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Journal of Bacteriology, February 2006, p. 1165-1168, Vol. 188, No. 3
0021-9193/06/$08.00+0     doi:10.1128/JB.188.3.1165-1168.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutations in the waaR Gene of Escherichia coli Which Disrupt Lipopolysaccharide Outer Core Biosynthesis Affect Cell Surface Retention of Group 2 Capsular Polysaccharides

Clare M. Taylor, Marie Goldrick, Liz Lord, and Ian S. Roberts*

Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Received 22 September 2005/ Accepted 31 October 2005


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ABSTRACT
 
On the basis of increased resistance to K5 capsule-specific bacteriophage, a waaR transposon mutant defective in the biosynthesis of lipopolysaccharide outer core was isolated. In a K1-expressing strain the mutation equally affected sensitivity to K1 capsule-specific bacteriophage, indicating a general effect on group 2 capsules. The waaR mutation affected retention on the cell surface of the K5 polysaccharide, with increased polysaccharide accumulating in the culture supernatant. This indicates that interactions between the outer core of lipopolysaccharide and group 2 capsular polysaccharides are important for the stabilization of group 2 capsular polysaccharides on the cell surface.


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TEXT
 
The expression of cell surface polysaccharides is a common feature of many bacterial pathogens. In the case of Escherichia coli the cell surface may be decorated with lipopolysaccharide (LPS), enterobacterial common antigen, as well as capsular polysaccharides or K antigens (17). It is likely that interactions between these polysaccharide molecules will be important in stabilizing and retaining the cell surface organization of these polysaccharides.

The LPS of E. coli has a tripartite structure consisting of highly conserved lipid A moiety inserted in the outer membrane, a phosphorylated core oligosaccharide, and a long polysaccharide chain that comprises the serotypic O antigen (9). The core oligosaccharide can be divided into inner and outer core; the inner core is more invariant among members of the family Enterobacteriaceae and is comprised of heptose and 3-deoxy-D-manno-oct-2-ulosonic acid residues which are phosphorylated (6, 9). The generic structure of the E. coli outer core is of a backbone oligosaccharide made up of three hexoses linked to the terminal heptose with two branch substitutions (6). Variation occurs in terms of the component hexoses, their linkage, and the position of the side chain substitutions (6, 9).

In E. coli there are five outer core types, R1, R2, R3, R4, and K-12 (6). Analysis of the distribution of outer core types in E. coli showed that the R1 core type was the most predominant, being associated with virulent extraintestinal pathogenic isolates, whereas the R3 core type was the only core type found in verotoxigenic E. coli (1). The waaQ gene cluster encodes the proteins for the biosynthesis of core oligosaccharide, and there is variation in the genetic organization between the five different waa gene clusters encoding the different outer core types (1, 6). The best studied is the K-12 core type, and functions have been assigned or predicted based on homology to the majority of the Waa proteins (Fig. 1) (6).


Figure 1
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  		FIG. 1. A. Organization of the waaQ operon in E. coli K-12. The primers K12-1 and K12-2a used to amplify the 916-bp fragment spanning the waaL and waaU genes are shown. B. Structure of the E. coli K-12 core, indicating the steps catalyzed by individual Waa proteins. The reactions performed by the WaaR, WaaU, WaaY, and WaaZ proteins are highlighted in boxes. Phosphate groups are depicted with the letter P. C. Site of the EZ::TN transposon insertion in the waaR gene in strain MSZA08. The small arrow depicts the EZ::TN forward sequencing primer (see text for details) used to determine the site of insertion in the waaR gene. The PstI restriction site used to rescue the transposon is shown by the letter P.

Escherichia coli can express over 80 chemically and serologically distinct capsular K antigens. Based on a number of biochemical and genetic criteria these K antigens have been divided into four groups (1 to 4) (17). Group 2 capsules are often expressed by pathogenic E. coli isolates causing extraintestinal disease (5) and resemble those found on the surfaces of Neisseria meningitidis and Haemophilus influenzae (12). The group 2 capsule gene cluster is termed kps and is located proximal to the serA locus on the E. coli chromosome (11). Molecular analysis of the K1 and K5 capsule gene clusters has demonstrated that kps gene clusters have a segmental organization in which two conserved regions (1 and 3) encoding proteins involved in the transport of group 2 capsular polysaccharides flank a central serotype-specific region that encodes proteins for the synthesis of the particular K antigen (17).

In this study we used transposon mutagenesis and sensitivity to K5 capsule-specific bacteriophage to identify mutants altered in K5 capsule expression. A waaR mutant defective in outer core synthesis was isolated and shown to be affected in the cell surface expression of both the K5 and K1 capsules, probably by altering retention of the capsular polysaccharide on the cell surface.

