Molecular Cloning of Genes Involved with Expression of A-Band ...

JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5624-5630 0021-9193/91/185624-07$02.00/0 Copyright X 1991, American Society for Microbiology

Vol. 173, No. 18

Molecular Cloning of Genes Involved with Expression of A-Band Lipopolysaccharide, an Antigenically Conserved Form, in Pseudomonas aeruginosa JEFF LIGHTFOOT AND JOSEPH S. LAM* Department of Microbiology' and Canadian Bacterial Diseases Network,2 University of Guelph, Guelph, Ontario, Canada NIG 2WI Received 21 March 1991/Accepted 1 July 1991 Most strains of Pseudomonas aeruginosa can express two chemically and immunologically distinct types of lipopolysaccharide (LPS), an antigenically conserved form called A band and the serotype-specific form called B band. To study the molecular controls regulating expression of the A-band LPS antigen, we have cloned the genes involved with A-band LPS expression. Strain AK1401, a phage-resistant mutant of PAO1 which was shown previously to produce only A-band LPS and not the 0-antigen-containing B-band LPS, was mutagenized by using ethyl methanesulfonate to generate an A-band-deficient mutant called rd7513. A cosmid clone bank of P. aeruginosa PAO1 whole genomic DNA was constructed in Escherichia coli. The gene bank was mobilized en masse into strain rd7513, and detection of complementation of synthesis of A band was done by screening transconjugants in a colony immunoblot assay with the A-band-specific monoclonal antibody N1F10. One recombinant cosmid, pFV3, complemented synthesis of A-band polysaccharide in rd7513. Silver-stained polyacrylamide gel and Western immunoblot analyses of LPS extracted from the transconjugant rd7513(pFV3) showed that the A band produced had a higher molecular weight than the A band of AKl401. Analysis of the plasmid pFV3 showed that it contained a chromosomal insert of 27 kb. Two subclones of pFV3, namely, pFV35 and pFV36, containing chromosomal inserts of 5.3 and 4.2 kb, respectively, also complemented A-band expression in rd7513. The LPS banding profile of rd7513(pFV35) was similar to that of AK1401, while the LPS profile of rd7513(pFV36) more closely resembled that of rd7513(pFV3). pFV3 complemented A-band expression in five of the six P. aeruginosa 0 serotypes which lack A band as well as in rough strain AK44 but failed to complement A-band expression in core mutants AK1012 and AK1282, suggesting that pFV3 contains genes for A-band expression and that synthesis of a complete core region in isogenic mutant strains is required for A-band synthesis. the unusually high proportion of D-rhamnose, with lesser amounts of 3-O-methyl-rhamnose, ribose, glucose, and mannose (1). This finding is similar to the data reported by Yokota et al. (48) and Kocharova et al. (22), who also isolated neutral polysaccharides from P. aeruginosa containing predominantly D-rhamnan residues and lesser proportions of rhamnose, 3-O-methyl-6-deoxyhexose, glucose, and xylose. Interestingly, by using an A-band-specific monoclonal antibody (MAb), N1C9, we were able to establish that A-band LPS is present as a common LPS antigen among the majority of standard serotype strains and clinical isolates from cystic fibrosis patients (27). As a first step toward understanding A-band LPS synthesis, we report here the cloning of genes involved with the expression of A-band

Lipopolysaccharide (LPS) is a complex group of macromolecules which constitute a major portion of the outer membranes of gram-negative bacteria. Like LPS of the members of the family Enterobacteriaceae, the LPS of Pseudomonas aeruginosa is chemically and structurally heterogeneous (24, 36). The chemical heterogeneity of the 0 polysaccharide (O antigen) repeat units distinguishes this species into 20 different 0 serotypes (29, 30). LPS is also heterogeneous in size, which is attributed to the variable number of 0-antigen repeat units substituted onto the lipid A-core oligosaccharide (24, 38). LPS is a major virulence factor of P. aeruginosa, particularly in wild-type strains, where LPS is fully capped with long 0-antigen side chains. It has been shown in several studies that organisms with 0-antigen-containing LPS have a lower 50% lethal dose in a burned-mouse sepsis model (6), are less serum sensitive (15, 42), and are more resistant to phagocytosis than mutant strains without 0 antigens (10). Recent studies (38, 39) indicated that P. aeruginosa could produce two immunologically and chemically distinct forms of LPS known as A-band and B-band LPS. B-band LPS is the 0-antigen-containing LPS and determines the 0 specificity of the bacterium, while A-band LPS contains shorter chains of predominantly neutral polysaccharide and lacks phosphate but contains stoichiometric amounts of sulfate. The high neutral-sugar content of the A band is attributed to *

