Characterization of Two β-1,3-Glucosyltransferases from Escherichia coli Serotypes O56 and O152

ABSTRACT The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-β1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-β1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

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Escherichia coli O123 O antigen genes and polysaccharide structure are conserved in some Salmonella enterica serogroups

Journal of Medical Microbiology ◽

10.1099/jmm.0.007187-0 ◽

2009 ◽

Vol 58 (7) ◽

pp. 884-894 ◽

Cited By ~ 5

Author(s):

Clifford G. Clark ◽

Andrew M. Kropinski ◽

Haralambos Parolis ◽

Christopher C. R. Grant ◽

Keri M. Trout-Yakel ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Salmonella Enterica ◽

Gene Clusters ◽

E Coli ◽

Antigen Gene ◽

O Antigen ◽

Antigenic Polysaccharide ◽

O Antigens

The serotyping of O and H antigens is an important first step in the characterization of Salmonella enterica. However, serotyping has become increasingly technically demanding and expensive to perform. We have therefore sequenced additional S. enterica O antigen gene clusters to provide information for the development of DNA-based serotyping methods. Three S. enterica isolates had O antigen gene clusters with homology to the Escherichia coli O123 O antigen region. O antigen clusters from two serogroup O58 S. enterica strains had approximately 85 % identity with the E. coli O123 O antigen region over their entire length, suggesting that these Salmonella and E. coli O antigen regions evolved from a common ancestor. The O antigen cluster of a Salmonella serogroup O41 isolate had a lower level of identity with E. coli O123 over only part of its O antigen DNA cluster sequence, suggesting a different and more complex evolution of this gene cluster than those in the O58 strains. A large part of the Salmonella O41 O antigen DNA cluster had very close identity with the O antigen cluster of an O62 strain. This region of DNA homology included the wzx and wzy genes. Therefore, molecular serotyping tests using only the O41 or O62 wzx and wzy genes would not differentiate between the two serogroups. The E. coli O123 O-antigenic polysaccharide and its repeating unit were characterized, and the chemical structure for E. coli O123 was entirely consistent with the O antigen gene cluster sequences of E. coli O123 and the Salmonella O58 isolates. An understanding of both the genetic and structural composition of Salmonella and E. coli O antigens is necessary for the development of novel molecular methods for serotyping these organisms.

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Immunological characterization of antibody responses to Escherichia coli O antigens in bacteraemic patients

Serodiagnosis and Immunotherapy in Infectious Disease ◽

10.1016/0888-0786(88)90016-9 ◽

1988 ◽

Vol 2 (4) ◽

pp. 291-299 ◽

Cited By ~ 1

Author(s):

Annelie Brauner ◽

Stefan B. Svenson ◽

Bengt Wretlind

Keyword(s):

Escherichia Coli ◽

Antibody Responses ◽

Immunological Characterization ◽

O Antigens

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Functional Characterization of the C-Terminus of YhaV in the Escherichia coli PrlF-YhaV Toxin-Antitoxin System

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.1803.03010 ◽

2018 ◽

Vol 28 (6) ◽

pp. 987-996 ◽

Cited By ~ 2

Author(s):

Wonho Choi ◽

Min-Ho Yoon ◽

Jung-Ho Park

Keyword(s):

Escherichia Coli ◽

Functional Characterization ◽

C Terminus

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Soluble neutral maltase–glucoamylase from the small intestine: separation and characterization of components with differing affinity for concanavalin A

Canadian Journal of Biochemistry ◽

10.1139/o82-129 ◽

1982 ◽

Vol 60 (11) ◽

pp. 1007-1013 ◽

Cited By ~ 2

Author(s):

G. Forstner ◽

A. Salvatore ◽

L. Lee ◽

J. Forstner

Keyword(s):

