Inefficient translocation of a truncated O unit by a Salmonella Wzx affects both O-antigen production and cell growth

Liu, Michael A.; Stent, Thomas L.; Hong, Yaoqin; Reeves, Peter R.

doi:10.1093/femsle/fnv053

Cited by 25 publications

(40 citation statements)

References 30 publications

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“…The loss of WzxB causes the accumulation of Und-PPlinked O-antigen intermediates (49), which in turn limits the availability of Und-P for PG synthesis. Because of this, wzxB mutants are notoriously difficult to isolate (38,49,50) and wzxB mutations are deleterious in the presence of a fully functional O-antigen-producing machinery (51,52). We recreated both of these circumstances in the present study, and in both instances the accumulation of Und-PP-linked O-antigen intermediates induced morphological defects that were reversed by eliminating the production of O antigen.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, it is not surprising that wzxB flippase mutants acquire suppressing mutations that prevent the formation of O-antigen intermediates (38). Indeed, investigators who study WzxB function in Salmonella enterica do so in a genetic background where expression of O antigen is controlled by a galE mutation, which removes the first committed step in the biosynthetic pathway so that no Und-PP-linked O-antigen intermediates can accumulate until galactose is supplied (49,51,52). The deleterious effects of Und-P sequestration have also been noted for other transport mutants (53,54), as well as mutants that are unable to ligate O-antigen intermediates to the lipid A core (24) or that truncate the core oligosaccharide (55).…”

Section: Discussionmentioning

confidence: 99%

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Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate

2016

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Undecaprenyl phosphate (Und-P) is a member of the family of essential polyprenyl phosphate lipid carriers and in the Gramnegative bacterium Escherichia coli is required for synthesizing the peptidoglycan (PG) cell wall, enterobacterial common antigen (ECA), O antigen, and colanic acid. Previously, we found that interruption of ECA biosynthesis indirectly alters PG synthesis by sequestering Und-P via dead-end intermediates, causing morphological defects. To determine if competition for Und-P was a more general phenomenon, we determined if O-antigen intermediates caused similar effects. Indeed, disrupting the synthesis of O antigen or the lipopolysaccharide core oligosaccharide induced cell shape deformities, which were suppressed by preventing the initiation of O-antigen biosynthesis or by manipulating Und-P metabolism. We conclude that accumulation of O-antigen intermediates alters PG synthesis by sequestering Und-P. Importantly, many previous experiments addressed the physiological functions of various oligosaccharides and glycoconjugates, but these studies employed mutants that accumulate deleterious intermediates. Thus, conclusions based on these experiments must be reevaluated to account for possible indirect effects of Und-P sequestration. IMPORTANCEBacteria use long-chain isoprenoids like undecaprenyl phosphate (Und-P) as lipid carriers to assemble numerous glycan polymers that comprise the cell envelope. In any one bacterium, multiple oligosaccharide biosynthetic pathways compete for a common pool of Und-P, which means that disruptions in one pathway may produce secondary consequences that affect the others. Using the Gram-negative bacterium Escherichia coli as a model, we demonstrate that interruption of the biogenesis of O antigen, a major outer membrane component, indirectly impairs peptidoglycan synthesis by sequestering Und-P into dead-end intermediates. These results strongly argue that the functions of many Und-P-utilizing pathways must be reevaluated, because much of our current understanding is based on experiments that did not control for these unintended secondary effects. L ong-chain isoprenoids are widely conserved lipid carriers that translocate glycan intermediates across biological membranes, where they are assembled onto other polymers or released (reviewed in reference 1). In bacteria, the primary lipid carrier is undecaprenyl phosphate (Und-P), a 55-carbon isoprenoid (C 55 -P) also referred to as bactoprenol, which is the dephosphor ylated product of undecaprenyl pyrophosphate (Und-PP), as synthesized by UppS (2, 3). Homologues with shorter chains serve the same function in species of mycobacteria and corynebacteria (C 50 -P) (4, 5) and Paracoccus denitrificans (C 45 -P) (6). Und-P is required for synthesizing numerous bacterial glycans, including cell wall peptidoglycan (PG) (7,8), wall teichoic acids (WTA) (9), enterobacterial common antigen (ECA) (10), O antigen (11), capsular polysaccharides (12), several commercially important exopolysaccharides (13), and a variety of other...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate

