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Alginate synthesis in Pseudomonas aeruginosa the role of AlgL ...

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     JOURNAL OF BACTERIOLOGY, Feb. 1996, p. 625?C632 0021-9193/96/$04.00 0 Copyright 1996, American Society for Microbiology

     Vol. 178, No. 3

     Alginate Synthesis in Pseudomonas aeruginosa: the Role of AlgL (Alginate Lyase) and AlgX

     STEVEN R. MONDAY

     AND

     NEAL L. SCHILLER*

     Division of Biomedical Sciences, University of California, Riverside, Riverside, California 92521-0121

     Received 27 July 1995/Accepted 20 November 1995

     Previous studies localized an alginate lyase gene (algL) within the alginate biosynthetic gene cluster at 34 min on the Pseudomonas aeruginosa chromosome. Insertion of a Tn501 polar transposon in a gene (algX) directly upstream of algL in mucoid P. aeruginosa FRD1 inactivated expression of algX, algL, and other downstream genes, including algA. This strain is phenotypically nonmucoid; however, alginate production could be restored by complementation in trans with a plasmid carrying all of the genes inactivated by the insertion, including algL and algX. Alginate production was also recovered when a merodiploid that generated a complete alginate gene cluster on the chromosome was constructed. However, alginate production by merodiploids formed in the algX::Tn501 mutant using an alginate cluster with an algL deletion was not restored to wild-type levels unless algL was provided on a plasmid in trans. In addition, complementation studies of Tn501 mutants using plasmids containing speci?c deletions in either algL or algX revealed that both genes were required to restore the mucoid phenotype. Escherichia coli strains which expressed algX produced a unique protein of 53 kDa, consistent with the gene product predicted from the DNA sequencing data. These studies demonstrate that AlgX, whose biochemical function remains to be de?ned, and AlgL, which has alginate lyase activity, are both involved in alginate production by P. aeruginosa. Pseudomonas aeruginosa is one of the most important opportunistic human pathogens, causing septicemia and severe, often lethal infections of the respiratory tract, urinary tract, burn wounds, eyes, and intestines, as well as other sites (11). P. aeruginosa is ubiquitous and exhibits innate resistance to a wide range of antimicrobial agents, making infections with this pathogen both common and dif?cult to treat. Patients with cystic ?brosis (CF), the most common lethal genetic metabolic disease among Caucasians, have a multisystem

    disease due to a biochemical defect in the regulation of epithelial chloride transport (52). This defect leads to the accumulation of thick mucus in the lungs causing respiratory congestion and increased susceptibility to bronchopulmonary disease (27, 59). Despite an aggressive host immune response (57), patients with CF usually have chronic pulmonary infections with P. aeruginosa which remain intractable to antibiotic treatment (45, 56). The incidence of P. aeruginosa colonization in CF patients is very high (60 to 90%) and reaches almost 100% in some clinical studies (18, 33, 59). As a consequence, P. aeruginosa lung infections are the predominant cause of morbidity and mortality in CF patients (27, 33, 56, 59). The persistence of P. aeruginosa in the lungs of CF patients, as well as the bacterial resistance to antibiotic action and hostmediated clearance mechanisms, has been attributed to the production of an exopolysaccharide called alginate (see reference 40 for a review). Alginate production decreases the uptake and early bactericidal effect of aminoglycosides (4) and inhibits nonopsonic phagocytosis by monocytes and neutrophils both in vitro (4, 20) and in vivo (3). Baltimore et. al (1, 2) demonstrated that the mucoid coating also inhibits opsonic phagocytosis by concealing opsonic immunodeterminants on the bacterial surface. Moreover, the alginate coat increases bacterial adherence to the respiratory epithelia (19, 46), thereby increasing the rate of colonization within the respiratory tract. P. aeruginosa alginate is a linear, acetylated polymer consisting of (1-4)-linked D-mannuronate and L-guluronate residues (26). Alginate synthesis is regulated by a complex process involving at least three distinct regions of the bacterial chromosome. A small cluster of genes including algR (algR1), algQ (algR2), and algP (algR3) at 9 min (see reference 15 for a review) and algB located at 13 min (30) are required for highlevel alginate production. These genetic loci express proteins which are thought to act primarily through DNA binding and bending (14, 15, 60), similar to other proteins known to be members of bacterial two-component signal transduction systems. Located at 68 min is another gene cluster containing algU (algT), mucA (algS), and mucB (algN), which collectively interact to form a molecular switch which is ultimately responsible for the nonmucoid-to-mucoid conversion via the expression of an alternative RNA polymerase sigma factor from algU (see reference 16 for a review). Mutational analysis of algT, which expresses a protein found to have sequence homology with an E. coli global response sigma factor ( E) (17, 35, 38), has revealed that algT is involved in regulating expression of the signal transduction receivers algR and algB, which in turn are required for optimal activation of the alginate biosynthetic gene cluster at 34 min (61). AlgT (AlgU) appears to be inhibited by the gene products of mucA and mucB (algN), which have been proposed