Concanavalin A ◽

Gradient Elution ◽

Soluble Form ◽

Rat Intestine ◽

Ph Optimum ◽

Molecular Weights ◽

Neutral Ph ◽

Membrane Bound ◽

Sepharose 4B

Intestinal maltase with a neutral pH optimum exists in both a brush border membrane-bound form and a soluble form in suckling rat intestine. Previous experiments in our laboratory have shown that the soluble enzyme contains a component which binds much more tightly to concanavalin A (ConA) than solubilized forms of the membrane enzyme. We studied the origin of this component by subjecting neutral, soluble maltase activity to chromatography on Sepharose 4B at age 13, 18 (preweaning), and 25 (postweaning) days. At 13 days, two maltase peaks were obtained with approximate molecular weights of 400 000 (peak I) and 150 000 (peak II). Peak II was less prominent at 18 days and was absent at 25 days. At 13 days, the majority of peak I consisted of material which was bound between 0.025 and 0.05 M α-methyl mannoside on gradient elution chromatography of ConA-Sepharose. Peak II contained material which eluted between 0.075 and 0.3 M α-methyl mannoside. At 25 days, all of the soluble maltase eluted between 0.025 and 0.04 M α-methyl mannoside. Peak I and peak II maltases had similar pH optima and Km's for maltase. Peak II maltase had a fourfold greater activity toward glycogen than peak I maltase with approximately the same activity for palatinose, turanose, and trehalose. Both maltases were precipitated by an antibody raised against adult membrane-bound maltase. Soluble maltase with neutral pH activity in the suckling rat intestine, therefore, consists of two immunologically related isozymes which differ in their molecular weight, their binding by ConA, and their specificity for glycogen. The small isozyme disappears at or about the time of weaning.

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Structure and genetics of Escherichia coli O antigens

FEMS Microbiology Reviews ◽

10.1093/femsre/fuz028 ◽

2019 ◽

Vol 44 (6) ◽

pp. 655-683 ◽

Cited By ~ 7

Author(s):

Bin Liu ◽

Axel Furevi ◽

Andrei V Perepelov ◽

Xi Guo ◽

Hengchun Cao ◽

...

Keyword(s):

Escherichia Coli ◽

Genetic Basis ◽

Structural Diversity ◽

Gene Clusters ◽

Current Standard ◽

E Coli ◽

Antigen Gene ◽

O Antigen ◽

Or Groups ◽

O Antigens

ABSTRACT Escherichia coli includes clonal groups of both commensal and pathogenic strains, with some of the latter causing serious infectious diseases. O antigen variation is current standard in defining strains for taxonomy and epidemiology, providing the basis for many serotyping schemes for Gram-negative bacteria. This review covers the diversity in E. coli O antigen structures and gene clusters, and the genetic basis for the structural diversity. Of the 187 formally defined O antigens, six (O31, O47, O67, O72, O94 and O122) have since been removed and three (O34, O89 and O144) strains do not produce any O antigen. Therefore, structures are presented for 176 of the 181 E. coli O antigens, some of which include subgroups. Most (93%) of these O antigens are synthesized via the Wzx/Wzy pathway, 11 via the ABC transporter pathway, with O20, O57 and O60 still uncharacterized due to failure to find their O antigen gene clusters. Biosynthetic pathways are given for 38 of the 49 sugars found in E. coli O antigens, and several pairs or groups of the E. coli antigens that have related structures show close relationships of the O antigen gene clusters within clades, thereby highlighting the genetic basis of the evolution of diversity.

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Purification and Characterization of a Membrane Bound Neutral pH Optimum Magnesium-dependent and Phosphatidylserine-stimulated Sphingomyelinase from Rat Brain

Journal of Biological Chemistry ◽

10.1074/jbc.273.51.34472 ◽

1998 ◽

Vol 273 (51) ◽

pp. 34472-34479 ◽

Cited By ~ 93

Author(s):

Bin Liu ◽

Daniel F. Hassler ◽

Gary K. Smith ◽

Kurt Weaver ◽

Yusuf A. Hannun

Keyword(s):

Rat Brain ◽

Purification And Characterization ◽

Ph Optimum ◽

Neutral Ph ◽

Membrane Bound

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In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-020-01432-y ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Fabien Le Chevalier ◽

Isabelle Correia ◽

Lucrèce Matheron ◽

Morgan Babin ◽

Mireille Moutiez ◽

...

Keyword(s):

Escherichia Coli ◽

Biological Activities ◽

Gene Clusters ◽

Chemical Diversity ◽

E Coli ◽

Chemical Structures ◽

In Vivo Characterization ◽

Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

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