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1 Department of Microbiology, University of Washington, Seattle, WA 98195, USA
2 Unité de Glycobiologie Structurale et Fonctionnelle CNRS UMR 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France
Correspondence
Yannick Lequette
yannick.lequette{at}lille.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: LGPTA, CERTIA, INRA, 369 rue Guelde, 59651 Villeneuve d'Ascq, France.
Supplementary figures showing growth curves of PA14 parent and YL119 strains, and biofilm formation in the YL119 mutant complemented with pYL205-G are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). OPGs vary from 5 to 25 glucosyl residues per molecule and the glucose backbones show structural diversity among different species. There are four families of OPG: Family I consists of heterogeneous-sized OPGs with a linear backbone of β-1,2-linked glucosyl residues to which branches of glucosyl units are linked by β-1,6 bonds (Bohin & Lacroix, 2006); Family II consists of heterogeneous-sized OPGs with cyclic β-1,2-glucan backbones; Family III consists of homogeneous-sized OPGs with a cyclic backbone containing glucosyl residues linked by β-1,3, β-1,6 bonds and sometimes by β-1,4 bonds; Family IV members consist of homogeneous-sized OPGs with cyclic β-1,2-glucan backbones and one 
-1,6 bond. Additionally, substituents derived from membrane phospholipids (phosphoglycerol, phosphoethanolamine, phosphocholine residues) or from intermediary metabolism (succinate, acetate residues) can decorate the backbones independent of backbone structure (Bohin & Lacroix, 2006).
Studies of OPG synthesis in different species have revealed three distinct glycosyl transferases involved in the biosynthesis of the glucosyl backbone. Genomic sequencing suggests that the opgH-encoded glycosyl transferase is the most common among the three. The OpgH protein was initially named MdoH (membrane-derived oligosaccharide) (Lacroix et al., 1991) in Escherichia coli and HrpM (hypersensitive reaction and pathogenicity) in Pseudomonas syringae (Mukhopadhyay et al., 1988). OpgH homologues, which show a typical glycosyl transferase 2 domain, can catalyse the synthesis of linear Family I OPGs, as in P. syringae (Talaga et al., 1994), or cyclic Family IV OPGs, as in Rhodobacter sphaeroides (Talaga et al., 2002). opgH is the second gene of the opgGH operon. opgG encodes the periplasmic protein OpgG that is necessary for OPG biosynthesis, although at present the function of this protein is unknown. OpgG has been proposed to be involved in the formation of β,1-6 linkages and/or in periplasmic release of newly synthesized OPG (Bohin & Lacroix, 2006). Synthesis of Family II and III OPGs is dependent on proteins named NdvB in Sinorhizobium meliloti and Bradyrhizobium japonicum. However, the two NdvB polypeptides do not show significant sequence similarity.
The importance of OPGs in pathogenesis has been shown for many human, animal and plant pathogens (Bohin & Lacroix, 2006). Mutants defective in OPG biosynthesis display pleiotropic phenotypes, often including hyperproduction of exopolysaccharides, motility defects and hypersensitivity to antibiotics. An explanation for this pleiotropy is that OPGs are critical for normal organization of the cell envelope.
The opportunistic pathogen Pseudomonas aeruginosa can be found in a variety of moist environments, including natural and man-made environments (Hardalo & Edberg, 1997; Schwartz et al., 2006; Spiers et al., 2000). This bacterium can cause infections in a variety of animals and plants, and can cause acute infections or chronic biofilm infections in the lungs of cystic fibrosis patients (Burns et al., 1993; Costerton, 1995; Costerton et al., 1999; Hoiby, 1993; Singh et al., 2000; Smith & Iglewski, 2003). A report by Mah et al. (2003) demonstrated that the P. aeruginosa ORF PA1163 plays a role in the tolerance of biofilm cells to antibiotics. The protein encoded by PA1163 shows 58 % identity to NdvB of B. japonicum. The NdvB protein synthesizes cyclic β-glucans; however, the identity of the compound synthesized by this protein in P. aeruginosa has not been determined. Two other P. aeruginosa ORFs, PA5077 and PA5078, encode putative polypeptides showing sequence similarity with OpgH and OpgG, respectively. The polypeptide encoded by PA5077 in P. aeruginosa shows 74 and 76 % similarity to the OpgH polypeptides of P. syringae and E. coli, respectively. The polypeptide encoded by PA5078 shows 82 and 81 % similarity to the OpgG polypeptides of P. syringae and E. coli, respectively. An opgH mutant (PA5077) of P. aeruginosa PA14, obtained by screening a random transposon insertion library, showed impaired virulence in Caenorhabditis elegans, mouse and Arabidopsis virulence models (Mahajan-Miklos et al., 1999).