    to act in a manner analogous to that of anti- factors (37). Inactivation of mucA, either experimentally or as seen in CF-associated isolates (37, 50), or experimental inactivation of mucB (36) or algN (28), derepresses AlgT (AlgU) activity. The large cluster of alginate structural genes appears to function as an operon (8). Transcriptional activation of the promoter of algD, the ?rst gene in this cluster, is associated with conversion to the alginate-producing (Alg ) phenotype (13, 51). Contained within this operon are algA, which encodes a bifunctional enzyme acting as both a phosphomannose isom625

     * Corresponding author. Phone: (909) 787-4569. Fax: (909) 7875504. Electronic mail address: Neals@ucrac1.ucr.edu.

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     MONDAY AND SCHILLER TABLE 1. Bacterial strains and plasmids used in this study

     Strain or plasmid Phenotype, genotype, or description

     J. BACTERIOL.

     Source or reference

     P. aeruginosa FRD1 P. aeruginosa FRD1114 P. aeruginosa FRD1128 E. coli JM109 E. coli HMS174(DE3) pEMR2 pALG2 pSM4 pSM5 pUCP21 pRK415 pNLS18 pNLS42 pNLS43 pNLS44 pRK2013 pMF36 pMF39 pNLS30 pUC129 pRSET5A pLysS

     Prototrophic, Alg CF isolate Alg (Tn501 inserted immediately downstream of algL) Hgr Alg algX::Tn501-28 Hgr recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi (lac-proAB) F [traD36 proAB lacIq lacZ M15] F recA r k12 m k12 Rifr( DE3) pBR322 cos oriT Apr/Cbr Kmr pEMR2 with 35-kb BamHI fragment from FRD1 containing argF algDGLFA pALG2 algL pRSET5A with algX inserted into vector NdeI-PstI restriction sites Broad-host-range expression vector with lac promoter; Apr/Cbr mob Broad-host-range expression vector with lac promoter; Tcr mob ca. 1.6-kb algL DNA fragment ligated into HindIII-EcoRI sites of pRK415 for AlgL expression ca. 8.8-kb HindIII-SstI (algXLFA ) DNA fragment ligated into pUCP21; mob Apr/Cbr ca. 8.4-kb HindIII-SstI (algLFA algX) DNA fragment ligated into pUCP21; mob Apr/Cbr ca. 8.35-kb HindIII-SstI (algXFA algL) DNA fragment ligated into pUCP21; mob Apr/Cbr ColE1-tra (RK2) Kmr Broad-host-range vector containing a tac promoter; mob Apr/Cbr pMF36 vector containing algG, algX, and algL on a ca. 4.5-kb NcoI-XbaI fragment properly oriented for expression from the vector tac promoter; Apr/Cbr mob pUC129 containing algX and algL on a ca. 3.5-kb HindIII-XbaI fragment from pMF39 pUC119-derivative cloning vector; Apr pBluescript backbone containing bacteriophage T7 expression elements of pET3; Apr pACYC184 with bacteriophage T7 lysozyme gene inserted into vector BamHI site; gene is inserted with start site away from vector Tcr promoter; Cmr

