The wclY gene of Escherichia coli serotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity

Glycobiology ◽  
2020 ◽  
Vol 30 (12) ◽  
pp. 9003-9014
Author(s):  
Alexander Kocev ◽  
Jacob Melamed ◽  
Vladimir Torgov ◽  
Leonid Danilov ◽  
Vladimir Veselovsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Clusters ◽  
Enterotoxigenic Escherichia Coli ◽  
Triple Mutant ◽  
E Coli ◽  
Glcnac Residue ◽  
Acceptor Specificity ◽  
Biosynthesis Gene ◽  
Glcnac Transferase

Abstract The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-4]n. A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6-tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1,4-Glc-transferase that transfers Glc to the Gal moiety of Galβ1-3GalNAcα-OPO3-PO3-phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1,4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.

scholarly journals Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Atsushi Iguchi ◽  
Hironobu Nishii ◽  
Kazuko Seto ◽  
Jiro Mitobe ◽  
Kenichi Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Strong Association ◽  
Gene Clusters ◽  
Epidemiological Studies ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
Wide Range ◽  
Biosynthesis Gene ◽  
Almost All

ABSTRACT The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

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

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.

scholarly journals Genotypic characterization of Egypt enterotoxigenic Escherichia coli isolates expressing coli surface antigen 6

2013 ◽  
Vol 7 (02) ◽  
pp. 090-100 ◽  
Author(s):  
Atef M El-Gendy ◽  
Adel Mansour ◽  
Hind I Shaheen ◽  
Marshall R Monteville ◽  
Adam W Armstrong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Antigen ◽  
Protective Immunity ◽  
Surface Antigens ◽  
Enterotoxigenic Escherichia Coli ◽  
Mechanisms Of Resistance ◽  
E Coli ◽  
Colonization Factor ◽  
Factor Expression

Introduction: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on inducing protective immunity against surface expressed antigenic factors. One such factor is coli surface antigen 6 (CS6); ETEC isolates expressing CS6 may also simultaneously co-express surface antigens CS4 or CS5. However, there is little information regarding the inter-relationships of isolates expressing the CS6 antigen alone or in combination with CS4 or CS5. Methodology: A total of 62 CS6-associated ETEC isolates were evaluated for their antimicrobial susceptibility, mechanisms of resistance, toxin genes, colonization factor expression, and XbaI-pulsed-field gel electrophoretic profiles. Results: We observed 46 XbaI profiles; 31 were exclusive to ETEC expressing CS6 alone and 15 among the ETEC co-expressing CS4 or CS5. Nearly half (47%) of these isolates were resistant to ampicillin, a third (37%) of the isolates were resistant to trimethoprim-sulfamethoxazole, and 24% of the isolates were tetracycline-resistant. A blaTEM gene was detected in 24 (83%) ampicillin-resistant isolates. Trimethoprim-sulfamethoxazole-resistant isolates (n = 23) carried either sulI (n = 1, 4%), sulII (n = 8, 35%) or both genes (n = 10, 43%); 4 had no detectable sul gene. Conclusions: Our results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface antigen.

Characterization of enterovirulent Escherichia coli isolated from yak calves died of diarrhoea in India

2016 ◽  
Author(s):  
Samiran Bandyopadhyay ◽  
Achintya Mahanti ◽  
Indranil Samanta ◽  
Subhasis Batabyal
Keyword(s):  
Escherichia Coli ◽  
Nalidixic Acid ◽  
Shiga Toxin ◽  
Enterotoxigenic Escherichia Coli ◽  
Drug Resistant ◽  
Arunachal Pradesh ◽  
E Coli ◽  
Clonal Relationship

Shiga toxin producing (STEC), enteropathogenic (EPEC) and enterotoxigenic Escherichia coli (ETEC) were isolated from 29 diarrhoeic yak calves from two districts of Arunachal Pradesh, India (West Kameng and Tawang) during 2005 to 2011. The STEC (28) and EPEC (12) isolates belonged to 25 different O serogroups. Among the 28 STEC isolates, 8 (28.5%) isolates carried only stx1, 10 (35.7%) isolates were positive for both stx1 and stx2, 10 (35.7%) isolates carried only stx2. The stx variants such as stx1c, stx2c, stx2d and stx2e were detected in 7 (25%), 9 (32.1%), 1 (3.5%) and 4 (14.28%) isolates, respectively. Among the 12 EPEC isolates, 2 (4.65%) strains were ‘typical’ possessing bfpA gene. The ehxA and saa genes were present in 19 and 4 isolates, respectively. Among the 14 ETEC isolates, 6 (42.8%), 7 (50%), 5 (35.7%), 5 (35.7%), 7 (50%) and 6 (42.8%) isolates carried genes coding for STa, STb, LT, F5, F41 and EAST1, respectively. The present study detected LT, STb, F41 as the most prevalent type in yaks associated with diarrhoea. Further, the STEC, EPEC and ETEC isolates showed resistance against furazolidone (40%), nitrofurantoin (39%), nalidixic acid (38%), erythromycin (38%), kanamycin (38%), amikacin (37%). The dendrogram revealed clonal relationship among the isolates after RAPD/ERIC analysis. The study could not detect any specific RAPD/ERIC cluster associated with origin, serotypes or virotypes of the isolates. Thus, it was concluded that genetically diverse, multi-drug resistant enterovirulent E. coli were associated with death due to diarrhoea in yak calves in India.

