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Journal of Bacteriology, March 2009, p. 1716-1718, Vol. 191, No. 5
0021-9193/09/$08.00+0     doi:10.1128/JB.01371-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

Thomas Hudson, Marie Goldrick, and Ian S. Roberts^*

Faculty of Life Sciences, The Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom

Received 1 October 2008/ Accepted 8 December 2008

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The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the Escherichia coli K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.

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The polysaccharide capsules of bacteria have been studied in most detail in Escherichia coli (13). E. coli has over 80 chemically and serologically distinct polysaccharide capsules, which are designated K antigens and classified into four groups (13). Group 2 polysaccharide capsules, of which K1 and K5 have been most studied (12, 13), are commonly expressed in pathogenic extraintestinal E. coli (1, 3, 7) and closely resemble the capsules of Neisseria meningitidis and Haemophilus influenzae (13). Group 2 capsule gene clusters have a conserved genetic organization comprising three regions. Regions 1 and 3 are common to all group 2 capsule gene clusters and encode the Kps proteins involved in polysaccharide export across the inner membrane, periplasm, and outer membrane. Region 2, flanked by regions 1 and 3, contains the highly variable serotype-specific genes involved in the biosynthesis of the particular polysaccharide (12, 13). In the case of the K5 capsule gene cluster, this involves the kfiABCD genes (9).

The biosynthesis of the K5 polysaccharide occurs through the sequential addition of GlcA and GlcNAc residues to the nonreducing end of the polysaccharide chain catalyzed by two glycosyltransferases, KfiA and KfiC (4, 6). Polysaccharide biosynthesis occurs at the cytoplasmic face of the inner membrane and involves a hetero-oligomeric complex, consisting of proteins involved in both biosynthesis and export, that is localized at the pole of the cell (8). Such a complex would facilitate a linkage between polysaccharide synthesis and export, although at this stage the mechanism by which synthesis and export are linked is unclear. A recent paper in which the K1-specific endosialidase was expressed in the cytoplasm of a K1-expressing strain indicated that K1 polysaccharide synthesis may occur within a protected cytoplasmic compartment that is inaccessible to endosialidase cleavage (10). To test whether this was also true for the synthesis of the K5 polysaccharide, we expressed the K5-specific lyase, an enzyme that specifically degrades K5 and is associated with the tail spike of K5-specific bacteriophage (2, 5), in the cytoplasm of a K5-encoding strain. In contrast to the situation with K1, we found that expression of the K5 lyase in the cytoplasm reduced the cell surface expression of K5 polysaccharide, suggesting that unlike K1 polysaccharide synthesis, K5 polysaccharide is not synthesized within a protected cytoplasmic compartment.

Expression and purification of K5 lyase within MS101. To investigate whether K5 lyase could be expressed and purified from E. coli expressing K5 polysaccharide, the K5-positive strain MS101 (11) was transformed with plasmid pLYA100, an arabinose-inducible plasmid containing the K5 lyase gene (2), induction of which results in the accumulation of K5 lyase in the cytoplasm of the cell (2). Subsequently, a 500-ml cell culture was grown to an optical density at 600 nm (OD₆₀₀) of 0.5 at 37°C in LB medium, supplemented with 100 µg liter^–1 ampicillin and 50 µg liter^–1 streptomycin. Cultures were induced with 0.1% (wt/vol) arabinose, and the K5 lyase was purified by Ni affinity chromatography essentially as described previously (2). Samples at different stages of the purification process were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1). A large amount of K5 lyase was observed in the elution fractions, indicating that K5 lyase was efficiently expressed in MS101 and that the production of K5 polysaccharide had no negative effect on the expression of K5 lyase. Western blot analysis of lysate and elution fractions using antisera to either the KpsE or KpsD proteins that are involved in group 2 polysaccharide export (8) demonstrated that these two proteins were still being made in the presence of the K5 lyase (Fig. 1). Therefore, expression of the K5 polysaccharide capsule gene cluster was not significantly disrupted during the production of K5 lyase.

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FIG. 1. Purification of the K5 lyase from MS101(pLYA100). Samples were analyzed by Coomassie blue staining (top panel) and Western blotting using anti-KpsE and anti-KpsD antibodies (middle and bottom panels, respectively). Lane 1, lysate; lane 2, flowthrough; lane 3, 10 mM imidazole column wash; lane 4, 50 mM imidazole column wash; lane 5, 100 mM imidazole column wash; lanes 6 to 8, 250 mM imidazole column elution fractions.