polysaccharide. (Parts of this study were presented as a poster at The First Congress of the International Endotoxin Society in San Diego, Calif., 1990 [28].) MATERIALS AND METHODS

Strains and plasmids. The bacterial strains and plasmids used in this study are shown in Table 1. Media and cultural conditions. All bacterial strains were cultured on Luria-Bertani broth (1% Bacto-Tryptone [Difco Laboratories, Detroit, Mich.], 0.5% yeast extract [Difco], 1% NaCl). Minimal medium (35) or Pseudomonas Isolation Agar (Difco) was used for selection of transconjugants following triparental mating experiments, with amino acids

Corresponding author. 5624

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TABLE 1. Bacterial strains and plasmids Strain or plasmid

Genotype, phenotype, or relevant properties

Reference and/or source

P. aeruginosa

PA01 AK1401a rd7513a AK44 AK1012 AK1282 PAC556 PAC557 IATS 07 (ATCC 33353) IATS 012 (ATCC 33359) IATS 013 (ATCC 33360) IATS 014 (ATCC 33361) IATS 015 (ATCC 33362) IATS 016 (ATCC 33363)

Wild type; 05 0 antigen, A band 0-antigen-deficient mutant of OT684b A-band-deficient mutant of AK1401 0-antigen-deficient mutant of PAO1, complete core Rough mutant of PAO1 Deep rough mutant of AK1012, deficient in entire outer core Rough mutant of PACMR, core lacks L-rhamnose Rough mutant of PACMR, core contains L-rhamnose 07 0 antigen, lacks A bandc 012 0 antigen, lacks A bandc 013 0 antigen, lacks A bandc 014 0 antigen, lacks A bandc 015 0 antigen, lacks A bandc 016 0 antigen, lacks A bandc

R. Hancock (14) A. Kropinski (2) This study A. Kropinski (23) A. Kropinski (18) A. Kropinski (19) 40 40 29 29 29 29 29 29

DH5a

supE44 hsdR17 recAl endAl gyrA96 thi-l relAl

LE392

supE44 supF58 hsdR514 galK2 gaIT22 metBi trpR55 lacYl

GIBCO/Bethesda Research Laboratories 32

IncPl Tcr Kmr rlxX cos+ ColE1-Tra(RK2)+ Kmr IncPl Tcr A' cosmid clone in pCP13; Tcr 6.5-kb BamHI fragment from pFV3 cloned in pRK404; A' Tcr 2.3-kb HindIll deletion from pFV35; A' Tcr

A. Chakrabarty; 7, 12 A. Chakrabarty; 11 D. Helinski (8) This study This study This study

E. coli

Plasmids pCP13 pRK2013 pRK404 pFV3 pFV35 pFV36

a AK1401 and rd7513 are auxotrophic for leucine, lysine, and threonine. b OT684 (leu-J lys-I res4), the immediate progenitor strain of AK1401, is a restrictionless mutant of PAO1 (37). ' These six IATS 0-serotype strains were previously found to lack the A-band antigen (27).