     44 48 48 47 55 22 7 This study This study 58 31 This study This study This study This study 21 24 M. Franklin This study 31 49 55

     a Abbreviations: Alg , mucoid because of alginate production; Alg , nonmucoid; Tcr, tetracycline resistance; Rifr, rifampin resistance; Hgr, mercury resistance; Kmr, kanamycin resistance; Apr, ampicillin resistance; Cbr, carbenicillin resistance; Cmr, chloramphenicol resistance; Tra , transfer by conjugation.

     erase and a GDP-mannose pyrophosphorylase (53); algD, which expresses a GDP-mannose dehydrogenase (13); algF, a gene involved in alginate acetylation (24, 54); and algG, which encodes a C-5 epimerase (7, 23). Several other open reading frames (ORFs) found within the operon, including algE (9), alg-44 (34), and alg-8 (34), have also been described; however, the roles of the proteins encoded by these genes in alginate biosynthesis are unknown. Alginates are enzymatically depolymerized by alginate lyases (EC 4.2.2.3), which cleave the 1-4 glycosidic linkage by -elimination, resulting in an unsaturated, nonreducing terminus (25). We previously showed that algL, the P. aeruginosa gene for alginate lyase (48), is located within the alginate biosynthetic gene cluster, between algG and algF. The location of this gene within the biosynthetic gene cluster and its coregulation with genes involved in alginate production suggested that AlgL may be required for synthesis of the alginate polymer by P. aeruginosa. This paper presents the results obtained from two complementary molecular strategies, utilizing transposon-mutagenized, nonmucoid variants of the CF isolate FRD1, which conclusively demonstrate the involvement of both algL and algX, the gene immediately upstream of algL, in alginate production by P. aeruginosa. Expression of algX in Escherichia coli produced a unique protein of 53 kDa consistent with the gene product predicted from its DNA sequence. MATERIALS AND METHODS

     Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are described in Table 1. Strains were routinely cultured in L broth (1.0% Bacto-tryptone, 0.5% yeast extract, 0.5% NaCl) or on L agar plates (L broth with 1.5% agar) with antibiotics as needed. For some studies, a 1:1 mixture of L agar and Pseudomonas Isolation Agar (Difco Laboratories, Detroit, Mich.) (P/L agar plates) was used. Unless otherwise indicated, antibiotics were used at the following concentrations (in micrograms per milliliter): ampicillin, 100 for E. coli; carbenicillin, 300 for P. aeruginosa; kanamycin, 50 for E. coli; chloramphenicol,

     50 for E. coli; mercuric chloride, 18 for P. aeruginosa; tetracycline, 25 for E. coli and 100 for P. aeruginosa. Recombinant-plasmid construction. General procedures for plasmid preparation (including restriction digests, ligations, and transformations) were performed as described by Sambrook et al. (47) with E. coli JM109. When needed, plasmids were isolated from E. coli by using the Wizard mini plasmid preparation kit (Promega Corporation, Madison, Wis.). Construction of

    plasmid vectors pNLS42, pNLS43, and pNLS44. Each of the plasmid vectors pNLS42, pNLS43, and pNLS44 (see Fig. 1) was derived from pNLS37, a pRK415 vector containing an 8.8-kb HindIII-SstI fragment from pALG2 expressing the biosynthetic gene cluster from algX through algA. pNLS42 was constructed by isolating the entire 8.8-kb HindIII-SstI fragment from pNLS37 and ligating it into pUCP21, a vector chosen for enhanced expression of these genes in P. aeruginosa (58). pNLS44 is comparable to pNLS42 except that during its construction, the 465-bp PstI fragment internal to algL was removed. pNLS43 is comparable to pNLS42 except that during its construction, the 384-bp XcmI fragment located at the 3 terminus of the algX region was removed (this fragment encompasses the site into which the Tn501 transposon had inserted into the FRD1128 chromosome). Construction of pSM4. The wild-type algL gene of pALG2 was exchanged for the algL::cat gene of pNLS14 (previously described in reference 48) by an interplasmid exchange technique (41). Rare double crossover, homologous recombination events occurred in RecA E. coli C600. Following overnight incubation, the cosmid vector of the double transformants was packaged by making a cI857 lysate which was used to transduce E. coli JM109. Bacteria containing double-crossover recombinants were identi?ed as ampicillin-resistant,

