasmid in P. aeruginosa; therefore, successful growth under selective conditions requires plasmid integration at sites of chromosomal homology (here, the algC gene). This plasmid was transferred to P. aeruginosa PAO1 (serotype 05) and PAClR (serotype 03), and Tcr integrants (from the Tcr cartridge in algC) were selected on centrimide agar and screened for resistance to carbenicillin (Cbr, conferred by the Apr marker carried by the plasmid). In the context of these experiments, the Tcr Cbr antibiotic resistance pattern is indicative of merodiploid strains. Resolution of the merodiploids was promoted by growth of the strains in the presence of 5% sucrose to select for homologous excision and loss of the plasmid-encoded sacB gene. The resulting colonies were screened for concurrent loss of carbenicillin resistance. Chromosomal DNA was recovered from parental strains and from tetracycline- and sucroseresistant, carbenicillin-sensitive derivative colonies of these strains, digested with Notl and Sacl, and separated by gel electrophoresis. Southern blot hybridization experiments using the 2.2-kb NotI-SacI fragment from pLPS188 as a probe were performed to confirm that true algC allelic replacement had occurred. The probe hybridized, as expected, to a 2.2-kb fragment (the size of the algC-containing DNA fragment recovered from pLPS188) of DNA from the parental strains PAO1 and PAClR and to a 3.6-kb fragment (the algCcontaining DNA increased in size by insertion of the 1.4-kb Tcr gene) of chromosomal DNA recovered from the tetracyclineand sucrose-resistant, carbenicillin-sensitive isolates of these strains. The gene-replaced strains of PAO1 and PAClR are referred to as PAO1 algC::tet and PAClR algC::tet, respec-

tively. Characterization of P. aeruginosa PAO1 algC::tet and PACIR algC::tet. Strains PAO1 algC::tet and PAClR algC::tet were compared with the parental strains PAO1 and PACIR for serum resistance. Each of the parental strains was resistant to serum at a concentration of 100%, while the algC::tet mutant strains were killed by exposure to a concentration of serum as low as 5%. These strains were also tested for sensitivity to an 0 side chain-specific bacteriophage, E79 (25). The parental strains were sensitive to E79, while the algC::tet mutant strains were resistant to the bacteriophage. Since serum resistance and bacteriophage E79 sensitivity are both conferred on the bacteria by the presence of LPS 0 side chains (16, 25), these results confirm that the algC gene is required for the expression of 0 side chains by P. aeruginosa PAO1


VOL. 176, 1994 1 2 3

4 5 6

A 1

...

3503

B 2

3 4 5 6

7 8

1 2

3 4 5

6

A.... a w

..

FIG. 2. LPS was isolated from P. aeruginosa strains, subjected to Tricine-SDS-PAGE, and silver stained. (A) Lanes: 1, PAC1R; 2, PAClR algC::tet; 3, PAC605; 4, PAC612; 5, PAC556; 6, PAC557; 7, PAC610; 8, PAC608. (B) Lanes: 1, PAO1; 2, PAO1 aIgC::tet; 3, AK1012; 4, PACIR; 5, PAClR algC::tet; 6, PAC605. C

FIG. 1. LPS was isolated from P. aeruginosa strains. (A) Silverstained SDS-12% PAGE gel. (B) Immunoblot reacted with antiserum to P. aeruginosa serotype 05 LPS (lanes 1 to 3) or serotype 03 LPS (lanes 4 to 6). (C) Immunoblot reacted with MAb NIFIO, specific to P. aeruginosa A-band LPS. Lanes: 1, PA01; 2, PAG1 algC::tet; 3, PAO1 a/gC::tet(pLPS188); 4, PACIR; 5, PAClR algC::tet; 6, PAClR algC::tet(pLPS188).

