scholarly journals Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome: isolation of unique sequences and demonstration of significant similarity to the chromosome of E. coli K-12

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1041-1054 ◽  
Author(s):  
Qing Chen ◽  
Stephen J. Savarino ◽  
Malabi M. Venkatesan
Keyword(s):  
Escherichia Coli ◽  
Optical Mapping ◽  
Subtractive Hybridization ◽  
Enterotoxigenic Escherichia Coli ◽  
Mapping Technique ◽  
Sequence Information ◽  
Strain Mg1655 ◽  
E Coli ◽  
Etec Strain ◽  
K 12

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E. coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E. coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. blast searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E. coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain B7A, which is attenuated in virulence and lacked several H10407-specific sequences. Restriction-enzyme-based optical maps of strain H10407 were compared to in silico restriction maps of strain MG1655 and related E. coli pathogens. The 5·1 Mb ETEC chromosome was ∼500 kb greater in length than the chromosome of E. coli K-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H10407 chromosome is expected to demonstrate ∼96 % identity with that of E. coli K-12.

scholarly journals Gene Cluster for Assembly of Pilus Colonization Factor Antigen III of Enterotoxigenic Escherichia coli

2001 ◽  
Vol 69 (9) ◽  
pp. 5864-5873 ◽  
Author(s):  
Tooru Taniguchi ◽  
Yukihiro Akeda ◽  
Ayako Haba ◽  
Yoko Yasuda ◽  
Koichiro Yamamoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Gene Cluster ◽  
Enterotoxigenic Escherichia Coli ◽  
Human Colon Carcinoma Cell ◽  
E Coli ◽  
Colonization Factor ◽  
Type Iv ◽  
K 12 ◽  
Factor Antigen

ABSTRACT The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, includingcofA and cofP. Several proteins encoded bycof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing thecof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.

scholarly journals CS5 Pilus Biosynthesis Genes from Enterotoxigenic Escherichia coli O115:H40

1999 ◽  
Vol 181 (18) ◽  
pp. 5847-5851 ◽  
Author(s):  
Thomas G. Duthy ◽  
Lothar H. Staendner ◽  
Paul A. Manning ◽  
Michael W. Heuzenroeder
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Surface Expression ◽  
Open Reading Frames ◽  
Enterotoxigenic Escherichia Coli ◽  
Cell Surface Expression ◽  
E Coli ◽  
Pilus Biogenesis ◽  
K 12 ◽  
And Function

ABSTRACT We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designatedcsfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC,csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.

Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

1980 ◽  
Vol 29 (2) ◽  
pp. 704-713 ◽  
Author(s):  
Henk Gankema ◽  
Jan Wensink ◽  
Pieter A. M. Guinée ◽  
Wim H. Jansen ◽  
Bernard Witholt
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Sucrose Density Gradient ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Freeze Fracture Electron Microscopy ◽  
K 12

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.

scholarly journals The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1316-1331 ◽  
Author(s):  
Christophe Beloin ◽  
Kai Michaelis ◽  
Karin Lindner ◽  
Paolo Landini ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Mg1655 ◽  
Upec Strain ◽  
E Coli ◽  
Initial Adhesion ◽  
State Production ◽  
K 12

ABSTRACT We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5 Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

scholarly journals Expression of fnr Is Constrained by an Upstream IS5 Insertion in Certain Escherichia coli K-12 Strains

2005 ◽  
Vol 187 (8) ◽  
pp. 2609-2617 ◽  
Author(s):  
R. Gary Sawers
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Transcription Initiation ◽  
Regulatory Region ◽  
Initiation Site ◽  
Strain Mg1655 ◽  
Transcript Levels ◽  
E Coli ◽  
K 12 ◽  
Strain Mc4100

ABSTRACT FNR is a global transcriptional regulator that controls anaerobic gene expression in Escherichia coli. Through the use of a number of approaches it was shown that fnr gene expression is reduced approximately three- to fourfold in E. coli strain MC4100 compared with the results seen with strain MG1655. This reduction in fnr expression is due to the insertion of IS5 (is5F) in the regulatory region of the gene at position −41 relative to the transcription initiation site. Transcription of the fnr gene nevertheless occurs from its own promoter in strain MC4100, but transcript levels are reduced approximately fourfold compared with those seen with strain MG1655. Remarkably, in strains bearing is5F the presence of Hfq prevents IS5-dependent transcriptional silencing of fnr expression. Thus, an hfq mutant of MC4100 is devoid of FNR protein and has the phenotype of an fnr mutant. In strain MG1655, or a derivative of MC4100 lacking is5F, mutation of hfq had no effect on fnr transcript levels. This finding indicates that IS5 mediates the effect of Hfq on fnr expression in MC4100. Western blot analysis revealed that cellular levels of FNR were reduced threefold in strain MC4100 compared with strain MG1655 results. A selection of FNR-dependent genes fused to lacZ were analyzed for the effects of reduced FNR levels on anaerobic gene expression. Expression of some operons, e.g., focA-pfl and fdnGHJI, was unaffected by reduction in the level of FNR, while the expression of other genes such as ndh and nikA was clearly affected.

scholarly journals A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407

2010 ◽  
Vol 192 (21) ◽  
pp. 5822-5831 ◽  
Author(s):  
Lisa C. Crossman ◽  
Roy R. Chaudhuri ◽  
Scott A. Beatson ◽  
Timothy J. Wells ◽  
Mickael Desvaux ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Sequence ◽  
Epidemiological Data ◽  
Coli Strain ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Extraintestinal Disease ◽  
K 12 ◽  
Genetic Context ◽  
Complete Genomic

ABSTRACT In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.

scholarly journals Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

2001 ◽  
Vol 69 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Christoph Lindenthal ◽  
Eric A. Elsinghorst
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Sodium Dodecyl ◽  
Enterotoxigenic Escherichia Coli ◽  
Human Intestine ◽  
Glycoprotein Synthesis ◽  
E Coli ◽  
Etec Strain ◽  
Link Type

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407 . The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinantE. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis.

scholarly journals Definition of the Escherichia coli MC4100 Genome by Use of a DNA Array

2003 ◽  
Vol 185 (6) ◽  
pp. 2017-2021 ◽  
Author(s):  
Joseph E. Peters ◽  
Timothy E. Thate ◽  
Nancy L. Craig
Keyword(s):  
Escherichia Coli ◽  
Experimental System ◽  
Exact Nature ◽  
Chromosomal Dna ◽  
Strain Mg1655 ◽  
E Coli ◽  
Genome Array ◽  
Definition Of ◽  
K 12 ◽  
Relationship Of

ABSTRACT We have used an Escherichia coli K-12 whole-genome array based on the DNA sequence of strain MG1655 as a tool to identify deletions in another E. coli K-12 strain, MC4100, by probing the array with labeled chromosomal DNA. Despite the continued widespread use of MC4100 as an experimental system, the specific genetic relationship of this strain to the sequenced K-12 derivative MG1655 has not been resolved. MC4100 was found to contain four deletions, ranging from 1 to 97 kb in size. The exact nature of three of the deletions was previously unresolved, and the fourth deletion was altogether unknown.

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