    chloramphenicol-resistant, and tetracycline-sensitive colonies. Restriction digests of the vectors isolated from these bacteria con?rmed the proper construction, designated pSM3, which was next digested with XbaI to remove the algL::cat region and all but 87 bp of the chromosomal algL gene. After religation, constructs recovered from transformed bacteria were digested with XbaI to ensure that the algL region of pSM3 had been deleted. This plasmid was designated pSM4. Triparental matings. Triparental matings were used to mobilize recombinant plasmids from E. coli to P. aeruginosa with the conjugative helper plasmid pRK2013 by methods detailed elsewhere (29), with the following minor modi?cation. After a 6-h incubation at 37 C, the ?lter containing the mating mixtures was placed into a sterile tube containing 5 ml of 0.85% NaCl and vortexed to dislodge the bacteria. P/L agar plates containing carbenicillin were inoculated with 100 l of a 1:10 dilution of the bacterial suspension and incubated at 37 C until colonies appeared. Individual transconjugants were scored for reversion to the mucoid phenotype. Electroporation. Electroporation-competent P. aeruginosa FRD1114 and FRD1128 cells were prepared by harvesting 100-ml overnight cultures by cen-

     VOL. 178, 1996

     PSEUDOMONAS ALGINATE SYNTHESIS: ROLE OF AlgL AND AlgX

     627

     trifugation and washing them three successive times with 15 ml of ice-cold 10% glycerol. After the ?nal wash, the bacteria were

    resuspended in 5 ml of 10% glycerol, aliquoted, and frozen at 80 C. Electroporation was performed by the Pseudomonas putida protocol as described by the manufacturer of the electroporator, BTX Electronic Genetics (San Diego, Calif.). A 100- l aliquot of thawed competent bacteria was mixed with 1 g of plasmid DNA and loaded into prechilled 2-mm cuvettes. The bacteria were electroporated at 2.5 kV/cm, 129 W, and 0 capacitance for 4.6 ms. Immediately following electroporation, 500 l of KMB medium (2% proteose peptone, 1% glycerol, 6 mM MgSO4 7H2O, 6.5 mM K2HPO4 3H2O, pH 7.0] was added to each cuvette. Suspensions were transferred from the cuvettes to microcentrifuge tubes, where bacteria recovered during a 3-h incubation at 37 C with gentle inversion. P/L agar plates containing carbenicillin were inoculated with 100 l of these bacteria and incubated at 37 C until colonies appeared. Individual transformants were transferred to P/L agar plates containing carbenicillin, previously coated with 80 l of 100 mM