Here, we report that PA5077 and PA5078 in P. aeruginosa PA14 are responsible for the synthesis of OPGs similar to those present in a variety of bacterial species (Bohin & Lacroix, 2006). The glucans are linear with β-1,2-linked glucose units branched with β-1,6 glucose units. Succinyl residues substitute the glucose backbone. We also show that opgGH and ndvB do not co-operate in the biosynthesis of these linear OPGs. Although strains with opgG and opgH defects form abnormal biofilms, the linear glucans do not appear to participate in the tolerance of biofilm cells to antibiotics.
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METHODS |
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. The parent strain was P. aeruginosa PA14. Strain YL119 contained a PA5077 and PA5078 deletion and was constructed as follows. The intermediate construct pYL177 was generated by flanking a gentamicin resistance cassette with a 1.58 kb DNA fragment containing the region upstream of the PA5078 start codon and a 1.89 kb DNA fragment containing the region downstream of the PA5077 stop codon. A KpnI fragment containing the mobilization cassette from pKV69 was inserted into the KpnI restriction site of pYL177 to create pYL178, a mobilizable suicide vector. We moved pYL178 from E. coli SM10 into P. aeruginosa PA14 by conjugation. To obtain P. aeruginosa YL119 we selected gentamicin-resistant colonies and then screened for carbenicillin sensitivity. Strain YL119 has a deletion encompassing the entire operon PA5078-PA5077, from 250 bp upstream of the PA5078 start codon to 171 bp downstream of the PA5077 stop codon. The deletion was confirmed by PCR analysis. A DNA fragment containing 1.58 kb upstream of the PA5078 start codon, the two ORFs PA5078 and PA5077 and 171 bp downstream of the PA5077 stop codon was cloned into pUCP18 to generate pYL205.
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). The osmolarity of LOS medium was increased by addition of sucrose or NaCl as indicated. Antibiotics were used as appropriate at the following concentrations (ml–1): carbenicillin, 150 µg, gentamicin, 100 µg, for P. aeruginosa; ampicillin, 50 µg, gentamicin, 10 µg, for E. coli.
Real-time PCR.
The following primers were designed with Primer Express software (Taqman) and used for real-time PCR analyses: 5'-CGTTACACCCCGGTCCTGAA-3' and 5'-ACCGGCTTCTTCGGATCCT-3' for PA5078, and 5'-ATCCAGTGGCTGATCGAACAG-3' and 5'-CGGGTCAGGTGGTATTCGAA-3' for nadB. Total RNA, extracted from cultures when they reached an OD600 of 0.8, was purified using the RNeasy kit (Qiagen) and cDNA was synthesized as described by Schuster et al. (2003). PCR controls using designated primers were performed with cDNA or genomic DNA to verify the integrity of the PCR products. Real-time PCR reactions included 5 ng cDNA and primers at a concentration of 300 nM in 25 µl SYBR Green PCR amplification Master Mix (Applied Biosystems). Conditions for real-time PCR were 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C (denaturation) and 1 min at 60 °C (annealing and extension). Genomic DNA was used as a standard and nadB (PA0761) was used as an internal control. No amplification was measured when RNA was added. Real-time PCR reactions were performed on an ABI 7900HT apparatus and associated software, SDS 2.1, was used to assess cDNA levels. Experiments were done three times with three replicates in each experiment.
Biofilm experiments in flow chambers.