serotype 05, as was previously determined (12), and by P. aeruginosa PACIR serotype 03 and demonstrate that the mutation introduced into the algC gene prevents these bacteria from expressing LPS 0 side chain molecules. To confirm that the insertion in the algC gene was not simply producing a polar effect on downstream genes required for LPS expression, we transferred the plasmids pUCP18 (43) and pLPS188 (which contains the wild-type algC gene cloned in pUCP18 [12]) to PAO1 algC::tet and PAClR algC::tet. Strains PAO1 algC::tet(pLPS188) and PAClR algC::tet(pLPS188) each reacquired the serum resistance, bacteriophage E79sensitive phenotypes of the parental strains, while each of the algC::tet strains containing pUCP18 alone remained serum sensitive and resistant to the 0 side chain-specific bacteriophage. These results indicate that the phenotypes observed in the algC::tet mutants are due specifically to the defect introduced into the algC gene and not to polar effects on downstream genes. algC mutants do not express LPS

0 side chain antigens or A-band LPS. LPS was isolated from the parental wild-type strains PAO1 and PAC1R, from mutant strains PA01 algC::tet and PACIR algC::tet, and from the complemented mutant strains PA01 algC::tet(pLPS188) and PAClR algC::tet (pLPS188). These LPS samples were separated by SDS-PAGE with a 12% gel and visualized by silver staining. As seen in Fig. 1A, both the parental strains and the complemented mutant strains produce a laddering pattern, visible after silver staining, typical of smooth LPS. The LPS from the algC mutants, PAO1 algC::tet and PAClR algC::tet, did not produce this laddering pattern. To confirm that the laddering observed was due to the expression of 0-antigenic LPS, parallel gels were blotted onto nylon membranes and reacted with polyclonal rabbit antiserum raised to purified P. aeruginosa serotype 05 or serotype 03 LPS. These antisera did not react to LPS recovered from the

algC::tet mutant strains, while strong reactions occurred to LPS from the parental and complemented strains, again demonstrating the pattern typical of LPS-smooth strains (Fig. 1B). These results indicate that strains PAO1 algC::tet and PAClR algC::tet each lack the 0 side chain serotype epitopes. We also reacted a parallel immunoblot with MAb NIF10, which is specific to A-band LPS (27). This MAb reacted with LPS from both parental strains and both algC::tet(pLPS188) strains but failed to react to LPS samples recovered from either of the algC::tet mutants (Fig. IC). This finding indicates that the algC mutation also prevents these bacteria from expressing A-band LPS. Comparison of LPS from the algC::tet mutant strains to spontaneously derived LPS-rough mutants. In order to compare the LPS core samples isolated from PA01 algC::tet and PAClR algC::tet with samples from spontaneously derived LPS-rough isolates of the same parental strains, we used a Tricine-SDS-PAGE system capable of resolving slight differences in molecular size (28). To assess the sensitivity of this system, LPS from PACIR and PAClR algC::tet was applied to a Tricine-SDS-PAGE gel and compared with samples isolated from various rough mutants of PACIR (Fig. 2A). These PAClR mutants are genetically undefined, but the structure of the core LPS of each is known; these mutants differ from one another in the number of sugar residues in the LPS core (39). In the Tricine-SDS-PAGE system, the LPS from PACIR algC::tet migrates most similarly to LPS recovered from strain PAC605 (Fig. 2A). LPS isolated from PAO1, PAO1 algC::tet, and AK1012 was compared with samples isolated from PAC1R, PACIR algC::tet, and PAC605. The two defined algC mutant strains, PA01 algC::tet and PACIR algC::tet, and the two spontaneously derived LPS mutant strains, AK1012 and PAC605, all displayed similar migration patterns on silverstained Tricine gels (Fig. 2B) and in each case differed from samples recovered from the parental strains. These data indicate that the LPS core of the algC::tet mutant strains is altered from that of the wild type in a manner that results in a core similar in size to that of the structurally defined mutants. Strains PAO1 algC::tet and PACIR algC::tet have no detectable PMM or PGM activity. We tested crude extracts of strains PA01, PAOI algC::tet, PAC1R, and PACIR algC::tet for PMM activity. Extracts of PAO1 and PAClR had an average PMM specific activity of 0.48 and 0.28 mU/mg of protein, respectively, while neither of the algC::tet mutants had detectable PMM activity. We also tested these extracts for PGM activity, using glucose 1-phosphate as a substrate. Extracts of the parental strains each contained measurable PGM specific activity, 0.71 and 0.92 mU/mg of protein for PAOI and

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COYNE ET AL.

