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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.sub.EcO157]) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that [Wzx.sub.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-PP-linked saccharide substrate across the membrane. doi: 10.1128/JB.00141-10

We reported previously that a Salmonella enterica serovar Enteritidis dam mutant expressing a truncated Dam protein does not agglutinate in the presence of specific antibodies against 09 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in Salmonella. The LPS O-antigen profiles of a dam null mutant (SE[DELTA]dam) and the Salmonella serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SE[DELTA]dam produced LPS with shorter O-antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen, we investigated whether Dam methylation is involved in regulating wzz expression. Densitometry analysis showed that the amount of Wzz produced by SE[DELTA]dam is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the wzz promoter in SE[DELTA]dam was reduced nearly 50% in logarithmic phase and 25% in stationary phase. These results were further confirmed by reverse transcription-PCR showing that wzz gene expression was threefold lower in the dam mutant than in the parental strain. Our results demonstrate that wzz gene expression is downregulated in a dam mutant, indicating that Dam methylation activates expression of this gene. This work indicates that wzz is a new target regulated by Dam methylation and demonstrates that DNA methylation not only affects the production of bacterial surface proteins but also the production of surface polysaccharides. doi: 10.1128/JB.00839-09

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded [WZZ.sub.pHS-2] proteins, respectively. In this study, genes wzzB, [wzz.sub.pHS-2] and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose-and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and [wzz.sub.pHS-2] revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and [wzz.sub.pHS-2] proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors. DOI 10.1099/mic.0.028944-0

Wzz is a membrane protein that determines the chain length distribution of the O-antigen lipopolysaccharide by an unknown mechanism. Wzz proteins consist of two transmembrane helices separated by a large periplasmic loop. The periplasmic loop of Escherichia coli K-12 Wzz (244 amino acids from K65 to A308) was purified and found to be a monomer with an extended conformation, as determined by gel filtration chromatography and analytical ultracentrifugation. Circular dichroism showed that the loop has a 60% helical content. The Wzz periplasmic loop also contains three regions with predicted coiled coils. To probe the function of the predicted coiled coils, we constructed amino acid replacement mutants of the E. coli K-12 Wzz protein, which were designed so that the coiled coils could be separate without compromising the helicity of the individual molecules. Mutations in one of the regions, spanning amino acids 108 to 130 (region I), were associated with a partial defect in O-antigen chain length distribution, while mutants with mutations in the region spanning amino acids 209 to 223 (region III) did not have an apparent functional defect. In contrast, mutations in the region spanning amino acids 153 to 173 (region II) eliminated the Wzz function. This phenotype was associated with protein instability, most likely due to conformational changes caused by the amino acid replacements, which was confirmed by limited trypsin proteolysis. Additional mutagenesis based on a three-dimensional model of region I demonstrated that the amino acids implicated in function are all located at the same face of a predicted [alpha]-helix, suggesting that a coiled coil actually does not exist in this region. Together, our results suggest that the regions predicted to be coiled coils are important for Wzz function because they maintain the native conformation of the protein, although the existence of coiled coils could not be demonstrated experimentally.

WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in AwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the AwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1 -P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by [WZZ.sub.pHS-2]. This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a [DELTA]rfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the [DELTA]rfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or [wzz.sub.pHS-2] also altered OAg chain-length distribution. Transcription of wzzB and [wzz.sub.pHS-2] genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and [wzz.sub.pHS-2] genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.

Undecaprenyl phosphate (Und-P) is a universal lipid carrier of glycan biosynthetic intermediates for carbohydrate polymers that are exported to the bacterial cell envelope. Und-P arises from the dephosphorylation of undecaprenyl pyrophosphate (Und-PP) molecules produced by de novo synthesis and also from the recycling of released Und-PP after the transfer of the glycan component to other acceptor molecules. The latter reactions take place at the periplasmic side of the plasma membrane, while cytoplasmic enzymes catalyse the de novo synthesis. Four Und-PP pyrophosphatases were recently identified in Escherichia coli. One of these, UppP (formerly BacA), accounts for 75% of the total cellular Und-PP pyrophosphatase activity and has been suggested to participate in the Und-P de novo synthesis pathway. Unlike UppP, the other three pyrophosphatases (YbjG, YeiU and PgpB) have a typical acid phosphatase motif also found in eukaryotic dolichyl-pyrophosphate-recycling pyrophosphatases. This study shows that double and triple deletion mutants in the genes uppP and ybjG, and uppP, ybjG and yeiU, respectively, are supersensitive to the Und-P de novo biosynthesis inhibitor fosmidomycin. In contrast, single or combined deletions including pgpB have no effect on fosmidomycin supersensitivity. Experimental evidence is also presented that the acid phosphatase motifs of YbjG and YeiU face the periplasmic space. Furthermore, the quadruple deletion mutant [DELTA]uppP-[DELTA]ybjG-[DELTA]yeiU-[DELTA]waaL has a growth defect and abnormal cell morphology, suggesting that accumulation of unprocessed Und-PP-linked O antigen polysaccharides is toxic for these cells. Together, the results support the notion that YbjG, and to a lesser extent YeiU, exert their enzymic activity on the periplasmic side of the plasma membrane and are implicated in the recycling of periplasmic Und-PP molecules.

