Membrane topology of the<i>Salmonella enterica</i>serovar Typhimurium Group B O-antigen translocase Wzx

Cunneen, Monica M.; Reeves, Peter R.

doi:10.1111/j.1574-6968.2008.01295.x

Cited by 26 publications

(26 citation statements)

References 48 publications

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“…Similar features appear in the topological models of Wzx proteins from S. enterica (Wzx Se ) (12) and R. leguminosarum (PssL) (35). In both proteins there are also four transmembrane domains, each containing lysine or arginine residues, and two periplasmic loops with short stretches of hydrophobic amino acids.…”

Section: Discussionmentioning

confidence: 53%

“…Another complication to investigate functionally the members of these families is the lack of solid topological models that accurately predict transmembrane helices and solvent-exposed loops. Currently, experimentally based topological models have only been established for the Salmonella enterica serovar Typhimurium group B Wzx protein (12), and the Wzx-like protein PssL from Rhizobium leguminosarum (35), which is involved in exopolysaccharide capsule production. However, these studies did not identify any regions or specific amino acids from the protein that could play a functional role in the translocation process.…”

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confidence: 99%

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Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Marolda¹,

Li²,

Lung³

et al. 2010

J Bacteriol

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Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx EcO157 ) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletionfusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx EcO157 consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PPlinked saccharide substrate across the membrane.Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, plays critical roles in bacterial cell physiology (36) and in disease (53). The structure of LPS is complex and consists at a minimum of lipid A and core oligosaccharide (OS) (42). Many Gram-negative bacteria also have an O-specific antigen polysaccharide (or O antigen) attached to one of the terminal residues of the core OS (42). The O antigen is the most variable portion of the LPS molecule and arises from the polymerization of discrete oligosaccharide units (42, 54).The biosynthesis of LPS requires many enzymes and assembly proteins and generally involves two separate pathways. One pathway results in the synthesis of the lipid A-core OS (42), which is translocated across the inner membrane by the lipid A flippase MsbA, an ABC transporter (14, 15, 60). The other pathway involves the synthesis and assembly of the O-antigen polysaccharide, which also begins at the cytosolic side of the inner membrane resulting in the formation of a lipid-linked molecule that is further translocated across the inner membrane. The formation of a complete LPS molecule containing O antigen is catalyzed by ...

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Section: Discussionmentioning

confidence: 53%

mentioning

confidence: 99%

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Marolda¹,

Li²,

Lung³

et al. 2010

J Bacteriol

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“…Alkaline phosphatase (PhoA) and b-galactosidase (LacZ) have been used widely as reporter proteins to study protein localization (Cunneen & Reeves, 2008;Daniels et al, 1998;Haardt & Bremer, 1996;Sarsero & Pittard, 1995). PhoA is enzymically active when it is translocated to the periplasm but inactive when it is retained in the cytoplasm (Derman & Beckwith, 1991).…”

Section: Resultsmentioning

confidence: 99%

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

et al. 2014

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Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)–vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

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“…Therefore, serotyping by PCR targeting wzx and wzy for E. coli O antigen and Streptococcus pneumoniae K antigen has been developed (DebRoy et al, 2005; Kong et al, 2005). Due to the low-level of similarity of wzx and wzy genes among different gene clusters, sequence similarity and transmembrane helix profiles have been used in wzx and wzy identification (Jiang et al, 2001;Mazur et al, 2005;Nakhamchik et al, 2007;Cunneen & Reeves, 2008).…”

Section: H-y Shu and Others 4174mentioning

confidence: 99%

Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates

et al. 2009

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Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 59 end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 39 end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.

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Membrane topology of theSalmonella entericaserovar Typhimurium Group B O-antigen translocase Wzx

Cited by 26 publications

References 48 publications

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates

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