PAC1R, respectively. There was no detectable PGM activity in extracts of either algC::tet strain. These results indicate that the wild-type strains PAO1 and PAClR each contain PMM and PGM activity, and insertional inactivation of the algC gene in either of these serotype strains results in mutants with neither PMM nor PGM activity. Complementation of an E. coli pgm mutant with the P. aeruginosa algC gene. To confirm that the P. aeruginosa algC gene product was responsible for the observed PGM activity, we used a defined pgm mutant of E. coli (30). This strain, E. coli W1485 pgmA::tet (kindly provided by M. Lu and N. Kleckner, Harvard University, Cambridge, Mass.), was constructed by replacing the wild-type pgm gene in strain W1485 with a copy in which a central deletion is replaced by a Tcr gene. We transferred pNZ49 (containing the P. aeruginosa algC gene cloned downstream from an inducible tac promoter [49]) or its vector, pMMB66HE, to the parental E. coli W1485 and the pgm mutant W1485 pgmA::tet. The ability of E. coli to ferment galactose is dependent on the presence of PGM activity (1, 32). Colonies were streaked onto MacConkey galactose agar plates to test for PGM activity. All wild-type W1485 strains containing plasmids grew as bright red colonies on MacConkey galactose agar, as did the parental strain, indicating fermentation of galactose. All W1485 pgmA::tet derivatives grew as pink colonies on this agar. As a control for this experiment, the E. coli pgm gene contained within plasmid pML14 (kindly provided by M. Lu and N. Kleckner) (30) was transferred to W1485 pgmA::tet; this complemented strain produced bright red colonies on MacConkey galactose agar. Since IPTG induces the tac promoter of the cloned algC gene on pNZ49, we supplemented the MacConkey galactose agar plates with IPTG and repeated the experiment. Strain W1485 pgmA::tet(pNZ49) now appeared bright red, while W1485 pgmA::tet(pMMB66HE) remained pink, indicating that the cloned P. aeruginosa algC gene has PGM activity that can complement thepgm mutation in E. coli W1485pgm/A::tet. The necessity for strong promotion of this gene in order to visualize complementation is not surprising, as the algC gene is expressed at low levels even in P. aeruginosa (49). DISCUSSION It is intriguing that the product of the algC gene is common to the biosynthesis pathways of both LPS and alginate in P. aeruginosa. In infections in cystic fibrosis patients, the two most prominent changes associated with the conversion of acutely infecting P. aeruginosa strains to those capable of producing long-term infections involve alteration in the expression of these polysaccharides. While the pathway for alginate biosynthesis is now well understood (6, 7, 31), less is known about the genes required for LPS biosynthesis in these organisms. Typical strains of P. aeruginosa found in the environment and in acute infection produce long LPS 0 side chains (LPS-smooth) and are nonmucoid. In contrast, strains of P. aeruginosa isolated from the lungs of chronically infected cystic fibrosis patients tend to produce LPS with few or short 0 side chains and are mucoid because of the overproduction of alginate (16). Since loss of the LPS-smooth phenotype is concomitant with the overproduction of alginate, mutations eliminating expression of the algC gene or resulting in a drastically altered product cannot be responsible for these shifts, as this gene is required for the biosynthesis of both LPS core and alginate. In addition, A-band polysaccharides are frequently present on chronically colonizing strains of P. aeruginosa isolated from cystic fibrosis patients (27). Our results demonstrate that the

J. BACT1ERIOL. Glc

Glc

Rha

Gic

Glc--

GaIN

AK1012

KDO Hep

Hep

KDO

PAO1

...

Ala

Gic

Rha .

.....

..

.

.

.

..