    isopropyl-D-thiogalactopyranoside (IPTG), incubated at 37 C, and scored for alginate production. Alginate assay. To measure alginate production, strains were grown on L agar plates with appropriate antibiotics for 28 h at 37 C. The bacteria were then swabbed into 10 ml of 0.85% NaCl, and tubes containing the mixture were vortexed vigorously and centrifuged to remove the bacteria for subsequent weighing. The alginate remaining in the supernatant was precipitated by the addition of 25 ml of 95% ethanol. The alginate precipitates were collected by centrifugation and resuspended in 2 ml of 0.85% NaCl. The uronic acid concentration was determined by the colorimetric assay described by Knutson and Jeanes (32). DNA sequencing. Double-stranded DNA sequencing was done by the dideoxynucleotide chain termination method with Sequenase (Amersham Corp., Arlington Heights, Ill.) and deoxyadenosine 5 [ -35S]thiotriphosphate (speci?c activity, 1,000 Ci/mmol; Amersham Corp.) to completely sequence both strands of the DNA between algG and algL. Sequence reactions were initially performed with dGTP nucleotide label and termination mixes. Resolution of GC compression artifacts, a complication commonly observed when pseudomonad DNA is sequenced, was achieved by replacing the dGTP nucleotides used in the sequencing reactions with either dITP or 7-deaza-dGTP nucleotide analogs. Custom oligonucleotide primers were obtained from Gibco BRL, Grand Island, N.Y. PCR. PCR employing the plasmid template pNLS30 with primers designed to introduce the NdeI (CATATG) and PstI (CTGCAG) restriction sites encompassing algX was performed in a ?nal volume of 50 l which contained 2 mM Mg2 , 400 M deoxynucleoside triphosphate, 0.4 M each primer, 750 ng of plasmid template, 2.5 U of Vent DNA polymerase (New England Biolabs), and 10% formamide. Following an initial incubation of 95 C for 5 min, insert ampli?cation was performed in 30 cycles of 95 C for 1.5 min, 56 C for

    2 min, and 75 C for 3 min. The reaction was concluded with a ?nal incubation of 72 C for 15 min. PCR products were electrophoresed on a 0.8% Tris-acetate-EDTA agarose gel and visualized with ethidium bromide. Expression of AlgX and polyacrylamide gel electrophoresis. E. coli HMS174 (DE3) was sequentially transformed with both pLysS and pSM5. A single ampicillin- and chloramphenicol-resistant colony was used to inoculate 5 ml of L broth containing ampicillin (200 g/ml) and chloramphenicol (50 g/ml). After incubation at 37 C for 2 h, the suspension was centrifuged, the supernatant was replaced with 5 ml of L broth containing the two antibiotics at identical concentrations, and the culture was incubated at 37 C. This medium exchange process was repeated at 1.5-h intervals until the bacterial culture reached an A600 of 0.7. At this time, expression of the endogenous T7 polymerase carried on the DE3 bacteriophage was induced by adding IPTG to 1 mM and incubating for another 2 h at 37 C. After centrifugation, the pellet was resuspended in 200 l of doubly distilled H2O and frozen at 20 C. A negative-control sample was prepared by the same procedure with the HMS174(DE3) lysogen carrying both pLysS and pRSET5A. Equal volumes of the cell suspension and 2.5 Laemmli sample solution were mixed and heated to 95 C for 5 min. The samples were then run on a 9%

    polyacrylamide?Csodium dodecyl sulfate (SDS) gel, 1.5 mm thick, and stained with Coomassie blue R250 as previously described (12). Nucleotide sequence accession number. The nucleotide sequence for algX (see Fig. 6) has been deposited in the DDBJ, EMBL, and GenBank DNA databases, and the entire nucleotide sequence of algG-algX-algL has been given the accession no. U27829.

     RESULTS Cloning and sequencing of algX. In our previous study (48), transposon mutagenesis was used to inactivate chromosomal algL in mucoid (Alg ) P. aeruginosa FRD1. Restriction mapping studies identi?ed the relative locations of these insertions, which were later con?rmed by DNA sequence analysis. Transposon insertion Tn501-28 was located in a putative gene (algX) 150 nucleotides upstream of the ATG codon initiating the algL sequence (Fig. 1). In order to sequence the algX locus, a

     3.5-kb HindIII-XbaI fragment, containing the 3 region of algG, the algX locus, and algL, was isolated from pMF39, ligated into a similarly digested pUC129 cloning vector, and used to transform E. coli JM109. Plasmids were isolated from ampicillin-resistant transformants and analyzed for proper construction by HindIII-XbaI restriction digests. Vectors found to contain the proper insert were subsequently designated pNLS30. The parameters of the algX gene were established by sequencing the DNA located between algG and algL (GenBank accession numbers U06720 and L09724, respectively). Doublestranded-DNA sequencing of pNLS30 was initiated by designing oligonucleotide primers that annealed to the

