Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and cross-linking: Wzz is able to oligomerize

The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (WzzSF) and Salmonella typhimurium Wzz (WzzST). These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination. Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of WzzSF. Some of the WzzSF variants resulted in O-antigen modal chain lengths much shorter than those of wild-type WzzSF, whereas other mutants inactivated WzzSF function entirely and a third class had a longer O-antigen chain length distribution. The data indicate that amino acids throughout the length of the WzzSF protein are important in determination of O-antigen modal chain length. In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the WzzSF protein is able to form dimers and oligomers of at least six WzzSF proteins. A carboxy-terminal-truncated WzzSF protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the amino-terminal region is sufficient for the Wzz-Wzz interaction observed. Shortened WzzSF proteins having internal deletions in the amino-terminal region were also able to oligomerize, suggesting that residues 59-194 are not essential for oligomerization. Cross-linking of WzzSF proteins with mutationally altered residues showed that loss of WzzSF function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects WzzSF-WzzSF dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis.     

Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control

The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with nonrandom (modal) chain length distribution. The number of the repeat O units in each individual O-antigen polymer depends on the Wzz chain length regulator, an inner membrane protein belonging to the polysaccharide copolymerase (PCP) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to >100 repeat units), despite having remarkably conserved structural folds. The molecular mechanism responsible for the selective preference for a certain number of O units is unknown. Guided by the three-dimensional structures of PCPs, we constructed a panel of chimeric molecules containing parts of two closely related Wzz proteins from Salmonella enterica and Shigella flexneri which confer different O-antigen chain length distributions. Analysis of the O-antigen length distribution imparted by each chimera revealed the region spanning amino acids 67 to 95 (region 67 to 95), region 200 to 255, and region 269 to 274 as primarily affecting the length distribution. We also showed that there is no synergy between these regions. In particular, region 269 to 274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269 to 274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by cross-linking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex.     

Investigation of the conformational states of Wzz and the Wzz.O-antigen complex under near-physiological conditions

Chain length determinant protein (Wzz) has been postulated to terminate the polymerization and regulate the chain length of the O-polysaccharide (O-antigen), an important component for constructing lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. The investigation to understand the mechanism of Wzz has been largely slowed down due to lack of structural information. In this report, we have applied small-angle X-ray scattering (SAXS) to study the conformational state and molecular properties of Wzz and the Wzz.O-antigen complex under near-physiological conditions. No concentration-dependent aggregation or structural changes, but repulsive intermolecular interactions between Wzz molecules, are suggested in the concentration series studies. The SAXS studies suggest that Wzz protein appears to be elongated and exists as a tetramer in solution. The reconstructed model built from SAXS data indicates that the middle regime of Wzz, most likely representing the periplasmic domain, contributes to the Wzz oligomerization, which has been proposed to be correlated to the function of Wzz. The immunoblotting analyses also demonstrate that the putative coiled-coil region in the periplasmic region contributes to the oligomerization. Further, the SAXS data corresponding to Wzz and the Wzz.O-antigen complex indicate an apparent substrate (O-antigen)-induced conformational change, consistent with previous circular dichroism studies. Our finding may shed light on the biological mechanism of Wzz as a chain length determinant of O-antigen.     

The Wzz (Cld) Protein in Escherichia coli: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.     

The double helix coiled coil structure of murein lipoprotein from Escherichia coli.

A rod-like structure is proposed for the murein lipoprotein of Escherichia coli, built of two parallel unbroken α-helices arranged in a coiled coil of the same type as in the muscle protein tropomyosin. The amino acid sequence has the required regular pattern of hydrophobic amino acids at intervals of three and four residues and the secondary structure predicted from the sequence is 80% helical. A space-filling model confirms that the coiled coil model is stereochemically reasonable, and energy calculations for a series of coils with different radii suggest that the best structure is one with the helix axes 8.25 Å apart. Energyrefined atomic co-ordinates have been calculated which show that the hydrophobic side-chains form a series of close-packed unstrained contacts between the two helices along the entire length of the sequence. On the basis of this study the hexagonal membrane pore model and the segmented helix model proposed by others seem unlikely. The coiled coil has a strongly hydrophilic outer surface, suggesting that the protein has a watery environment within the E. coli cell envelope and is not strictly a membrane protein. Probably only the fatty acid portion of the lipoprotein penetrates into the lipid region of the outer membrane, so that the protein may act as a tie or a spacer between the lipid and the murein wall.     