leucine, lysine, and threonine (Sigma, St. Louis, Mo.) (20 pug/ml each) added to minimal medium for growth of AK1401. Bacto-Agar (Difco) was added to 1.5% for all solid media. When necessary, broth and agar were supplemented with antibiotics (Sigma) as follows (per milliliter): for culturing P. aeruginosa, tetracycline, 250 ,ug (300 to 500 ,ig for Pseudomonas Isolation Agar; 100 jig for minimal medium); and for culturing Escherichia coli, tetracycline, 15 ,ug, and kanamycin, 50 ,ug. Strains were stored at -70°C in medium containing either 15% glycerol or 7% dimethyl sulfoxide (Sigma). Mutagenesis of AK1401 with EMS. Mutagenesis of AK1401 with ethyl methanesulfonate (EMS; Sigma) was done by the method of Darzins and Chakrabarty (7), except that cells were incubated with 15 ,ul of EMS per ml and mutagenized cells were suspended in Luria-Bertani broth instead of minimal medium. These cells were then screened for deficiency of A-band polysaccharide. Isolation of mutants of AK1401 deficient in expression of A-band polysaccharide. Screening of EMS-mutagenized cells for deficiency of A-band polysaccharide was done by a colony immunoblot assay that was facilitated by the use of ISO-GRID hydrophobic grid membrane filters (HGMFs) (QA Laboratories, Toronto, Ontario, Canada) as described by Lo and Cameron (31). Membranes were incubated at 37°C for 2 h with culture supernatants of MAb N1F10, which is specific for A-band polysaccharide (27). Membranes were treated with alkaline phosphatase-conjugated goat-antimouse immunoglobulin G (Jackson Immunoresearch Laboratories, West Grove, Pa.) and developed in a substrate solution consisting of 30 ,ug of Nitro Blue Tetrazolium and 15 jig of 5-bromo-4-chloro-3-indolyl phosphate toluidine in 100

ml of 0.1 M bicarbonate buffer (pH 9.8) (27). The membranes were dried and examined for any reactions which were not as intense in color as the positive reactions, indicating a lack of reactivity with MAb NlF10. Colonies which corresponded with lighter-blue reactions were selected and further characterized by reacting them with MAb NlFlO in slide agglutination tests. Those colonies which did not agglutinate with MAb NlFlO were chosen for further study. DNA isolations and general procedures. Plasmid DNA was isolated in preparative amounts from 1-liter cultures of P. aeruginosa and E. coli as described by Maniatis et al. (32). Plasmid minipreparations were done by the alkaline lysis method of Birnboim and Doly (4). Whole genomic P. aeruginosa DNA was isolated by the method of Goldberg and Ohman (13). All agarose gel electrophoresis was done by standard procedures using slab gels of 0.7% agarose in either Tris-borate or Tris-acetate buffer (32). All restriction enzymes were purchased from either GIBCO/Bethesda Research Laboratories or Boehringer (Mannheim, Germany). T4 DNA ligase and alkaline phosphatase were purchased from Boehringer-Mannheim. Plasmid DNA was transformed into E. coli strains following treatment with CaCl2 (32). Construction of a P. aeruginosa gene library, mobilization of recombinant plasmids, and screening for complementation of A-band expression. A gene library of P. aeruginosa PA01 chromosomal DNA was constructed in E. coli LE392 essentially as described by Goldberg and Ohman (13), using the broad-host-range cosmid vector pCP13 and partial XhoI fragments of 20 to 27 kb. Packaging in lambda particles and infection of LE392 was also done according to the method of Goldberg and Ohman (13). Ligation mixtures were mixed with lambda packaging extracts prepared and supplied by