    3 end of algG (nucleotides 1680 to 1697) and the 5 end of algL (nucleotides 37 to 21) which, when used in a sequencing reaction, would extend sequence information from algG and algL into the region containing the putative algX ORF. New primers were synthesized on the basis of the acquired sequence, allowing both strands of the region to be completely sequenced in a ????walking???? fashion. Analysis of strand complementarity con?rmed the sequence data obtained (Fig. 2). The algX ORF, initiating at the ATG start codon at nucleotide 40 and extending to a TAA stop codon at nucleotide 1462, encodes a polypeptide of 474 amino acids with a computer-determined molecular weight of 52,553 and a predicted pI of 7.52. The ORF is 66.7% G C overall and 94% G C in the third codon position. Such a high G C content is similar to that of other P. aeruginosa genes, including algL (48). A nonredundant database search of the Brookhaven Protein Data Bank, the Swiss-Prot (release 31.0, March 1995) database, and the GenBank database was done at the National Center for Biotechnology Information by using the Blast Network service. The search revealed that the AlgX polypeptide does not share any signi?cant sequence homology with any of the proteins currently contained within these databases. Role of algL in alginate production. Because of the operonic nature of the biosynthetic gene cluster (8), Tn501-28 (algX:: Tn501) is a polar mutation which inactivates algX, algL, and other downstream biosynthetic genes (such as algF and algA), thus rendering this FRD1 mutant (designated FRD1128) phenotypically nonmucoid (Alg ). This phenotype was con?rmed quantitatively by measuring the uronic acid content of each strain; whereas the Alg parental strain FRD1 typically produces 50 g of uronic acid per mg (wet weight) of bacteria, FRD1128 produced 1.15 1.99 g of uronic acid per mg (wet weight) of bacteria (Table 2). Another transposon insertion, Tn501-14, located 350 nucleotides below the 3 end of the algL coding region (Fig. 1), did not affect algL expression but still rendered this FRD1 mutant (designated FRD1114) Alg because of its polar effect on algA, an essential gene encoding the enzyme which catalyzes the ?rst reaction of alginate biosynthesis (53). Con?rmation that the nonmucoid phenotypes of FRD1128 and FRD1114 were due to the indicated transposon insertions, and not spontaneous mutations of algT (algU) (17, 37) or algR (61), was achieved by mating the plasmid pALG2, a ColE1-based replicon carrying the entire wildtype alginate biosynthetic operon, into the mutant strains. This vector, which is unable to autonomously replicate within P. aeruginosa, integrates into the host chromosome by homologous recombination, generating a merodiploid. Following conjugation and selection on

    carbenicillin-containing P/L agar plates, 95% of the pALG2 merodiploids were Alg within 24 h (Table 2), indicating that the Alg phenotypes of FRD1128 and FRD1114 are due to the mutational effect of the transposon

    insertions within the biosynthetic operon. Moreover, these studies demonstrated that these strains can be converted to the

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     MONDAY AND SCHILLER

     J. BACTERIOL.

     FIG. 1. Plasmid constructs used for FRD1114 and FRD1128 complementation analysis. The locations of the transposons in FRD1114 and FRD1128 are shown at the top. The alginate biosynthetic genes contained within each construct and their orientations relative to the various vector promoters are also depicted (vectors used for preparation of speci?c constructs are identi?ed in parentheses beneath the construct designations). The shaded segments denote regions which were deleted from the plasmid constructs.