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.     

Mutational analysis of the large periplasmic loop 7-8 of the putrescine transporter PotE in Escherichia coli

The PotE protein is a putrescine-ornithine antiporter found in many gram-negative bacteria. It is a member of the APA family of transporters and has 12 predicted alpha-helical transmembrane spanning segments (TMS). While the substrate binding site has previously been mapped to a region near the surface of the cytoplasmic lipid layer, no structural feature within the periplasmic domains of PotE have been shown to be important for function. We examined the role of the only large outer loop, situated between transmembrane spanning segment 7 and 8, in putrescine uptake. Deletion of the highly conserved amino acids in the region closest to transmembrane spanning segment 7 produced a protein with little activity. Glycine-scanning mutagenesis of this region showed that Val(249) and Leu(254) were required for optimal transporter function. The V249G mutant transported putrescine at a lower maximal rate compared to wild-type (WT) but with the same substrate binding affinity. In contrast, the L254G mutant had a higher substrate affinity. A series of Val(249) mutants indicated that the hydrophobicity of this residue, which is located at or near the membrane surface, is important for PotE function. Secondary structure predictions of the large outer loop indicated the presence of a hydrophobic alpha-helix in the centre with a hydrophobic region at each end suggesting that the loop was not entirely exposed to the aqueous periplasmic space. The study shows that loop 7-8 is important for PotE function, possibly by forming a re-entrant loop in the channel of the transporter.     

Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.     

The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ)

The Rhizobium meliloti dctA gene encodes the C₄-dicarboxylate permease which mediates uptake of C₄-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.     

The Symmetrical Structure of Structural Maintenance of Chromosomes (SMC) and MukB Proteins: Long,Antiparallel Coiled Coils,Folded at a Flexible Hinge

Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH₂-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180°, separating the terminal domains by 100 nm, or close to near 0°, bringing the terminal globular domains together.A surprising observation is that the ∼300–amino acid–long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35–45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH₂- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.     

Identification of active site residues in the Shigella flexneri glucosyltransferase GtrV

Abstract

The serotype-specific glucosyltransferase, GtrV, is responsible for glucosylation of the O-antigen repeating unit of Shigella flexneri serotype 5a strains. GtrV is an integral inner membrane protein with two essential periplasmic loops: the large Loop 2 and the C-terminal Loop 10. In this study, the full length of the Loop 2 was shown to be necessary for GtrV function. Site-directed mutagenesis within this loop revealed that conserved aromatic and charged amino acids have a critical role in the formation of the active site. Sequential deletions of the C-terminal end indicated that this region may be essential for assembly of the protein in the cytoplasmic membrane. The highly conserved FWAED motif is thought to form the substrate-binding site and was found to be critical in GtrV and GtrX, a serotype-specific glucosyltransferase with homology to GtrV. The data presented constitutes a targeted analysis of the formation of the GtrV active site and highlights the essential role of the large periplasmic Loop 2 in its function.     

The Yeast Rad50 Gene Encodes a Predicted 153-Kd Protein Containing a Purine Nucleotide-Binding Domain and Two Large Heptad-Repeat Regions

The RAD50 gene of Saccharomyces cerevisiae is required for chromosome synapsis and recombination during meiosis and for repair of DNA damage during vegetative growth. The precise role of the RAD50 gene product in these processes is not known. Most rad50 mutant phenotypes can be explained by the proposal that the RAD50 gene product is involved in the search for homology between interacting DNA molecules or chromosomes, but there is no direct evidence for this model. We present here the nucleotide sequence of the RAD50 locus and an analysis of the predicted 153-kD RAD50 protein. The amino terminal region of the predicted protein contains residues suggestive of a purine nucleotide binding domain, most likely for adenine. The remaining 1170 amino acids consist of two 250 amino acid segments of heptad repeat sequence separated by 320 amino acids, plus a short hydrophobic carboxy-terminal tail. Heptad repeats occur in proteins such as myosin and intermediate filaments that form alpha-helical coiled coils. One of the two heptad regions in RAD50 shows similarity to the S-2 domain of rabbit myosin beyond that expected for two random coiled coil proteins.     

Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides

In vitro studies have suggested that the TatBC complex serves as the receptor for signal peptides targeted for export via the twin-arginine translocation (Tat) pathway. Substitution of the hallmark twin-arginine dipeptide with two lysines abrogates export of physiological substrates in all organisms. We report the isolation and characterization of suppressor mutations that allow export of an ssTor(KK)-GFP-SsrA tripartite fusion. We identified two amino acid suppressor mutations in the first cytoplasmic loop of TatC. In addition, two other amino acids in the first cytoplasmic loop exhibit epistatic suppression. Surprisingly, we also identified a suppressor mutation predicted to lie within the second periplasmic loop of TatC, a region that is not expected to interact directly with the signal peptide. The suppressor mutations allowed export of the native Esherichia coli Tat substrate trimethylamine N-oxide reductase with a twin-lysine substitution in its signal sequence. The cytoplasmic suppressor mutations conferred SDS sensitivity and partial filamentation, indicating that Tat export of authentic substrates was impaired.     

大肠杆菌和志贺氏菌O-抗原糖基转移酶与聚合酶的生物信息学研究

利用生物信息学手段对大肠杆菌和志贺氏菌的 1 1 0个O 抗原糖基转移酶与 39个O 抗原聚合酶的序列进行分析 ,探讨这两种酶的序列和结构特点。统计了其序列一致性 ,密码子使用和 (G C) %含量的特点 ;讨论了O 抗原糖基转移酶和聚合酶对底物的特异性 ;推测了 6组糖基转移酶的功能 ;通过对蛋白拓扑结构的预测 ,发现O 抗原聚合酶中广泛存在一个位于细胞周质中的亲水环 (Loop) ,是可能的功能区域 ;通过对蛋白高级结构的预测 ,发现O 抗原糖基转移酶属于两个不同的蛋白超家族。     

Overexpression and characterization of Wzz of Escherichia coli O86:H2

O-Antigen plays a critical role in the bacterium-host interplay, the chain length is an important factor in O-antigen functions. Wzz protein is responsible for O-antigen chain length regulation, but the mechanism is still unknown. Here, we overexpressed the Wzz of Escherichia coli O86:H2 in wzz mutant O86:H2 strain, the yield can achieve 15 mg/L. The recombinant Wzz was purified to 99% purity in dodecylmaltoside by sequential Ni-affinity chromatography and anion-exchange. Size exclusion chromatography and in vivo cross-linking experiments both showed that Wzz formed tetramer. Furthermore, analysis with circular dichroism revealed that the predominant structural composition in Wzz is alpha-helices, and incubation with O-antigen significantly changed Wzz conformation. The results suggested that Wzz protein can interact with O-antigen.     

Functional Properties of Pro Region of Escherichia coli Heat-Stable Enterotoxin

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.     

Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella

MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria. Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype). However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations. In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions. In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable. In the cytoplasmic loop region of MotA, deletions made the protein unstable. For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus. We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it. Further investigations in MotB were carried out by combining dispensable deletion segments. The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span. These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation. Part of the MotA C-terminal cytoplasmic region is not essential for torque generation. MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif. In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical. About 40 residues at the C-terminus are also unimportant.     

Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10–13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and β-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.     

Topological Analysis of DcuA, an Anaerobic C4-Dicarboxylate Transporter of Escherichia coli

Escherichia coli possesses three independent anaerobic C₄-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470–6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a β-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the “positive-inside rule.” Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.     

Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide o antigen

Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, WzxO16 and WzxE, and also corresponding polymerases (WzyO16 and WzyE) and O-antigen chain-length regulators (WzzO16 and WzzE), respectively. In this study, we show that the E. coli wzxE gene can fully complement a wzxO16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the WzyE polymerase or the WzzE chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of WzxE. We also show that this property is not unique to WzxE, since WzxO16 and WzxO7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.