Synthesis and Reactivity of Precolibactin 886

2019 ◽  
Author(s):  
Seth Herzon ◽  
Alan R. Healy ◽  
kevin wernke ◽  
Chung Sub Kim ◽  
Nicholas Lees ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gene Cluster ◽  
Amino Alcohol ◽  
Biomimetic Synthesis ◽  
Cross Linking ◽  
E Coli ◽  
Hplc Purification ◽  
Structural Assignment ◽  
Biosynthetic Precursor ◽  
Double Oxidation

<div>The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal E. coli, and clb metabolites are thought to initiate colorectal cancer via DNA cross-linking. Precolibactin 886 (1) is one of the most complex isolated clb metabolites; it contains a 15-atom macrocycle and an unusual 5-hydroxy-3-oxazoline ring. Here we report confirmation of the structural assignment via a biomimetic synthesis of precolibactin 886 (1) proceeding through the amino alcohol 9. Double oxidation of 9 afforded the unstable α-ketoimine 2 which underwent macrocyclization to precolibactin 886 (1) upon HPLC purification (3% from 9). Studies of the putative precolibactin 886 (1) biosynthetic precursor 2, the model α-ketoimine 25, and the α-dicarbonyl 26 revealed that these compounds are susceptible to nucleophilic rupture of the C36–C37 bond. Moreover, cleavage of 2 produces other known clb metabolites or biosynthetic intermediates. This unexpected reactivity explains the difficulties in isolating full clb metabolites and accounts for the structure of a recently identified colibactin–adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 (1) to form a non-genotoxic pyridone, suggesting precolibactin 886 (1) lies off-path of the major biosynthetic route.</div>

scholarly journals Synthesis and Reactivity of Precolibactin 886

2019 ◽  
Author(s):  
Seth Herzon ◽  
Alan R. Healy ◽  
kevin wernke ◽  
Chung Sub Kim ◽  
Nicholas Lees ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gene Cluster ◽  
Amino Alcohol ◽  
Biomimetic Synthesis ◽  
Cross Linking ◽  
E Coli ◽  
Hplc Purification ◽  
Structural Assignment ◽  
Biosynthetic Precursor ◽  
Double Oxidation

<div>The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal E. coli, and clb metabolites are thought to initiate colorectal cancer via DNA cross-linking. Precolibactin 886 (1) is one of the most complex isolated clb metabolites; it contains a 15-atom macrocycle and an unusual 5-hydroxy-3-oxazoline ring. Here we report confirmation of the structural assignment via a biomimetic synthesis of precolibactin 886 (1) proceeding through the amino alcohol 9. Double oxidation of 9 afforded the unstable α-ketoimine 2 which underwent macrocyclization to precolibactin 886 (1) upon HPLC purification (3% from 9). Studies of the putative precolibactin 886 (1) biosynthetic precursor 2, the model α-ketoimine 25, and the α-dicarbonyl 26 revealed that these compounds are susceptible to nucleophilic rupture of the C36–C37 bond. Moreover, cleavage of 2 produces other known clb metabolites or biosynthetic intermediates. This unexpected reactivity explains the difficulties in isolating full clb metabolites and accounts for the structure of a recently identified colibactin–adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 (1) to form a non-genotoxic pyridone, suggesting precolibactin 886 (1) lies off-path of the major biosynthetic route.</div>

scholarly journals Structure elucidation of colibactin

2019 ◽  
Author(s):  
Mengzhao Xue ◽  
Chung Sub Kim ◽  
Alan R. Healy ◽  
Kevin M. Wernke ◽  
Zhixun Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Structure Prediction ◽  
Structure Elucidation ◽  
Isotope Labeling ◽  
Degradation Products ◽  
Cross Linking ◽  
Tandem Ms ◽  
Cross Link ◽  
Structural Assignment ◽  
Unknown Structure

AbstractColibactin is a gut microbiome metabolite of unknown structure that has been implicated in colorectal cancer formation. Several studies now suggest that the tumorgenicity of colibactin derives from interstrand cross-linking of host DNA. Here we use a combination of genetics, isotope labeling, tandem MS, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster. DNA cross-link degradation products derived from synthetic and natural colibactin were indistinguishable by tandem MS analysis, thereby confirming the structure prediction. This work reveals the structure of colibactin, which has remained incompletely defined for over a decade.

scholarly journals Cytotoxic Escherichia coli strains encoding colibactin, cytotoxic necrotizing factor, and cytolethal distending toxin colonize laboratory common marmosets (Callithrix jacchus)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Colleen S. McCoy ◽  
Anthony J. Mannion ◽  
Yan Feng ◽  
Carolyn M. Madden ◽  
Stephen C. Artim ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cellular Differentiation ◽  
Gastrointestinal Disease ◽  
Phylogenetic Group ◽  
Cytolethal Distending Toxin ◽  
Whole Genome ◽  
Common Marmosets ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Inflammatory Bowel