Isolation of a waaR mutant with reduced sensitivity to the K5 capsule-specific bacteriophage. To identify mutants affected in K5 capsule expression, strain MS101 (13) was mutagenized using the EZ::TN <R6K{gamma}ori/KAN-2> Tnp Transposome kit (Epicenter) according to the manufacturer's instructions. Five thousand independent insertion mutants were screened for those altered in sensitivity to K5 capsule-specific bacteriophage (10). Ten mutants with altered sensitivity to K5 capsule-specific bacteriophage were isolated, of which one mutant (MAZA07), with a three-log reduction in bacteriophage sensitivity, could not be complemented using plasmid pGB118, which encodes the entire K5 capsule gene cluster (10). The transposon mutation was moved by P1 transduction from strain MSZA07 into MS101 to generate MSZA08. This strain displayed the same pattern of sensitivity to K5 capsule-specific bacteriophage as MSZA07, confirming that that the disruption of K5 capsule expression was a consequence of the transposon insertion.

The site of transposon insertion was determined following digestion of MSZA08 chromosomal DNA with PstI, which cleaves at one end of the EZ::TN <R6K{gamma}ori/KAN-2> (Fig. 1) and subsequent transformation of self-ligated DNA into strain SM10{lambda}pir, selecting for kanamycin-resistant transformants. Analysis of plasmid DNA from 12 transformants showed the presence of an 8-kb plasmid that contained 6 kb of chromosomal DNA flanking one end of the transposon insertion (Fig. 1). The nucleotide sequence of the flanking chromosomal DNA flanking was determined using the EZ::TN <R6K{gamma}ori/KAN-2> forward sequencing primer, 5'-ACCTACAACAAAGCTCTCATCAACC-3', and indicated that the transposon had inserted 124 bp 5' from the end of the waaR gene (Fig. 1).

The waaR gene is part of the waaQ operon, encoding proteins involved in core oligosaccharide synthesis (Fig. 1), and is organized differently depending on the R core type (6). To confirm the core type of MS101 and thereby the organization of the waaQ operon, colony PCR was performed on strains MS101 and MSZA08 using primers K12-2a, 5'-TAATGATAATTGGAATGCTGC-3',and K12-1, 5'-TTCGCCATTTCGTGCTACTT (1). These primers amplified a 916-bp fragment containing the 3' ends of the waaL and waaU genes diagnostic of a K-12 core type (1) in which the waaY, waaZ, and waaU genes are 3' to the waaR gene (Fig. 1).

Characterization of the waaR mutant. The WaaR protein is an {alpha}1,2-glycosyltransferase that adds the third glucose residue (GlcIII) to the K-12 outer core such that a waaR mutant will be predicted to express truncated core lacking both the GlcIII as well as the terminal HepIV residue (Fig. 1) (6). Analysis of the lipopolysaccharide profiles of strains MS101 and MSZA08 by T-sodium dodecyl sulfate (15) showed that the waaR mutation resulted in a truncated core lipopolysaccharide compared to MS101 (Fig. 2). No O antigen is detectable in strain MS101 since it has an rmlD mutation that blocks TDP-rhamnose biosynthesis essential for the synthesis of the K-12 (O16) antigen (14).


Figure 2
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  		FIG. 2. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide preparations from strains MS101 (lane A), MSZA08(pWaaR) (lane B), and MSZA08 (lane C). The waaR mutation results in a loss of glucose and heptose from the outer core, generating a faster-migrating lipopolysaccharide molecule. The presence of the cloned waaR gene restores the wild-type lipopolysaccharide profile.

To establish if the transposon insertion in the waaR gene was polar on expression of the waaY, waaZ, and waaU genes (Fig. 1), the waaR gene was amplified from MS101 using primers WaaRFB, 5'TGGGATCCGGGTAGCATTGTGGACTCATTTC-3', and WaaRRE, 5'-GCGGAATTCCACTAAAAGATG-3', and cloned into pBluescript (Stratagene). to generate plasmid pWaaR. Strain MSZA08(pWaaR) expressed a wild-type core lipopolysaccharide profile (Fig. 2) and was as sensitive to K5 capsule-specific bacteriophage as MS101, indicating that the phenotype of MSZA08 is due to a loss of WaaR function. In addition, MS101 and MSZA08 had identical MICs of 50 mg ml–1 for novobiocin and 100 mg ml–1 for sodium dodecyl sulfate, indicating that strain MSZA08 had no perturbation of the outer membrane which would have been predicted if there was a loss of either WaaY or WaaZ activity, both of which are involved in modifications to the inner core (4, 18, 19). In contrast, the WaaU protein acts after the WaaR protein to add the terminal HepIV residue to GlcIII in the outer core (Fig. 1) (4, 6).

To confirm that WaaU function was not essential for sensitivity to K5 capsule-specific bacteriophage, the waaU::kan mutation was moved by P1 transduction from strain CS2529 (1) into MS101. The introduction of the waaU mutation had no effect on sensitivity to K5 capsule-specific bacteriophage. Overall these data indicate that the effect on sensitivity to K5 capsule-specific bacteriophage is a consequence of a loss of WaaR function.

To establish if the effect of the waaR mutation was specific to expression of the K5 capsule the waaR mutation in strain MSZA08 was moved by P1 transduction into the K1 capsule-expressing strain EV1 (16). The introduction of the waaR mutation into EV1 caused a three-log reduction in sensitivity to K1E bacteriophage as assayed by bacteriophage titer. This indicates that the effect of the waaR transposon mutation was not specific to strains expressing the K5 capsule and sensitivity to the K5 capsule-specific bacteriophage.