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R. Y. C. Lo (University of Guelph, Guelph, Ontario, Canada). Tetracycline-resistant, kanamycin-sensitive colonies (2 x 103) were chosen at random. Triparental matings between A-band-deficient mutants, the pooled library, and E. coli HB101(pRK2013) were done by the method of Ruvkun and Ausubel (41). Mating mixtures were passed through HGMFs (as described above), plated onto selective media, and incubated for 24 to 48 h at 37°C. Colonies were transferred to nitrocellulose and screened for complementation of A-band expression by using MAb NlFlO in the colony immunoblot assay with HGMFs as described above. Colonies that reacted with MAb NlFlO were isolated for further study. Isolation of LPS. LPS was isolated by the method of Hitchcock and Brown (16) for small-scale preparations and by the hot-aqueous-phenol method of Westphal and Jann (46) for preparative amounts. SDS-PAGE analysis and silver staining. LPS was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) as described by Hancock et al. (15). Silver staining was done by the ammoniacal-silver method of Dubray and Bezard (9). Western immunoblotting. LPS samples were transferred from SDS-PAGE gels to nitrocellulose by the method of Towbin et al. (44) as modified by Burnette (5). Reaction of immunoblots with MAb MF15-4 (specific for serotype 05) (26) or MAb NlFlO (27) as well as reaction with the enzyme-conjugated second antibody and substrate was as described above. RESULTS Isolation of an A-band-deficient mutant of AK1401. Previous studies on the isolation of LPS-deficient mutants of P. aeruginosa have predominantly used LPS-specific phage. AK1401 is a mutant of strain OT684 (a restrictionless derivative of PA01) that is resistant to phage E79, which is specific for LPS of P. aeruginosa (17). In this study, we used EMS to generate A-band-deficient mutants of AK1401. The use of HGMFs greatly facilitated the screening of the mutagenized cells, allowing approximately 1,200 colonies per plate to be screened with each filter. Our studies were also aided by the use of MAb N1F10, which can be used to readily differentiate between colonies which express A band and those that do not. Of approximately 4 x 103 colonies screened by the colony immunoblot method, 1 colony showed lack of reactivity with MAb NlFlO (i.e., lighter-blue reaction). This mutant was named rd7513. rd7513 did not agglutinate with MAb NlFlO in slide agglutination tests. The colony morphology of rd7513 did not differ significantly from that of AK1401, which shows large, round, smooth, shiny colonies. Analysis of LPS from mutant rd7513. LPS from rd7513 was analyzed by SDS-PAGE and silver staining. The electrophoretic mobility of the lipid A-core band of rd7513 in SDSpolyacrylamide gels resembled that of AK1401 (Fig. 1). Analysis of silver-stained gels loaded with decreasing amounts of LPS from rd7513 revealed that it has the same profile as AK1401, i.e., lipid A-core plus one 0-antigen repeat unit (data not shown). LPS from rd7513 showed no reaction with MAb NlFlO in Western immunoblots (see Fig.

3b). Cloning of A-band LPS genes. A P. aeruginosa PAO1 gene library was constructed by infecting E. coli LE392 with lambda phage particles packaged with chromosomal DNA from P. aeruginosa PAO1 partially digested with XhoI and ligated to XhoI-digested cosmid vector pCP13. Cloning the

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FIG. 1. Silver-stained SDS-polyacrylamide gel of LPS from P. aeruginosa AK1401, mutant strain rd7513, and A-band clone strain rd7513(pFV3). Arrow indicates limit of visible resolution of A-band ladder pattern, showing that the clone strain expresses highermolecular-weight A band.

genes involved with the synthesis of A band was done by mating of this gene library with the A-band-deficient mutant rd7513 and subsequent screening of the tranconjugants with MAb NlFlO and HGMFs in colony immunoblots. Approximately 2 x 105 transconjugants were screened in this manner, resulting in six colonies that reacted with MAb NMFlO. When we attempted to recover all six of the transconjugants from the HGMFs, only one isolate, called pFV3, maintained the ability to react with MAb NlFlO in colony immunoblots. In slide agglutination tests, rd7513(pFV3) reacted strongly with MAb NlFlO but failed to react with MAb MF15-4, suggesting that only A-band expression and not B-band LPS (O antigen) expression was complemented in rd7513 by pFV3. The positive slide agglutination reaction with MAb NlFlO also suggested that the complemented A band was expressed on the cell surface. A restriction endonuclease map of pFV3 was constructed (Fig. 2). The chromosomal insert was found to be approximately 27 kb in size, judged by the sizes of restriction fragments in agarose gels. Three BamHI fragments of 11.8, 7.9, and 6.5 kb were used for initial subcloning into plasmid vector pRK404. These fragments were chosen because of their relatively large sizes, and, when combined, they represented practically the entire insert. These fragments were individually ligated to BamHI-digested pRK404. Recombinant plasmids were transformed into E. coli DH5a and were i i

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EXPRESSION OF A-BAND LPS IN P. AERUGINOSA

VOL. 173, 1991

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