.... _ _ _ .I

Glc

Glc

PAC6O5

*

GalN-

Hep

KDO

KDO

*....PAC1 R Ala

FIG. 3. Structures of the LPS cores of P. aeruginosa PAO1 and PAClR and their relevant LPS-rough mutants. KDO, 2-keto-3-deoxyoctonic acid; Hep, heptose; GalN, galactosamine; Ala, alanine; Rha, rhamnose; Glc, glucose. Dotted lines indicate deficiencies in the LPS-rough mutants AK1012 and PAC605. (Data from references 2 and 39, used with permission.)

loss of a functional algC gene results in a strain unable to express A-band LPS. LPS core samples recovered from the two structurally defined mutants, AK1012 and PAC605, and from the two genetically defined algC mutant strains, PAO1 algC::tet and PAClR algC::tet, all display similar migration patterns upon separation by Tricine-SDS-PAGE (Fig. 2B). It is significant that the LPS cores of AK1012 and PAC605 have both been shown to be lacking in glucose residues (21, 39) (Fig. 3). The deficiency of glucose residues in the LPS core of strain AK1012 was recently confirmed (2). In addition, when provided with either the P. aeruginosa algC gene (12) or the E. coli pgm gene (data not shown), strain AK1012 regains the ability to express both serotype 05 and common antigen LPS. These observations provide compelling evidence that the algC gene product catalyzes the production of an intermediate necessary for the addition of glucose residues to the LPS core. Strains with mutations in this gene would be unable to synthesize a complete core and would not express 0 side chain antigens or A-band LPS. Our finding that the algC gene has enzymatic activity in addition to that of PMM-that of PGM-suggests that either glucose 6-phosphate or glucose 1-phosphate is required for completion of the LPS core. Since PGM is a bidirectional enzyme, the inability of the mutants studied to synthesize a complete LPS core when lacking the PGM activity of a functional algC gene product may indicate a requirement for glucose in either the 1- or 6-phosphate form. Glucose 6-phosphate, however, is a product or intermediate in a number of catabolic pathways in P. aeruginosa, including the utilization of mannitol and fructose. In these sugar utilization pathways, the enzyme phosphoglucose isomerase catalyzes the conversion of fructose 6-phosphate to glucose 6-phosphate. P. aeruginosa phosphoglucose isomerase mutants have been described previously (35); mutants lacking this enzyme are unable to grow on minimal

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medium containing mannitol as the sole carbon source. We found that PAO1 algC::tet was able to grow normally on minimal medium with mannitol as the sole carbon source (data not shown), thus indicating that this strain contains an intact phosphoglucose isomerase enzyme and therefore can synthesize glucose 6-phosphate. In E. coli, glucose 1-phosphate is produced not only by the action of PGM but also during the fermentative breakdown of many carbohydrates (1). P. aeruginosa, however, is a nonfermentative aerobe that dissimilates sugars by extracellular oxidation or phosphorylative degradation (29). Neither of these alternative pathways produces glucose 1-phosphate as an intermediate. Thus, P. aeruginosa apparently has no source of glucose 1-phosphate other than the conversion of glucose 6-phosphate via PGM. These facts, coupled with evidence that PAO1 algC::tet retains pathways capable of producing glucose 6-phosphate (as evidenced by its ability to grow on minimal medium containing mannitol), indicate that the LPS core defect in the algC mutants likely reflects an inability to produce glucose 1-phosphate. This lack of glucose 1-phosphate presumably precludes the synthesis of uridine diphosphoglucose, known to be crucial to the production of core LPS in other gram-negative bacteria (42). Our finding that the algC gene, previously shown to encode a protein with PMM activity, was able to complement the pgm mutation in the E. coli pgm mutant confirms the bifunctional nature of this gene product in the synthesis of both mannose 1-phosphate and glucose 1-phosphate. Padgett and Phibbs, reporting on the partial purification of PMM from mucoid and nonmucoid strains of P. aeruginosa, indicated that they were unable to separate PGM activity from partially purified PMM enzyme (34). More recently, a gene (xanA) that encodes both PMM and PGM activities was isolated and characterized from Xanthomonas campestris (24). X campestris is a gram-negative plant pathogen, closely related to P. aeruginosa, that produces both LPS and an exopolysaccharide, xanthan gum. Koplin et al. (24) analyzed a spontaneously exopolysaccharide-deficient isolate of X campestris and found it to contain an insertional mutation (ca. 0.3 kb) in the xanA gene. In this xanA mutant, both PMM and PGM activities were reduced to 0.2% of wild-type levels; however, this mutant still appeared by colony morphology to make LPS and synthesized low levels of an altered xanthan gum. These investigators hypothesized that xanA encodes an enzyme or regulatory factor necessary for the biosynthesis of both mannose 1-phosphate and glucose 1-phosphate in X campestris. Our observation of the P. aeruginosa algC gene's ability to complement a pgm mutation in a heterologous background minimizes the possibility that the algC gene product is simply a regulatory factor and indicates instead that it encodes an enzyme with PGM activity. Koplin et al. (24) also reported that the inferred amino acid sequence of the X campestris xanA product is similar to that of rabbit muscle PGM (37). The similarity between rabbit muscle PGM and the deduced amino acid sequence of the xanA gene was particularly apparent (47.8% identity) in the 23-aminoacid region identified as the active site in the rabbit muscle protein. Zielinski et al. (49) also noted well-conserved sequence similarity (42.8% identity) between the derived amino acid sequence of P. aeruginosa algC and this region of the rabbit muscle PGM protein. At the time these reports were published, neither Zielinski et al. (49) nor Koplin et al. (24) had the benefit of the others' work; thus, a direct comparison of these two sequences was unavailable. A comparison of the derived amino acid sequences of the algC and xanA genes by using the GAP program included in the Genetics Computer Group sequence analysis software package (version 7.2, Octo-