Biofilms were grown in flow chambers as described elsewhere (Davies et al., 1998). We used LOS medium for flow chamber experiments. Overnight cultures were used to inoculate chambers with 5x106 bacteria in 500 µl LOS medium. One hour after inoculation of the chambers, flow was initiated at a rate of 0.17 ml min–1. Biofilm images were acquired using a Zeiss scanning confocal laser microscope (SCLM) system (Axioplan2 confocal microscope and Laser scanning module LSM 5.10; Carl Zeiss MicroImaging). The excitation wavelength for green fluorescent protein (GFP) was 488 nm and emission was observed at 505 nm with an LP 505 filter. Three distinct objective lenses were used: a plan-apochromat x63 oil objective lens, a C-apochromat x40 water objective lens, and a plan-neofluar x20 objective lens. Three-dimensional images were generated with Volocity software (Improvision). All biofilm experiments were repeated a minimum of four times at room temperature (26–28 °C). Measurements of biofilm architectural features were determined by using COMSTAT v.1 software (Heydorn et al., 2000).
Isolation and purification of OPGs.
P. aeruginosa strains were grown overnight in 350 ml LOS medium. Cells were harvested by centrifugation and the OPGs were extracted from cell pellets with 5 % trichloroacetic acid followed by charcoal adsorption and elution with aqueous pyridine as described by Lacroix et al. (1989). OPGs present in the aqueous pyridine extracts were purified by gel filtration chromatography on a Bio-Rad Biogel P6 column (1.8x62 cm, flow rate 10 ml h–1). The column was pre-equilibrated with 0.05 % acetate solution and OPGs were eluted with 0.05 % acetate solution in fractions of 1 ml. Sugar content was determined by a colorimetric procedure using phenol/sulfuric acid reagent (Dubois et al., 1956). Fractions containing sugar were pooled and concentrated by rotary evaporation. Where indicated, fractions of OPG were treated with 0.05 M KOH at 37 °C for 1 h to remove O-ester-bound substituents. After neutralization with AG 50W-X8 (Bio-Rad; H+ form), samples were desalted on a Bio-Gel P2 column (Bio-Rad). Native and KOH-treated oligosaccharides were lyophilized.
Mass spectrometry.
Matrix-assisted laser desorption-ionization (MALDI)-MS experiments were done with a Vision 2000 (Finnigan MAT) time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm wavelength and 3 ns pulse width). A 2,5-dihydroxybenzoic acid matrix was used for carbohydrate analysis (10 g l–1 in water) (Stahl et al., 1991). Lyophilized oligosaccharide samples were dissolved in double-distilled water at a concentration of 0.1 µg µl–1, and then diluted with an appropriate volume of the matrix solution (1 : 2, v/v). One microlitre of the resulting solution was deposited on a stainless steel target, and the solvent was evaporated under a gentle stream of warm air. After microscope-assisted selection of the appropriate site on the target, laser light was focused onto the sample/matrix mixture at an angle of 1 ° and a power level of 106–107 W cm–2. Positive ions were extracted by a 5–10 keV acceleration potential and focused with a lens. Masses were separated by a reflectron time-of-flight instrument. Ions were post-accelerated to 20 keV for maximum detection efficiency. Resulting signals were recorded by using a fast transient digitizer with a maximum of 2.5 ns channel resolution. All MALDI mass spectra are the result of 20 single-shot accumulations.
Methylation analysis.
Oligosaccharides were treated with sodium borodeuteride to reduce the reducing glycosyl termination. Glucosidic linkage analysis was performed by the methylation method of Parente et al. (1985). Methyl ether compounds were hydrolysed (4 M trifluoroacetic acid at 100 °C for 4 h), reduced with sodium borodeuteride and peracetylated. The partially methylated and acetylated glycosides were analysed by GLC-MS using a Delsi apparatus with a capillary column (25 mx0.2 mm) coated with DB-1 (0.5 µm film thickness). We applied a temperature gradient of 110–240 °C at 2 °C min–1 and a helium pressure of 40 kPa. Mass spectra were recorded on a Nermag 10-10B mass spectrometer (Rueil Malmaison) using an electron energy of 70 eV and an ionizing current of 0.2 mA. Specific standards of glucosyl residues were used to calibrate the GLC column and identify the eluted glycosides. The identity of each eluted glycoside was confirmed by MS coupled to GLC (Fournet et al., 1981).