Effect of K5 lyase on the cell surface expression of the K5 polysaccharide capsule. To determine whether K5 polysaccharide biosynthesis was taking place in a protected compartment in the cytoplasm, we studied the effects of expression of the K5 lyase on the cell surface expression of K5 polysaccharide in strain MS101(pLYA100). We assayed the cell surface expression of K5 polysaccharide by measuring the sensitivity to K5-specific bacteriophage and measuring the amount of cell surface K5 polysaccharide (Fig. 2), while the level of K5 lyase expression was determined by Western blot analysis of the total cell lysates (Fig. 3). Cultures of MS101(pLYA100) were grown to an OD₆₀₀ of 0.4 in LB medium at 37°C, and the expression of the K5 lyase was induced by the addition of a 0.1% (wt/vol) concentration of arabinose and incubation for a further 2 h at 37°C. Two additional controls were also included. First, to control for any effects of arabinose on K5 capsule production, MS101 was grown in the presence of 0.1% (wt/vol) arabinose. Second, to exclude any indirect effects on K5 expression due to protein overexpression, an inactive lyase containing a Y229A mutation that abolishes catalytic activity (J. Thompson and I. S. Roberts, unpublished results) was induced by growth in 0.1% (wt/vol) arabinose. After induction, all the cultures were subjected to a K5 bacteriophage assay as described previously (11). The presence of arabinose had no effect on the sensitivity of MS101 to K5 bacteriophage (Fig. 2) or on the level of cell surface K5 polysaccharide (data not shown). The susceptibility of strain MS101(pLYA100) to K5 bacteriophage was equal to that of MS101 following growth in the absence of arabinose, although the plaques for MS101(pLYA100) were smaller (Fig. 2). In contrast, when strain MS101(pLYA100) was grown in the presence of 0.1% (wt/vol) arabinose, it became completely resistant to K5 bacteriophage, with no detectable plaques (Fig. 2). This was equally true at lower dilutions of the K5 bacteriophage (data not shown). However, the sensitivity to K5 bacteriophage of strain MS101(pLYA100[Y229A]), which makes inactive lyase, was unaffected by the presence of 0.1% (wt/vol) arabinose in the growth medium, although the presence of arabinose reduced the plaque size (Fig. 2). The presence or absence of arabinose had no effect on the sensitivity of strain MS101(pBAD33) to K5 bacteriophage, with this strain being as sensitive as MS101 under both conditions (Fig. 2). Overall, these data clearly demonstrate that the effect on sensitivity to K5 bacteriophage requires the expression of a functional K5 lyase enzyme. To confirm the level of expression of the K5 lyase following induction with 0.1% (wt/vol) arabinose, cell lysates of cultures were analyzed by Western blotting. These data show that in the absence of arabinose, there was no detectable expression of the K5 lyase, and that in the presence of arabinose, the levels of induction of recombinant protein were comparable for both MS101(pLYA100) and MS101(pLYA100[Y229A]) (Fig. 3). Therefore, the effect of arabinose induction on the sensitivity to K5 bacteriophage is not merely a consequence of overexpression of recombinant protein in the cell; rather, it also requires that the K5 lyase be functionally active. Our interpretation of these data are that induction of K5 lyase expression reduces the cell surface expression of the K5 polysaccharide and, therefore, the sensitivity of the strain to K5-specific bacteriophage.

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FIG. 2. Effect of K5 lyase production on K5-specific bacteriophage sensitivity. Cultures were grown to an OD600 of 0.4, induced with arabinose where appropriate, and plated out with approximately 140 PFU of K5 phage. (A) MS101; (B) MS101 plus 0.1% (wt/vol) arabinose; (C) MS101(pLYA100); (D) MS101(pLYA100) plus 0.1% (wt/vol) arabinose; (E) MS101 (pLYA100[Y229A]); (F) (pLYA100[Y229A]) plus 0.1% (wt/vol) arabinose; (G) MS101(pBAD 33); (H) MS101(pBAD33) plus 0.1% (wt/vol) arabinose.

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FIG. 3. Western blot analysis showing arabinose induction of the K5 lyase enzyme. Duplicate cultures were grown in L broth at 37°C to an OD600 of 0.4 before the addition of arabinose to a final concentration of 0.1% (wt/vol) to one of each pair of cultures. The cultures were then incubated for a further 2 hours before 1 ml was extracted and the cells were harvested by centrifugation at 13,000 x g. The cells were resuspended in 100 µl of SDS loading buffer (91 mM Tris [pH 6.8], 2% [vol/vol] SDS, 0.2% [vol/vol] glycerol, 0.2% [vol/vol] bromophenol blue, and 7% [vol/vol] 2-mercaptoethanol), and the proteins were analyzed by SDS-PAGE and Western blotting as described previously (9), using antisera to the five-His tag (Qiagen, United Kingdom) on the lyase protein. The samples were MS101 (lane 1), MS101 plus 0.1% (wt/vol) arabinose (lane 2), MS101(pLYA100[Y229A]) (lane 3), MS101(pLYA100[Y229A]) plus 0.1% (wt/vol) arabinose (lane 4), MS101(pLYA100) (lane 5), and MS101(pLYA100) plus 0.1% (wt/vol) arabinose (lane 6).