AbstractCyclomodulins are virulence factors that modulate cellular differentiation, apoptosis, and proliferation. These include colibactin (pks), cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt). Pathogenic pks+, cnf+, and cdt+ E. coli strains are associated with inflammatory bowel disease (IBD) and colorectal cancer in humans and animals. Captive marmosets are frequently afflicted with IBD-like disease, and its association with cyclomodulins is unknown. Cyclomodulin-encoding E. coli rectal isolates were characterized using PCR-based assays in healthy and clinically affected marmosets originating from three different captive sources. 139 E. coli isolates were cultured from 122 of 143 marmosets. The pks gene was detected in 56 isolates (40%), cnf in 47 isolates (34%), and cdt in 1 isolate (0.7%). The prevalences of pks+ and cnf+ E. coli isolates were significantly different between the three marmoset colonies. 98% of cyclomodulin-positive E. coli belonged to phylogenetic group B2. Representative isolates demonstrated cyclomodulin cytotoxicity, and serotyping and whole genome sequencing were consistent with pathogenic E. coli strains. However, the presence of pks+, cnf+, or cdt+ E. coli did not correlate with clinical gastrointestinal disease in marmosets. Cyclomodulin-encoding E. coli colonize laboratory common marmosets in a manner dependent on the source, potentially impacting reproducibility in marmoset models.

scholarly journals Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

2010 ◽  
Vol 76 (8) ◽  
pp. 2500-2508 ◽  
Author(s):  
S. D. Braun ◽  
J. Hofmann ◽  
A. Wensing ◽  
M. S. Ullrich ◽  
H. Weingart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Pentanoic Acid ◽  
Promising Candidate ◽  
Biosynthesis Gene Cluster ◽  
E Coli ◽  
Highly Active ◽  
Putative Aminotransferase

ABSTRACT The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km , 7 mM; k cat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

scholarly journals Organization of the Flagellar Switch Complex ofBacillus subtilis

2018 ◽  
Vol 201 (8) ◽  
Author(s):  
Elizabeth Ward ◽  
Eun A Kim ◽  
Joseph Panushka ◽  
Tayson Botelho ◽  
Trevor Meyer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interaction ◽  
Protein Complex ◽  
Cross Linking ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Middle Domain ◽  
Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

scholarly journals A Bacterial Microcompartment Is Used for Choline Fermentation byEscherichia coli536

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Taylor I. Herring ◽  
Tiffany N. Harris ◽  
Chiranjit Chowdhury ◽  
Sujit Kumar Mohanty ◽  
Thomas A. Bobik
Keyword(s):  
Electron Microscopy ◽  
Heart Disease ◽  
Gene Cluster ◽  
Transcriptional Regulator ◽  
Human Gut ◽  
Content Type ◽  
E Coli ◽  
Bacterial Microcompartment ◽  
New Type ◽  
Insight Into

ABSTRACTBacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization byProteusandDesulfovibriospecies. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogenEscherichia coli536. Growth studies indicatedE. coli536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization (cut) gene cluster ofE. coli536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that thecutXgene encodes a positive transcriptional regulator required for induction of the maincutoperon in response to choline supplementation. Each of the 16 genes in thecutcluster was individually deleted, and phenotypes were examined. ThecutX,cutY,cutF,cutO,cutC,cutD,cutU, andcutVgenes were required for choline degradation, but the remaining genes of thecutcluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed.IMPORTANCEHere, we investigate choline degradation inE. coli536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation inE. coli536, an organism for which sophisticated genetic analysis methods are available. In addition, thecutgene cluster ofE. coli536 is located in pathogenicity island II (PAI-II536) and hence might contribute to pathogenesis.

scholarly journals Cross‐linking study of membrane subunits K, L, M and N in E. coli complex I (997.10)

2014 ◽  
Vol 28 (S1) ◽  
Author(s):  
Steven Vik ◽  
Shaotong Zhu ◽  
Ablat Tursun
Keyword(s):  
Complex I ◽  
Cross Linking ◽  
E Coli

scholarly journals Prevalence of the “High-Pathogenicity Island” of Yersinia Species among Escherichia coliStrains That Are Pathogenic to Humans

1998 ◽  
Vol 66 (2) ◽  
pp. 480-485 ◽  
Author(s):  
S. Schubert ◽  
A. Rakin ◽  
H. Karch ◽  
E. Carniel ◽  
J. Heesemann
Keyword(s):  
Gene Cluster ◽  
Yersinia Pseudotuberculosis ◽  
Pathogenicity Island ◽  
Uptake System ◽  
Highly Pathogenic ◽  
E Coli ◽  
Yersinia Species ◽  
The Family ◽  
High Pathogenicity ◽  
Dna Sequence Comparison

ABSTRACT The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis,Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this “high pathogenicity island” (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregativeEscherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella entericastrains investigated. Polypeptides encoded by the fyuA,irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess thehms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the fyuA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.

scholarly journals Altered Utilization of N-Acetyl-d-Galactosamine by Escherichia coli O157:H7 from the 2006 Spinach Outbreak

2007 ◽  
Vol 190 (5) ◽  
pp. 1710-1717 ◽  
Author(s):  
Amit Mukherjee ◽  
Mark K. Mammel ◽  
J. Eugene LeClerc ◽  
Thomas A. Cebula
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Culture Collection ◽  
Base Substitution ◽  
Phosphotransferase System ◽  
Single Nucleotide ◽  
E Coli ◽  
Single Nucleotide Change ◽  
Phenotype Comparison ◽  
In Silico Analyses

ABSTRACT In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-d-galactosamine (Aga) and d-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)Aga/Gam, EIIBAga, EIICAga, and EIIDAga of the Aga PTS are present, E. coli O157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIICGam, and consequently, E. coli O157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates of E. coli O157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+ Gam− phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associated E. coli O157:H7 outbreak, were analyzed, all (194/194) displayed an Aga− Gam− phenotype. Comparison of aga/gam sequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C→A:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIAAga/Gam of the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associated E. coli O157:H7 isolates harbored this same G:C→A:T substitution. Notably, when agaF + was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga− phenotype in these isolates.

scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

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