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RESULTS |
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). To determine the function of OpgH (PA5077) and OpgG (PA5078) in P. aeruginosa, we generated a deletion mutant, YL119, which is devoid of the opgGH locus, in a strain PA14 background. Extracts from the parent strain PA14 and YL119 were separated by gel filtration. A large peak containing sugar residues was eluted in fractions 80–100 from PA14 extracts (Fig. 1). This peak was absent in extracts from the opgGH mutant strain YL119. For each strain, fractions 80–100 were pooled for further analyses. MS of the pooled fractions from PA14 extracts revealed the presence of 8 molecular ions (Fig. 2). Five molecular ions, m/z=1013, 1175, 1337, 1499 and 1661, correspond to five distinct linear polymers containing 6–10 glucose residues, respectively. Three molecular ions with m/z=1275, 1437 and 1599 correspond to linear polymers of 7–9 glucosyl residues, respectively, with a mass increment of 100 Da each (Fig. 2). A mild alkaline treatment of OPGs eliminates the substitutent responsible of this increment. The mass increment of 100 Da may correspond to a succinate residue or an isomer. MS of the pooled fractions 80–100 from strain YL119 did not reveal any polymers of glucose (data not shown). To confirm that the opgGH locus is necessary for the biosynthesis of OPGs, we complemented the opgGH mutation in strain YL119 with pYL205, which contains opgGH and 1.58 kb upstream of the opgG translation start codon. Strain YL119(pYL205) produced 14 times more OPGs than wild-type PA14 (27x10–15 and 2x10–15 g equivalent glucosyl residues per cell, respectively, taking into account only the second peak in Fig. 1). The overexpression of OPGs caused by the presence of opgGH on a multicopy plasmid did not influence the growth of planktonic culture (supplementary Fig. S1, available with the online version of this paper).
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In other bacteria the expression of opgGH homologues is generally repressed by high environmental osmolarity (Bohin & Lacroix, 2006). To determine if this is true of opgGH in P. aeruginosa, we measured opgGH transcript levels by real-time PCR in P. aeruginosa PA14 grown in LOS medium (low osmolarity medium, 70 mOsm) and LOS medium supplemented with 300 mM sucrose or 150 mM NaCl (370 mOsm). In LOS medium, the level of opgGH RNA reached 54±10 fg opgGH RNA (ng RNA)–1, while in LOS medium with added sucrose or NaCl, opgGH RNA levels were only 13±2 or 10±3 fg opgGH RNA (ng RNA)–1, respectively. The expression of opgGH in LOS medium was four times higher (P<0.01) than in LOS medium with added sucrose and five times higher (P<0.002) than in LOS medium supplemented with NaCl. These results confirm that OPG biosynthesis in P. aeruginosa is repressed under high osmolarity conditions.
Involvement of linear OPGs in biofilm formation
An earlier report indicated that cyclic OPGs were critical for the tolerance of P. aeruginosa biofilm cells to antibiotics (Mah et al., 2003). To determine whether the opgGH-dependent linear OPG influenced biofilm formation or biofilm tolerance to antibiotics, we first measured biofilm formation in LOS medium by using a microtitre dish assay (O'Toole & Kolter, 1998) and we detected no significant difference in the amount of biofilm formed by the mutant opgGH and the parent using LOS medium with or without added NaCl (150 mM) or sucrose (300 mM), or M63 medium (data not shown). We then measured biofilm sensitivity to tobramycin using the microtitre dish assay described by Mah et al. (2003). We also detected no difference in sensitivity of the opgGH mutant and the parent to tobramycin. The minimal bactericidal concentration (MBC) was 400 µg ml–1 for both strains. As a control we tested a mutant ndvB strain, described by Mah et al. (2003) as sensitive to tobramycin during biofilm growth. This mutant showed a reduced MBC in the biofilm mode of growth (50–100 µg ml–1), but was unaffected in planktonic growth (MBC for all three strains was 4–8 µg ml–1). Based on these results, the OPGs described here do not appear to play a significant role in attachment of cells to a substratum or in P. aeruginosa biofilm resistance to tobramycin.
