Model for the Controlled Synthesis of O-Antigen Repeat Units Involving the WaaL Ligase

Assessment of O-antigen mutant growth in a Gal-free M9-based medium.Assessment of growth of S. enterica O-antigen mutants requires a Gal-free medium, with the major carbon source not leading to catabolic repression (e.g., glucose). This allows controllable O-antigen synthesis by the addition of Gal (see the next section for details). Here we used 1× M9 minimal medium supplemented with 100 µM CaCl₂, 1.6 mM MgSO₄, 0.32% (wt/vol) Casamino Acids, 0.4% (vol/vol) glycerol, 1 µg ⋅ liter⁻¹ thiamine, and 8 µg ⋅ liter⁻¹ tryptophan. Overnight cultures were diluted 1:40 and grown at 37°C with aeration. Tetracycline was added after the first 30 min to induce expression of genes carried by either the pWQ552 or the pWQ572 vector (7). To allow for O-unit expression, Gal (0.2%, wt/vol) was added after another 30 min. Samples were taken every 30 min for 6 h for measurement of optical density at 600 nm (OD₆₀₀) with a BioPhotometer (Eppendorf). The growth of each strain under each tested condition was repeated three times (error bar shows standard errors of the means [SEM]). Samples were then transferred to test tubes and photographed to illustrate turbidity.

Accumulation of O-antigen repeat units affects growth only when they are on the cytoplasmic face of the membrane.We constructed our strains in a ΔgalE background (8), as described by Datsenko and Wanner (18) (see Table S3 in the supplemental material for the oligonucleotides used). This prevents the endogenous synthesis of UDP-Gal, the precursor substrate used by WbaP to give UndPP-Gal. This approach allows experimental control of O-antigen synthesis by addition of exogenous Gal (8). The abe, wzx, wzy, and waaL genes were deleted by allelic replacements (Fig. 1B). The sequences of the resulting constructs were confirmed by showing that complementation with a clone of the deleted gene restored the wild-type phenotype in all cases (Fig. S1 and S2).

All of the mutants grew well in the absence of Gal (Fig. S3). However, the Δabe and Δwzx mutants stopped growing after exposure to Gal for 30 min, whereas both the Δwzy and ΔwaaL mutants were unaffected (Fig. 3). The normal growth in the absence of Gal shows that the growth effects are directly related to O-unit synthesis. Both Wzy and WaaL act after translocation (1), and mutants will accumulate any surplus O units on the periplasmic face of the membrane, whereas the Δwzx mutant will accumulate them on the cytoplasmic face (9), and it has been shown that the trisaccharide intermediates produced in Δabe mutants are mostly not translocated (6, 8). The growth defect is correlated with an accumulation of O units or intermediates on the cytoplasmic face of the membrane. We therefore propose that there is a feedback mechanism that regulates the synthesis of O units, based on the level of O units on the periplasmic face of the inner membrane.

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FIG 3

Disruption of O-antigen synthesis can be either conditionally lethal or nonlethal. Growth assays were performed as described in Materials and Methods (averages are from 3 experiments, and error bars show SEM). (Left) Growth curve; (right) turbidity.

WaaL may have a role in the regulation of O-unit synthesis.The proposed feedback mechanism requires detection of O units on the periplasmic face of the inner membrane and transmission of a signal. This most likely involves regulation of the functioning of O-unit synthesis machinery. Regulation commonly acts on the first step in the synthesis of the compound being regulated, which in this case is the synthesis of UndPP-Gal by WbaP. It is unlikely that regulation is by interaction of the end product, the UndPP-O unit, with the promoter, as synthesis occurs on the cytoplasmic face of the inner membrane and the end product is proposed to be effective in regulation only when it is on the periplasmic face of that membrane.