To confirm these data, a second independent waaR mutant strain (MS101{Delta}waaR) was generated using the PCR-based allelic replacement method of Datsenko and Wanner (3) in which the entire waaR coding sequence was removed. Strain MS101{Delta}waaR displayed the same phenotype as strain MSZA08 with regard to sensitivity to K5 capsule-specific bacteriophage and could be complemented by pWaaR. This confirms that a loss of WaaR function effects sensitivity to K5 capsule-specific bacteriophage.

waaR mutation does not affect K5 polysaccharide biosynthesis but does affect the retention of cell surface K5 polysaccharide. To determine if the reduced sensitivity to K5 capsule-specific bacteriophage of strain MSZA08 was a consequence of less K5 polysaccharide being synthesized, the total amount of K5 polysaccharide produced by MS101 and MSZA08 was quantified by measuring the increase in A232 following degradation of the purified polysaccharide using the K5 lyase (2). Strain MSZA08 synthesized more K5 polysaccharide than MS101 (Table 1) but significantly, the majority (77%) of K5 polysaccharide synthesized by strain MSZA08 was present in the culture supernatant, as opposed to 33% in the case of MS101 (Table 1).


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  		TABLE 1. Synthesis of K5 polysaccharide by strains MS101 and MSZA08 following overnight growth at 37°C in L broth

It is believed that the K5 polysaccharide is retained on the cell surface through lipid substitution at the reducing end of the polysaccharide to a phosphatidic acid molecule in the outer membrane (7). Polysaccharide present in the culture supernatant is believed to arise due to the lability of the phosphodiester linkage between the capsular polysaccharide and the phosphatidic acid (7). Analysis of the polysaccharide present in the culture supernatant of both MS101 and MSZA08 by polyacrylamide gel electrophoresis and alcian blue staining (2) demonstrated that in both cases the polysaccharide had the same molecular mass, within the range of 50 to 60 kDa (data not shown). This indicates that the waaR mutation was not causing the synthesis of lower-molecular-weight K5 polysaccharide that was being differentially shed from the cell surface and that the waaR mutation is affecting the retention of the K5 polysaccharide on the cell surface.

To examine the cell surface capsule, bacteria were subjected to transmission electron microscopy following labeling with cationized ferritin as described (8). In the case of MS101 an electron-dense area corresponding to polysaccharide capsule was detectable (Fig. 3) which was lacking in the acapsular laboratory strain PA360 (Fig. 3). In the case of MSZA08 the capsule appeared incomplete, with polysaccharide detached from the cell surface being sloughed off into the surrounding medium (Fig. 3); in contrast strain MSZA08(pWaaR) had a wild-type capsule (Fig. 3). These data indicate that the waaR mutation was affecting the retention of cell surface K5 polysaccharide.


Figure 3
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  		FIG. 3. Transmission electron microscopy of cationized ferritin-labeled cells. (A) MSZA08; (B) MSZA08(pWaaR); (C) PA360; (D) MS101. The arrows show the electron-dense cell surface capsule and in panel A the material sloughing off the cell surface. Bar, 1.2 mm.

Conclusions. The identification that mutations in the waaR gene affect sensitivity to K5 capsule-specific bacteriophage is surprising. The observation that waaR mutants are not blocked in K5 biosynthesis suggested that the effect of the waaR mutation was likely to be on the decoration of the cell surface with K5 polysaccharide. The transmission electron microscopy confirmed that the waaR mutations were perturbing the formation of a cell surface capsule, appearing to reduce the retention on the cell surface of K5 polysaccharide. This was supported by the increased proportion of K5 polysaccharide present in the culture supernatants of strain MSZA08 compared to the wild type. Since the WaaR protein is an {alpha}1,2-glycosyltransferase that adds the third glucose residue (GlcIII) to the K-12 outer core, the effect on the cell surface retention of K5 might suggest that interactions between the outer core and the K5 capsular polysaccharide are important in maintaining the overall cell surface architecture. The fact that waaU mutants that lack the terminal HepIV are unaffected in their sensitivity to K5 capsule-specific bacteriophage indicates that it is the addition of the GlcIII alone or the GlcIII and HepIV together that is important for this effect. The finding that waaR mutations also affect the sensitivity of a K1 strain to capsule-specific bacteriophage would suggest that this effect on capsule retention is not peculiar to the K5 capsule.


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ACKNOWLEDGMENTS
 
We acknowledge the kind gift of strain CS2529 from C. Whitfield at the University of Guelph.

This work was supported by grants from the BBSRC, United Kingdom.


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FOOTNOTES
 
* Corresponding author. Mailing address: Faculty of Life Sciences, 1.800 Stopford Building, University of Manchester, Manchester M13 9PT, United Kingdom. Phone: (0)161 275 5601. Fax: (0)161 275 5656. E-mail: i.s.roberts{at}manchester.ac.uk. Back


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Journal of Bacteriology, February 2006, p. 1165-1168, Vol. 188, No. 3
0021-9193/06/$08.00+0     doi:10.1128/JB.188.3.1165-1168.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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