All of the previously described biofilm experiments involve early biofilm development under static growth conditions. Thus we decided to study the influence of opgGH on biofilm development under a flow of culture medium by using scanning confocal microscopy (Fig. 3). Twenty-four hours after inoculation with the parent PA14, individual cells and some small cell clusters were evident on the glass surface of the flow cell. A similar pattern was observed with the opgGH mutant at 24 h. At 48 h, microcolonies of the parent had developed, but the mutant bacterial cells remained as individuals. At 72 h, microcolonies of the parent were larger than those observed at 48 h with a mean thickness of 25 µm and a maximum thickness of 65 µm (Table 2). The biofilm covered the entire glass surface. During the next 4 days, the biofilm formed the mushroom-like structures characteristic of mature P. aeruginosa biofilms grown under the conditions we used (Fig. 3). Mutant strain microcolonies were evident at 72 h. However, the mutant biofilm did not develop further during the next 4 days (Fig. 3). A statistical analysis with COMSTAT v.1 (Heydorn et al., 2000) confirmed our qualitative analysis (Table 2). For example, the mean thickness of a 5-day mutant biofilm was 25 % of the parent, and the total area of biomass was about half that of the parent. The mutation did not affect growth of P. aeruginosa in either LB broth or LOS medium (data not shown).
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and supplementary Fig. S2, available with the online version of this paper). Apparently, OPG molecules have subtle effects on biofilm development. The mutant is slow to develop microcolonies and does not develop into mature biofilms with mushroom-like structures. We studied biofilm formation of wild-type PA14 and the opgGH mutant under conditions of high osmolarity by adding sucrose (300 mM) or NaCl (150 mM) to LOS medium. Both wild-type PA14 and the opgGH mutant biofilms showed the same phenotype as the biofilm of the YL119 mutant grown under low osmolarity conditions (Table 2 and Fig. 3). No difference in growth rate was detected when planktonic growth of strains YL119 and PA14 under different conditions of osmolarity was studied (supplementary Fig. S1). This result shows that the biofilm growth defect in YL119 is specific to the biofilm growth mode.
Motility and rhamnolipid production are not affected by opgGH
Motility mediated by type IV pili and the polar monotrichous flagellum, respectively (Harshey, 2003), and rhamnolipid production are known to be involved in microcolony and mushroom-like structure development (Davey et al., 2003; Klausen et al., 2003a, b; Lequette & Greenberg, 2005). To determine whether abnormal biofilm development in the opgGH mutant resulted from altered motility, we compared flagellar and twitching motility of the mutant and the parent. We used LOS medium as the base medium with 0.3, 0.5 and 1 % agar for swimming, swarming and twitching motility measurements, respectively (Bradley, 1980; Kohler et al., 2000; Taylor & Koshland, 1974). The mutant and parent were indistinguishable (data not shown). We monitored rhamnolipid gene expression by using an rhlA-gfp fusion, and rhamnolipid production by a rhamnolipid plate assay (Kohler et al., 2000; Lequette & Greenberg, 2005). As with motility, we could not distinguish the parent and mutant (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Similarly, an opgH mutant of the plant pathogen P. syringae opgH was shown to be non-pathogenic (Mukhopadhyay et al., 1988). Genomic sequencing has shown that opgH is often located immediately downstream of opgG in a putative operon (Bohin & Lacroix, 2006). In P. aeruginosa the opgG (PA5078) and opgH (PA5077) homologues are contiguous and show a three-codon overlap. Thus, the two genes are probably co-transcribed as an operon.