WaaL, which ligates single O units or polymers to lipid A-core, the last step in LPS synthesis, is a potential candidate for involvement in this regulation. We note that the UndPP-O unit is also a substrate for O-antigen capsule synthesis but that control is likely to be based on O-antigen demand, as long-chain O antigen is present under all conditions, whereas the presence of O-antigen capsule is variable (4). We hypothesize that WaaL regulates O-unit synthesis by transmitting a signal determined by the availability of UndPP-linked single or polymeric O units, which controls the function of WbaP.

We constructed a ΔwaaL Δabe double mutant by allelic replacement of the abe gene in the ΔwaaL strain (see Table S1 in the supplemental material for strains and plasmids). The SDS-PAGE profile (Fig. S1, lane 11) was the same as for the ΔwaaL strain (Fig. S1, lane 9), as expected. Strikingly, the strain grew normally even in the presence of Gal, which allows initiation of O-unit synthesis (Fig. 4A). To exclude the possibility that this observation relates to suppressor mutations inactivating O-unit synthesis, we introduced a waaL clone that regenerates a typical Δabe strain in terms of its LPS SDS-PAGE profile (Fig. S1, lane 12) and growth defect pattern (Fig. 4A). We conclude that WaaL activity is required for the abe deletion to be deleterious.

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FIG 4

WaaL is required for the growth defect observed in a Δabe strain but not in a Δwzx strain. Growth assays were performed as described for Fig. 3. (A) WaaL is dependent for growth on the defect of abe deletion. (B) Deletion of waaL could not reverse the growth defect of wzx deletion. (Left) Growth curve; (right) turbidity.

It is commonly proposed that O-antigen synthesis membrane proteins, including Wzx, Wzy, and Wzz, form a complex (19–22), and recently Wzy and Wzz were isolated as a complex in a pulldown experiment (23). The probable presence of such a complex has important implications for our proposed model. WbaP, which initiates O-unit synthesis, is also a membrane protein and may be part of the proposed protein complex (Fig. 2) and affect the control of O-unit synthesis. We note that there are data suggesting that one class of mutant WbaP proteins retains association with the developing O-unit until its completion (24), which raises the possibility that control of O-unit synthesis may operate by WbaP not releasing completed UndPP-linked O units for translocation.

However, the situation is more complex, as in a ΔwaaL Δwzx strain, the absence of WaaL does not relieve the growth defect of the Δwzx mutant, which is still unable to grow when O-antigen synthesis is initiated (Fig. 4B). It may be that Wzx is required for transmission of a regulatory signal from WaaL to WbaP. It is too early to speculate on details, as the nature of any complex is unknown, but it is clearly possible that if one component (e.g., Wzx) is missing from a complex, then another component (e.g., WaaL) might lose contact with the target protein, perhaps WbaP.

Conclusions.We propose that maintaining the balance between supply and demand of O units for O-antigen synthesis requires a specific regulatory process and that this involves a sufficiency of O units being detected by the periplasm-exposed part of the WaaL ligase. This is a novel hypothesis based on limited data, but those data are all consistent with the proposed model at this stage. We studied only S. enterica, but the findings from P. aeruginosa mutants referred to above, namely, that they accumulate second-site mutations which either restore synthesis of normal LPS or abolish O-antigen expression completely (12), suggest that similar regulatory processes occur in other species. The ability to adjust O-unit supply based on its levels on the periplasmic side of the membrane allows O-antigen chain length to be varied depending on circumstances and may thus have very important implications, for example in the tradeoff between phage resistance and virulence (25)

Our growth curve data give a clear demonstration of the effect of mutation in wzx on growth and also the effect of blocking completion of the O unit. The finding that a waaL mutation allows a strain lacking a functional abe gene to grow well when making an incomplete O unit shows that WaaL has some novel activity in addition to its ligase action and is consistent with the proposed model. However, the recognition that WaaL may play an important role is but the first step in understanding this physiological process, which most likely involves a protein complex with multiple functions.

Possible directions.We have proposed a novel model for the regulated synthesis of O units based on interactions within a hypothetical membrane protein complex. Many fundamental aspects of this model will need to be rigorously tested and validated. In Table 1, we provide some sample questions yet to be addressed.