scholarly journals The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3260-3269 ◽  
Author(s):  
Javier A. Carter ◽  
Juan C. Jiménez ◽  
Mercedes Zaldívar ◽  
Sergio A. Álvarez ◽  
Cristina L. Marolda ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Chain Length ◽  
Shigella Flexneri ◽  
Length Distribution ◽  
Direct Interaction ◽  
Cellular Level ◽  
Chain Length Distribution ◽  
Inducible Promoters ◽  
O Antigen

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 WzzpHS-2 proteins, respectively. In this study, genes wzzB, wzz 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 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 WzzpHS-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.

scholarly journals The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

2012 ◽  
Vol 45 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Anilei Hoare ◽  
Denisse Bravo ◽  
Mara Martinic ◽  
Miguel A Valvano ◽  
Inés Contreras ◽  
...  
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Length Distribution ◽  
Chain Length Distribution ◽  
O Antigen ◽  
Shigella Flexneri 2A

O-antigen chain-length distribution in Salmonella enterica serovar Enteritidis is regulated by oxygen availability

2016 ◽  
Vol 477 (4) ◽  
pp. 563-567 ◽  
Author(s):  
Cecilia A. Silva-Valenzuela ◽  
Felipe Velásquez ◽  
Johany Peñailillo ◽  
Héctor Garcias-Papayani ◽  
Paulina Fernández ◽  
...  
Keyword(s):  
Chain Length ◽  
Salmonella Enterica ◽  
Length Distribution ◽  
Oxygen Availability ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Chain Length Distribution ◽  
O Antigen

scholarly journals Mutagenesis and Chemical Cross-Linking Suggest that Wzz Dimer Stability and Oligomerization Affect Lipopolysaccharide O-Antigen Modal Chain Length Control

2010 ◽  
Vol 192 (13) ◽  
pp. 3385-3393 ◽  
Author(s):  
Magdalene Papadopoulos ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Three Dimensional ◽  
Class Ii ◽  
Cross Linking ◽  
Wild Type ◽  
Class V ◽  
O Antigen ◽  
Class Iv

ABSTRACT In Shigella flexneri, the polysaccharide copolymerase (PCP) protein WzzSF confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within WzzSF, random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzzi proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzzi proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzzi mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100°C in the presence of SDS. Unlike the WzzSF wild type and class IV and V Wzzi mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.

scholarly journals Functional Analysis of Predicted Coiled-Coil Regions in the Escherichia coli K-12 O-Antigen Polysaccharide Chain Length Determinant Wzz

2008 ◽  
Vol 190 (6) ◽  
pp. 2128-2137 ◽  
Author(s):  
Cristina L. Marolda ◽  
Emily R. Haggerty ◽  
Michael Lung ◽  
Miguel A. Valvano
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Chain Length ◽  
Length Distribution ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Chain Length Distribution ◽  
O Antigen ◽  
Region I ◽  
K 12

ABSTRACT 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 α-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.

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

1998 ◽  
Vol 180 (10) ◽  
pp. 2670-2675 ◽  
Author(s):  
Agustin V. Franco ◽  
Dan Liu ◽  
Peter R. Reeves
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chain Length ◽  
Sequence Variation ◽  
Length Distribution ◽  
Chain Length Distribution ◽  
E Coli ◽  
O Antigen ◽  
Amino Acid Sequence Variation

ABSTRACT 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.

scholarly journals Dam Methylation Controls O-Antigen Chain Length in Salmonella enterica Serovar Enteritidis by Regulating the Expression of Wzz Protein

2009 ◽  
Vol 191 (21) ◽  
pp. 6694-6700 ◽  
Author(s):  
Sebastián H. Sarnacki ◽  
Cristina L. Marolda ◽  
Mariángeles Noto Llana ◽  
Mónica N. Giacomodonato ◽  
Miguel A. Valvano ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chain Length ◽  
Parental Strain ◽  
Salmonella Enterica ◽  
Length Distribution ◽  
Surface Proteins ◽  
Logarithmic Phase ◽  
Chain Length Distribution ◽  
O Antigen ◽  
Dam Methylation

ABSTRACT 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 O9 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in Salmonella. The LPS O-antigen profiles of a dam null mutant (SEΔdam) and the Salmonella serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEΔdam 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 SEΔdam is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the wzz promoter in SEΔdam 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.

Direct Observation of Chain Lengths and Conformations in Oligofluorene Distributions from Controlled Polymerization by Double Electron-Electron Resonance

2019 ◽  
Author(s):  
Dennis Bücker ◽  
Annika Sickinger ◽  
Julian D. Ruiz Perez ◽  
Manuel Oestringer ◽  
Stefan Mecking ◽  
...  
Keyword(s):  
Chain Length ◽  
Length Distribution ◽  
Catalyst System ◽  
Chain Conformation ◽  
Controlled Polymerization ◽  
Chain Length Distribution ◽  
Electron Resonance ◽  
Double Electron ◽  
The Individual ◽  
Double Electron Electron Resonance

Synthetic polymers are mixtures of different length chains, and their chain length and chain conformation is often experimentally characterized by ensemble averages. We demonstrate that Double-Electron-Electron-Resonance (DEER) spectroscopy can reveal the chain length distribution, and chain conformation and flexibility of the individual n-mers in oligo-(9,9-dioctylfluorene) from controlled Suzuki-Miyaura Coupling Polymerization (cSMCP). The required spin-labeled chain ends were introduced efficiently via a TEMPO-substituted initiator and chain terminating agent, respectively, with an in situ catalyst system. Individual precise chain length oligomers as reference materials were obtained by a stepwise approach. Chain length distribution, chain conformation and flexibility can also be accessed within poly(fluorene) nanoparticles.

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