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ber 1992) (8) reveals 56.5% similarity and 34.5% identity along the full lengths of these polypeptides. Furthermore, both of these sequences contain the PGM and PMM phosphohistidine signature, (G,A)(L,I,V,M)X(L,I,V,M)T(G,A)SHXPX4G, included in the PROSITE Dictionary of Protein Sites and Patterns (release 10.2, July 1993). In Salmonella enterica, two separate pmm genes have been described. The derived amino acid sequences of both of these S. enterica genes, as included in the GenPept data base (release 77.0, June 1993), contain this signature peptide sequence. One of these genes, rJbK, is involved in LPS 0 side chain synthesis (22), and the other, cpsG, is implicated in the synthesis of the M antigen capsular polysaccharide (47). It is intriguing that the biosynthesis pathways leading to the production of capsular polysaccharide and LPS in S. enterica utilize separate pmm genes, while P. aeruginosa has evolved such that the algC gene is used in both alginate and LPS syntheses. Whether either of the S. enterica PMM enzymes provides secondary PGM activity has not been tested. Both PMM and PGM are members of a family of enzymes that catalyze intramolecular phosphate transfer reactions by way of a phosphorylated enzyme intermediate. While members of this family have been shown to exhibit a broad specificity for hexose phosphates, these enzymes often demonstrate a marked preference for a particular substrate. For example, purified PMM also exhibits PGM activity (14) and purified PGM has been shown to convert mannose (38), but conversion of these alternate sugars proceeds at a vastly lower rate than that of the enzyme's primary substrate. However, our data, in agreement with those of Padgett and Phibbs (34), show that the PMM and PGM activity levels encoded by the P. aeruginosa algC gene are similar. In addition, we found that the P. aeruginosa algC gene apparently provides the only source of PMM and PGM (the algC mutants showed no PMM or PGM activity, while the wild-type strains had both). These findings suggest that P. aeruginosa minimizes redundancy by utilizing the algC gene in two biosynthesis pathways (LPS and alginate) and as the single enzymatic source for two functions, those of PMM and PGM. Koplin et al. (24) also reported the presence, adjacent to the X. campestris xanA gene, of another exopolysaccharide gene. This gene, xanB, encodes a bifunctional enzyme with phosphomannose isomerase and GDP-mannose pyrophosphorylase activities (24) and is similar to a P. aeruginosa alginate gene, algA, which also has both of these enzymatic activities (45). It is interesting that two alginate genes, algC and algA, both have a bifunctional nature. Whether alginate genes other than algC effect the production of LPS is currently under investigation. ACKNOWLEDGMENTS We thank Gerald Pier, Kazue Hatano, and Paul V. Phibbs, Jr., for helpful discussions; Min Lu and Nancy Kleckner for kindly providing us with the cloned E. coli pgm gene and the E. coli pgm mutant strain and for communicating unpublished results; J. Lam for kindly providing the MAb NlF10; and Drago Clifton for advice and assistance in PMM and PGM assays. This research was supported by National Institutes of Health grant A130050 from the National Institute of Allergy and Infectious Diseases. REFERENCES

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