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, [Wzx.sub.o16] and [Wzx.sub.E], and also corresponding polymerases ([Wzy.sub.o16] and [Wzy.sub.E]) and O-antigen chain-length regulators ([Wzz.sub.o16] and [Wzz.sub.E]), respectively. In this study, we show that the E. coli [wzx.sub.E] gene can fully complement a [wzx.sub.o16] 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 [Wzy.sub.E] polymerase or the [Wzz.sub.E] chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of [Wzx.sub.E]. We also show that this property is not unique to [Wzx.sub.E], since [Wzx.sub.o16] and [Wzx.sub.o7] 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.

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli 07 antigen lipopolysaccharide (LPS). In this work, [DELTA]toLA and [DELTA]pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQS02 (encoding the genes necessary for the synthesis of Salmonella enterica 0:54). Both [DELTA]toA and [DELTA]pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli 07 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in [DELTA]tolA and [DELTA]pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110[DELTA]to/A and W3110[DELTA]pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extraeytoplasmic stress responses in [tol.sup.+] and [pal.sup.+] genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 [DELTA] wzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.

Author(s): Jonne Helenius [1]; Davis T. W. Ng [2]; Cristina L. Marolda [3]; Peter Walter [4]; Miguel A. Valvano [3]; Markus Aebi (corresponding author) [1] N -linked glycosylation of proteins [...]

Research has been conducted on the ADP-L-glycerol-D-manno-heptose precursor of inner core lipopolysaccharide biosynthesis. The isolation of the mini-Tn10 insertion has been carried out.

The genetics of Escherichia coli VW187 (O7:K1) are discussed. Genetic organization of the 07-specific lipopolysaccharide biosynthesis cluster of the strain has been studied. The complete DNA sequence of one region is given and the O antigen chain length regulator gene was identified near the O7 polysaccharide cluster.

Research was conducted to examine the intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae. It was observed that clinical and environmental strains of the B. cepacia complex can survive within different isolates of the genus Acanthamoeba. Results indicate that free-living amoebae may be a reservoir for the acquisition, and perhaps the transmission of B. cepacia.

A study was conducted to characterize the structural and functional roles of a promoter region of the Escherichia coli o7-specific lipopolysaccharide gene cluster. The Escherichia coli strain was cultured in a Luria broth which was supplemented by ampicillin, kanamycin and tetracycline. Results revealed the existence of an untranslated leader messenger RNA segment of 173 bp whose structure may fold into a series of complex stem-loop structures.

Genes in the rfb polysaccharide gene cluster in Escherichia coli VW187 were sequenced, found to be extremely conserved and to have homologous genes in the rff cluster, which is involved in the production of an antigen. The rfb genes express enzymes involved in the synthesis of polysaccharide precursor dTDP-rhamnose. The rff homologs appear to express dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities. The location of the rfb and rff genes are discussed.

The rfb locus in Escherichia coli is involved in the biosynthesis of O-specific lipopolysaccharide (LPS). Localization of the E. coli gene encoding gluconate-6-phosphate dehydrogenase established the 3' endpoint of the rfb locus. Further sequence analysis of this region revealed the presence of two genes coding for phosphomannomutase and GDP-mannose pyrophosphorylase, enzymes involved in the conversion of mannose-6-phosphate to GDP-mannose, a nucleotide sugar precursor of the O7-specific repeating unit of LPS.

Arising from: J. Helenius et al. Nature 415, 447-450 (2002) Protein N-glycosylation requires flipping of the glycolipid Man5G1cNAC-diphosphate dolichol ([Man.sub.5]Glc[NAc.sub.2]-PP-Dol) across the endoplasmic reticulum (ER) (1-3). Helenius et al. (4) [...]

Research has been conducted on Salmonella enterica serovar Typhimurium rfaH gene. The study of the growth-dependent regulation of rfaH gene expression which occurs at the transcription initiation level is presented.

S taphylococcus aureus elaborates two citrate-containing siderophores, staphyloferrin A ( SA) and staphyloferrin B ( SB), that enhance growth under iron-restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron-regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S . aureus in iron-restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB-deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron-restricted media, and is significantly more impaired for virulence than either of the single-deletion mutants. We propose that SB is the more important of the two siderophores for S . aureus insofar as it is synthesized, and supports iron-restricted growth, without need of TCA cycle activity. [ABSTRACT FROM AUTHOR]

Organic acids are being used in animal husbandry due to their positive effects on overall health and growth rate in animal husbandry. However, little is known about the long-term effects of organic acids on the normal microbiota. In this work, an Escherichia coli strain isolated from a healthy pig was passed weekly for 20 months in medium with and without formic acid (FA) to investigate the effects of long-term exposure to organic acids. Passages were made using buffered medium at pH 5.25 and 6.1 with and without FA. Colonies showing reduced O antigen lipopolysaccharide production and increased susceptibility to FA were isolated after 12 months exposure to FA at pH 5.25 and after 20 months exposure to FA at pH 6.1. Adaptation to acid and bacterial growth was strongly affected by FA at pH 5.25. Also, after 2 months of serial passages, we observed colonies exhibiting multicellular behaviour, with production of fimbriae and an extracellular matrix. We conclude that long-term exposure to FA of an E. coli isolate from the normal intestinal microbiota is associated with various changes in the bacterial cell surface involving reduced O antigen expression and increased extracellular matrix production together with increased susceptibility to FA. [ABSTRACT FROM AUTHOR]

The article presents a study on the effect of varying the relative expression levels of the genes wzzB, wzzpHS-2 and wzy on O antigen polysaccharide chain length distribution in Shigella flexneri 2a. It states that the genes were cloned under the control of arabinose- and rhamnose-inducible promoters. Furthermore, no direct interaction between Wzy and WzzB was found, which suggests that they may not interact physically but by other means such as translocated O antigen precursors.

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.