     Alg phenotype by genes contained on pALG2. Presumably, those colonies not converted to the mucoid state by this merodiploid strategy had undergone spontaneous mutations in either the algT (algU) or algR regions, rendering them nonmucoid. To determine whether AlgL functions in alginate production, we used the same merodiploid strategy with the plasmid pSM4, an algL deletion variant of pALG2. (The construction of pSM4 is described in Materials and Methods.) That the biosynthetic operon in pSM4 was otherwise intact was con?rmed by demonstrating that 95% of the FRD1114::pSM4 recombinants were Alg within 24 h of incubation at 37 C (Table 2), similar to the FRD1114::pALG2 recombinants. In contrast to the results observed with FRD1114::pSM4, 95% of the FRD1128::pSM4 recombinants remained Alg (Table 2), suggesting that AlgL is involved in biosynthesis. To con?rm that the nonmucoid phenotype of these FRD1128:: pSM4 merodiploids was due to the absence of AlgL, pNLS18, an autonomously replicating, broad-host-range vector that contains algL below its lac promoter (Fig. 1), was mated into FRD1128::pSM4. Transconjugants, which appeared phenotypically identical to the FRD1128::pALG2 merodiploids, rapidly produced alginate (data not shown). The ability of pNLS18, which supplies only algL in trans, to restore wild-type levels of alginate synthesis in FRD1128::pSM4 implicates AlgL as a necessary component of normal alginate synthesis. Both algL and algX participate in alginate production. Complementation studies using FRD1114 and FRD1128 were performed to determine whether algL, algX, or both are needed to restore alginate synthesis in the nonmucoid transposon mutants FRD1114 and FRD1128.

     Transformation of FRD1114 with pCC75 (Fig. 1), which expresses algA under control of a tac promoter, converted this strain to Alg . The restoration of alginate synthesis in FRD1114, whose transposon is inserted immediately downstream of algL, therefore requires only algA (algF, involved in alginate acetylation, is not needed to build the

    polymer). In contrast, FRD1128, with Tn501-28 inserted in algX, upstream of algL, was not restored to the mucoid phenotype by transformation with pCC75 (Fig. 1). This observation suggested that either algL and/or algX is required for alginate synthesis, as these are the only genes blocked by the transposon in FRD1128 which are unaffected in FRD1114. Transformation of FRD1128 with pNLS42, a plasmid vector which expresses all genes affected by the Tn501-28 insertion, restored alginate production (Fig. 1 and Table 2), demonstrating that (i) in-trans complementation of the FRD1128 mutant to the mucoid state is achievable and (ii) either AlgL, AlgX, or both are required for the synthesis of alginate. To speci?cally determine whether algL and/or algX is required for alginate production, plasmid variants of pNLS42 individually containing algL or algX deletions were prepared and used to transform FRD1128 or FRD1114. (Plasmid constructions are described in Materials and Methods, and plasmid maps are depicted in Fig. 1). The ability of these transformants to synthesize alginate was then determined. pNLS44 is similar to pNLS42 except that a 465-bp PstI fragment internal to algL was deleted. Transformation of FRD1114 with pNLS44 converted this strain to the mucoid phenotype (Table 2), demonstrating the expression of algA. In contrast, colonies of FRD1128 transformed with pNLS44 remained phenotypically nonmucoid, with alginate concentra-

     FIG. 2. Nucleotide sequence of algX and amino acid sequence for AlgX. Locations of selected endonuclease restriction sites are denoted above the sequence. The Shine-Dalgarno sequence is shown in boldface type. Underlined sequences depict the nucleotides of the 3 end of algG (upstream of algX) and the 5 end of algL.

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     J. BACTERIOL.

     TABLE 2. Alginate production by various P. aeruginosa strains

     Study and strains g of uronic acid/ mg (wet wt) of bacteriaa

     DISCUSSION During our previous studies of algL (48), we were surprised to ?nd that the gene encoding alginate lyase, an alginatedegrading enzyme, was located within the alginate biosynthetic gene cluster and positively coregulated with alginate synthesis. This observation prompted our study to determine what role, if any, AlgL had in alginate production. By using a strain of the mucoid CF patient isolate FRD1 rendered nonmucoid by Tn501 insertion in algX, a gene immediately upstream of algL, restoration of alginate production by complementation in trans was found to require a plasmid carrying all of the genes inactivated by the insertion, including algL and algX. Alginate production was also recovered when a merodiploid that

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