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

2009 ◽  
Vol 58 (7) ◽  
pp. 884-894 ◽  
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.

scholarly journals Isolation and Characterization of Two Members of the Siderophore-Microcin Family, Microcins M and H47

2009 ◽  
Vol 54 (1) ◽  
pp. 288-297 ◽  
Author(s):  
Gaëlle Vassiliadis ◽  
Delphine Destoumieux-Garzón ◽  
Carine Lombard ◽  
Sylvie Rebuffat ◽  
Jean Peduzzi
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Posttranslational Modification ◽  
Gene Clusters ◽  
Genetic Studies ◽  
E Coli ◽  
Isolation And Characterization ◽  
Bactericidal Activities ◽  
Additional Member

ABSTRACT In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiu genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coli Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.

scholarly journals Isolation of PDX2, a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria

2001 ◽  
Vol 183 (11) ◽  
pp. 3383-3390 ◽  
Author(s):  
Marilyn Ehrenshaft ◽  
Margaret E. Daub
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Degenerate Primer ◽  
Biosynthesis Pathway ◽  
Protein Motifs ◽  
E Coli ◽  
Biosynthesis Gene ◽  
Targeted Gene Replacement

ABSTRACT In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to theE. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that containPDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.

scholarly journals Molecular characterization of enterotoxigenic Escherichia coli toxins and colonization factors in children under five years with acute diarrhea attending Kisii Teaching and Referral Hospital, Kenya

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Erick Kipkirui ◽  
Margaret Koech ◽  
Abigael Ombogo ◽  
Ronald Kirera ◽  
Janet Ndonye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acute Diarrhea ◽  
Enterotoxigenic Escherichia Coli ◽  
Medical Attention ◽  
Toxin Gene ◽  
E Coli ◽  
Heat Stable ◽  
Colonization Factors ◽  
Porcine Origin

Abstract Background Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infectious diarrhea in children. There are no licensed vaccines against ETEC. This study aimed at characterizing Escherichia coli for ETEC enterotoxins and colonization factors from children < 5 years with acute diarrhea and had not taken antibiotics prior to seeking medical attention at the hospital. Methods A total of 225 randomly selected archived E. coli strains originally isolated from 225 children with acute diarrhea were cultured. DNA was extracted and screened by multiplex polymerase chain reaction (PCR) for three ETEC toxins. All positives were then screened for 11 colonization factors by PCR. Results Out of 225 E. coli strains tested, 23 (10.2%) were ETEC. Heat-stable toxin (ST) gene was detected in 16 (69.6%). ETEC isolates with heat-stable toxin of human origin (STh) and heat-stable toxin of porcine origin (STp) distributed as 11 (68.8%) and 5 (31.2%) respectively. Heat-labile toxin gene (LT) was detected in 5 (21.7%) of the ETEC isolates. Both ST and LT toxin genes were detected in 2 (8.7%) of the ETEC isolates. CF genes were detected in 14 (60.9%) ETEC strains with a majority having CS6 6 (42.9%) gene followed by a combination of CFA/I + CS21 gene detected in 3 (21.4%). CS14, CS3, CS7 and a combination of CS5 + CS6, CS2 + CS3 genes were detected equally in 1 (7.1%) ETEC isolate each. CFA/I, CS4, CS5, CS2, CS17/19, CS1/PCFO71 and CS21 genes tested were not detected. We did not detect CF genes in 9 (39.1%) ETEC isolates. More CFs were associated with ETEC strains with ST genes. Conclusion ETEC strains with ST genes were the most common and had the most associated CFs. A majority of ETEC strains had CS6 gene. In 9 (39.1%) of the evaluated ETEC isolates, we did not detect an identifiable CF.

scholarly journals Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pengpeng Xia ◽  
Yunping Wu ◽  
Siqi Lian ◽  
Guomei Quan ◽  
Yiting Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Symptoms ◽  
Mucosal Damage ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Epithelial ◽  
Antibody Microarray ◽  
E Coli ◽  
Fimbrial Subunit ◽  
Induced Apoptosis ◽  
Weaning Piglets

AbstractEnterotoxigenic Escherichia coli (ETEC) F4ac is a major constraint to the development of the pig industry, which is causing newborn and post-weaning piglets diarrhea. Previous studies proved that FaeG is the major fimbrial subunit of F4ac E. coli and efficient for bacterial adherence and receptor recognition. Here we show that the faeG deletion attenuates both the clinical symptoms of F4ac infection and the F4ac-induced intestinal mucosal damage in piglets. Antibody microarray analysis and the detection of mRNA expression using porcine neonatal jejunal IPEC-J2 cells also determined that the absence of FaeG subunit alleviated the F4ac promoted apoptosis in the intestinal epithelial cells. Thus, targeted depletion of FaeG is still beneficial for the prevention or treatment of F4ac infection.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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