We have shown that, in P. aeruginosa, opgGH are involved in the biosynthesis of linear glucans of heterogeneous size, ranging from 6 to 10 glucosyl units per molecule. Expression of opgGH is repressed by elevating the osmolarity of the culture medium. Our analysis of opgGH-dependent glucans revealed that the glucosyl backbone is a linear chain of glucosyl units linked at positions 1 and 2, and branched with a few glucosyl residues linked at position 6. All OpgH homologues share motifs conserved in Family II glycosyl transferases that catalyse β anomer glycosyl linkages (Coutinho et al., 2003) (Carbohydrate-Active Enzymes server at http://afmb.cnrs-mrs.fr/CAZY/). OpgH homologues catalyse the formation of β-1,2 linkages using UDP--glucose as a substrate (Loubens et al., 1993; Therisod et al., 1986). One can assume that like other OpgHs, the P. aeruginosa OpgH catalyses the synthesis of a β-1,2-linked linear glucose chain by using UDP--glucose as well. Unlike OPG in P. syringae, our analysis indicates the glucosyl backbones of P. aeruginosa OPG are substituted, probably with succinate residues. We do not know what physiological significance such decoration might confer. OPG synthesis was not affected by inactivation of ndvB, and no cyclic glucans could be detected after extraction of the samples with 5 % trichloroacetic acid (data not shown), a procedure known to allow purification of cyclic OPG (Talaga et al., 2002).
Periplasmic glucans synthesized by P. aeruginosa OpgGH can represent 0.75 % of the dry cell weight in parent strain PA14. Moreover, in the opgGH overexpression strain, OPGs constituted nearly 10 % of the total cell dry weight. OPGs can represent 5–20 % of the total cell dry weight depending on the species and growth conditions (Bohin & Lacroix, 2006; Breedveld & Miller, 1994). Low osmolarity and high levels of available nutrients are two of the critical conditions for large amounts of glucans. In several species, the synthesis of large amounts of glucans does not disturb growth when cultures are grown under optimal conditions, such as in the laboratory (Bohin & Lacroix, 2006; Breedveld & Miller, 1994). However, we should be cautious about the significance of these quantities, considering the harsh conditions encountered by bacteria in the environment, where growth rate is low and nutrient availability is restricted.
We investigated the involvement of opgGH in biofilm development because a previous report suggested that OPGs might be involved in the innate tolerance of cells in P. aeruginosa biofilms to antibiotics (Mah et al., 2003). This earlier study involved a genetic screen for mutations that increased the sensitivity of biofilms to the clinically relevant antibiotic tobramycin. The screen revealed that mutations in ndvB decreased biofilm resistance to tobramycin. Using an assay similar to that used previously, we showed that, unlike ndvB, the opgGH locus is not involved in tolerance of biofilm-grown bacteria to tobramycin, even though the OPGs synthesized by OpgGH were significant in their quantity. This line of investigation led us to explore the potential relationship between opgGH and biofilm development more thoroughly. Our experiments indicate that initiation of biofilm development is not altered by an opgGH mutation, but there is a delay in the development of microcolonies which susbsequently do not develop into the normal mushroom-like structures characteristic of wild-type biofilms under low osmolarity conditions. Increasing the medium osmolarity also affected the biofilm growth rate and the development of mushroom-like structures in wild-type PA14 (Table 2). These results suggest that P. aeruginosa PA14 develops preferentially larger and more structured biofilms under low osmolarity conditions, and that OPGs are important for biofilm development under these conditions.
In summary, previous investigations have shown that opgH is involved in virulence of P. aeruginosa. We show here that, like in other bacteria, the P. aeruginosa opgGH operon is required for synthesis of abundant osmotically regulated glucans, which we assume to be periplasmic. We have characterized the structures of these glucans, but how they function in virulence remains to be determined.
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ACKNOWLEDGEMENTS |
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Edited by: P. Cornelis
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Received 12 April 2007;
revised 19 June 2007;
accepted 27 June 2007.
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), opgGH mutant strain YL119 (
) and YL119 with plasmid pYL205 (
) were eluted on a gel-filtration Biogel P6 column (see Methods). PA14 and YL119 data are plotted on the left-hand y-axis and YL119-pYL205 data are plotted on the right-hand y-axis.
