Membrane Topology Mapping of the O-Antigen Flippase (Wzx), Polymerase (Wzy), and Ligase (WaaL) from Pseudomonas aeruginosa PAO1 Reveals Novel Domain Architectures

Islam, Salim T.; Taylor, Véronique L.; Meng, Qiang; Lam, Joseph S.

doi:10.1128/mbio.00189-10

Cited by 99 publications

(172 citation statements)

References 47 publications

(81 reference statements)

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“…During LPS assembly, Wzy and RfaL/WaaL share the same substrate, undecaprenol-O-antigen polymer, which is subsequently transferred onto the sugar acceptor of the lipid A-core intermediate. Both Wzy and RfaL/WaaL proteins are highly hydrophobic; however, topology and biochemical studies point to distinct features (33,34,47). Wzy proteins encompass 14 TM segments with 2 large periplasmic loops, PL3 and PL5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…and Pseudomonas aeruginosa (33,34). The topology of P. aeruginosa WaaL has been experimentally determined with translational hybrids, revealing 12 transmembrane (TM) and 5 periplasmic loops (PL1 to -5) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The topology of P. aeruginosa WaaL has been experimentally determined with translational hybrids, revealing 12 transmembrane (TM) and 5 periplasmic loops (PL1 to -5) (Fig. 1B) (34). According to PFAM prediction, the PF13425 family S. pneumoniae protein with accession number CKE53299.1 and PF13425 proteins B. anthracis BAS0847 and BAS5274 are predicted to encompass 12 TM segments with one large extracytoplasmic loop between TM9 and TM10, reminiscent of WaaL PL5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis. Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan. IMPORTANCE The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡) with ␣-Gal and ␤-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.KEYWORDS Bacillus anthracis, S-layers, Wzy repeat polymerase, cell wall polysaccharide, envelope assembly T he peptidoglycan cell wall of Gram-positive microbes is a determinant of bacterial shape and integrity (1, 2). Peptidoglycan also serves as a scaffold for the attachment of secondary polymers, which support specific cell cycle functions of bacteria (3, 4). Some Gram-positive bacteria, for example, Bacillus subtilis and Staphylococcus aureus, attach wall teichoic acid (WTA) to peptidoglycan, thereby positioning peptidoglycan synthesis enzymes, division septa, and murein hydrolases for cell growth and division (5-7). Other microbes, including Streptococcus species and Bacillus cereus, attach a secondary cell wall polysaccharide (SCWP) to peptidoglycan, and these polymers presumably fulfill functions similar to that of WTA during the bacterial cell cycle (8, 9). While WTA synthesis has been studied in detail (10), little is known about the genetic determinants and assembly reactions for SCWP synthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Lunderberg

Château

et al. 2017

J Bacteriol

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“…Both are very hydrophobic with multiple trans-membrane segments (TMSs). Wzy has two major periplasmic loops, which are between TMS 5/6 and TMS 9/10 in the topology maps for Shigella flexneri and Pseudomonas aeruginosa PAO1, with 12 and 14 TMSs, respectively, and similar topology from TMS 1 to TMS 12 (Daniels et al, 1998;Islam et al, 2010). Both of these loops have been shown to be important for Wzy function in the two examples discussed (Islam et al, 2011(Islam et al, , 2013.…”

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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“…The putative Wzy proteins have 9-12 predicted transmembrane segments, with a large periplasmic loop of 36-88 amino acid residues. One or two large periplasmic loops are observed for a number of O antigen polymerases, and seem to be important in the recognition of the O unit and/or for catalytic activity (Daniels et al, 1998;Islam et al, 2010 Islam et al, , 2011. Hence, the Wzx/Wzy-dependent process is suggested to mediate the synthesis of all Providencia O antigens studied.…”

Section: O Antigen Processing Genesmentioning

confidence: 99%

Localization and molecular characterization of putative O antigen gene clusters of Providencia species

et al. 2012

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Enterobacteria of the genus Providencia are opportunistic human pathogens associated with urinary tract and wound infections, as well as enteric diseases. The lipopolysaccharide (LPS) O antigen confers major antigenic variability upon the cell surface and is used for serotyping of Gram-negative bacteria. Recently, Providencia O antigen structures have been extensively studied, but no data on the location and organization of the O antigen gene cluster have been reported. In this study, the four Providencia genome sequences available were analysed, and the putative O antigen gene cluster was identified in the polymorphic locus between the cpxA and yibK genes. This finding provided the necessary information for designing primers, and cloning and sequencing the O antigen gene clusters from five more Providencia alcalifaciens strains. The gene functions predicted in silico were in agreement with the known O antigen structures; furthermore, annotation of the genes involved in the three-step synthesis of GDP-colitose (gmd, colD and colC) was supported by cloning and biochemical characterization of the corresponding enzymes. In one strain (P. alcalifaciens O39), no polysaccharide product of the gene cluster in the cpxA-yibK locus was found, and hence genes for synthesis of the existing O antigen are located elsewhere in the genome. In addition to the putative O antigen synthesis genes, homologues of wza, wzb, wzc and (in three strains) wzi, required for the surface expression of capsular polysaccharides, were found upstream of yibK in all species except Providencia rustigianii, suggesting that the LPS of these species may be attributed to the so-called K LPS (K LPS ). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates. INTRODUCTIONThe genus Providencia of the family Enterobacteriaceae currently consists of eight species (Juneja & Lazzaro, 2009;Somvanshi et al., 2006;O'Hara et al., 2000), among which Providencia stuartii, Providencia rettgeri, Providencia rustigianii and Providencia alcalifaciens are the species that most commonly cause human infection. P. stuartii is often found in complicated urinary tract infections in patients with chronic indwelling urinary catheters (Rahav et al., 1994). Providencia species, especially P. rustigianii and P. alcalifaciens, are an infrequent cause of traveller's diarrhea (Yoh 3These authors contributed equally to this work.Abbreviations: 6dTal, 6-deoxytalose; Col, colitose; CPS, capsular polysaccharide; ECA, enterobacterial common antigen; ESI-MS, electrospray ionization-MS; Fuc3NAc, 3-acetamido-3,6-dideoxygalactose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; OGC, O antigen gene cluster; PLP, pyridoxal 59-phosphate; Qui, quinovose; TOCSY, total correlation spectroscopy; UndPP, undecaprenyl diphosphate.The GenBank/EMBL/DDBJ accession numbers for the cpxA-yibK region sequences of P. alcalifaciens O28, O36, O44 and O39 are JQ319041, HM583639, JN097784 and JN097785, respectively.Three supplementary figures and three supplem...

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Membrane Topology Mapping of the O-Antigen Flippase (Wzx), Polymerase (Wzy), and Ligase (WaaL) from Pseudomonas aeruginosa PAO1 Reveals Novel Domain Architectures

Cited by 99 publications

References 47 publications

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

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

Localization and molecular characterization of putative O antigen gene clusters of Providencia species

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