2016

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“…Removal of the terminal DDH residue from several S. enterica Gal‐initiated O antigens results in poor Wzx translocation (Hong et al., ; Liu et al., , ). To assess this situation for the Y. pseudotuberculosis Wzx 1 and Wzx 2 , we made O:2a Δ abe (pPR2297) and O:1a Δ prt‐wbyH (pPR2326) constructs that both produce the same O unit main‐chain without a DDH residue, but express wzx 2 or wzx 1 , respectively.…”

Section: Resultsmentioning

confidence: 99%

“…However it has since been demonstrated on several occasions that Wzx can exhibit a strong preference for residues beyond the first sugar (Hong et al., ). Furthermore, inefficient Wzx translocation of nonpreferred O‐unit substrates results in cytoplasmic O‐unit accumulation and sequestration of UndP, which in turn interferes with peptidoglycan synthesis, causing a loss of membrane integrity and ultimately cell lysis (Jorgenson & Young, ; Liu, Stent, Hong, & Reeves, ). It is now becoming clear that Wzx substrate specificity represents a key checkpoint in the O‐antigen biosynthesis pathway.…”

Section: Introductionmentioning

confidence: 99%

Wzx flippases exhibiting complex O‐unit preferences require a new model for Wzx–substrate interactions

2018

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The Wzx flippase is a critical component of the O-antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram-negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O-unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O-unit main-chain, despite both being able to translocate several other structurally-divergent O units. We also identified other Y. pseudotuberculosis Wzx that can translocate a structurally divergent foreign O unit with high efficiency, and thus exhibit an apparently relaxed substrate preference. It now appears that Wzx substrate preference is more complex than previously suggested, and that not all O-unit residues are equally important determinants of translocation efficiency. We propose a new "Structure-Specific Triggering" model in which Wzx translocation proceeds at a low level for a wide variety of substrates, with high-frequency translocation only being triggered by Wzx interacting with one or more preferred O-unit structural elements found on its cognate O unit(s).

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“…We have recently reported that Wzx translocases can translocate only their native O units at optimal efficiency (Hong et al, 2012;Hong & Reeves, 2014;Liu et al, 2015). Wzy polymerizes the Und-PP-linked O units to produce long-chain O antigen, and also exhibits enormous sequence variation.…”

Section: Introductionmentioning

confidence: 99%

Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units

et al. 2015

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The Wzx/Wzy-dependent pathway is the predominant pathway for O-antigen production in Gram-negative bacteria. The O-antigen repeat unit (O unit) is an oligosaccharide that is assembled at the cytoplasmic face of the membrane on undecaprenyl pyrophosphate. Wzx then flips it to the periplasmic face for polymerization by Wzy, which adds an O unit to the reducing end of a growing O-unit polymer in each round of polymerization. Wzx and Wzy both exhibit enormous sequence diversity. It has recently been shown that, contrary to earlier reports, the efficiency of diverse Wzx forms can be significantly reduced by minor structural variations to their native O-unit substrate. However, details of Wzy substrate specificity remain unexplored. The closely related galactose-initiated Salmonella O antigens present a rare opportunity to address these matters. The D1 and D2 O units differ only in an internal mannose-rhamnose linkage, and D3 expresses both in the same chain. D1 and D2 polymerases were shown to be specific for O units with their respective a or b configuration for the internal mannose-rhamnose linkage. The Wzy encoded by D3 gene cluster polymerizes only D1 O units, and deleting the gene does not eliminate polymeric O antigen, both observations indicating the presence of an additional wzy gene. The levels of Wzx and Wzy substrate specificity will affect the ease with which new O units can evolve, and also our ability to modify O antigens, capsules or secreted polysaccharides by glyco-engineering, to generate novel polysaccharides, as the Wzx/Wzydependent pathway is responsible for much of the diversity.

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Inefficient translocation of a truncated O unit by a Salmonella Wzx affects both O-antigen production and cell growth

Cited by 25 publications

References 30 publications

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate

Wzx flippases exhibiting complex O‐unit preferences require a new model for Wzx–substrate interactions

Three Wzy polymerases are specific for particular forms of an internal linkage in otherwise identical O units

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