To confirm the phage sensitivity data, we purified the K5 polysaccharide from the different cultures and quantified the amounts of cell surface K5 polysaccharide. The amount of K5 polysaccharide was determined by digesting to completion the purified K5 polysaccharide with the lyase enzyme and monitoring the increase in absorbance at 232 nm due to the generation of the ^4,5 bond formed during cleavage of the K5 polysaccharide by β-elimination (2). This assay is both reproducible and reliable (2). The levels of K5 polysaccharide were expressed per 10¹¹ cells and are the means of two independent experiments. Strain MS101 expressed 0.70 and 0.63 mg of K5 polysaccharide (mean, 0.66 mg). Strain MS101(pLYA100) grown in LB medium expressed 0.37 and 0.34 mg of K5 polysaccharide (mean, 0.35 mg), which dropped to 0.09 and 0.11 mg of K5 polysaccharide (mean, 0.1 mg) following growth in 0.1% (wt/vol) arabinose and induction of K5 lyase expression. In strain MS101(pBAD33) grown in LB medium, there were 0.31 and 0.25 mg of K5 polysaccharide (mean, 0.28 mg), indicating that the vector alone had some effect on the level of K5 polysaccharide. For a negative control, we measured the amount of K5 polysaccharide produced by strain MS101kfiC that has a deletion mutation in one of the K5 glycosyltransferase genes essential for K5 biosynthesis (4). In this case, we were unable to detect any K5 polysaccharide. This confirms the K5 bacteriophage sensitivity data and demonstrates that induction of expression of the K5 lyase in the cytoplasm of the cell reduces the expression of cell surface K5 polysaccharide. The observation that there was still a detectable low level of cell surface K5 polysaccharide in strain MS101(pLYA100) grown with 0.1% (wt/vol) arabinose yet the strain was completely resistant to K5 bacteriophage suggests that there is a minimum of cell surface K5 polysaccharide for K5 bacteriophage infection.

Overall, our interpretation of these data is that the presence of the K5 lyase enzyme in the cytoplasm results in the degradation of the intracellular K5 polysaccharide that is being synthesized on the cytoplasmic face of the inner membrane, although at this stage we cannot preclude the possibility that the K5 lyase is interfering with the synthesis of the K5 polysaccharide. However, what is clear is that the K5 polysaccharide is unlikely to be synthesized and exported within a protected cytoplasmic compartment that is inaccessible to K5 lyase. This conclusion is different from that observed for K1 polysaccharide production in an earlier report, whose authors proposed that K1 synthesis and export occur in a subcellular compartment termed the sialisome (10). This difference in accessibility may be a consequence of significant differences in the biosynthesis of K1 polysaccharide and that of K5 polysaccharide. The K5 polysaccharide is a heteropolymer and requires the alternate action of two glycosyltransferases, KfiA and KfiC (4, 6). In contrast, K1 is a homopolymer that requires a single transferase, NeuS (10). It is possible that the successive cycles of reengagement of KfiC and KfiA with the growing K5 polysaccharide chain expose it to degradation by the K5 lyase, while the presence of the NeuS enzyme effectively blocks the access of the endosialidase to the growing K1 polysaccharide. Alternatively, the difference in accessibility may reflect differences between the K5 lyase and the K1 endosialidase in terms of binding their respective substrates. Currently, the minimum size of polysaccharide chain that is required for these enzymes to bind and cleave their substrate is unknown. As such, any differences in the size of the polysaccharide chain required for enzyme binding will affect the apparent sensitivity of the nascent polysaccharide chain to degradation in the cytoplasm. However, what is clear from these results is that the synthesis of the K5 polysaccharide is not in a protected compartment within the cytoplasm. Because of this, one should be cautious in extrapolating the K1 data to the biosynthesis of other group 2 capsules.

 
The work in the laboratory of I.S.R. is supported by the BBSRC and MRC of the United Kingdom. T.H. gratefully acknowledges receipt of a BBSRC studentship.

 
* Corresponding author. Mailing address: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Dover Street, Manchester M13 9PT, United Kingdom. Phone: 0161-275 5753. Fax: 0161-275 5082. E-mail: I.s.Roberts{at}manchester.ac.uk

Published ahead of print on 12 December 2008.

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Journal of Bacteriology, March 2009, p. 1716-1718, Vol. 191, No. 5
0021-9193/09/$08.00+0     doi:10.1128/JB.01371-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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