scholarly journals Sequence Analysis of Tn10 Insertion Sites in a Collection of Escherichia coli Strains Used for Genetic Mapping and Strain Construction

1998 ◽  
Vol 180 (23) ◽  
pp. 6408-6411 ◽  
Author(s):  
Brian P. Nichols ◽  
Obaid Shafiq ◽  
Victoria Meiners
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Hot Spots ◽  
Insertion Site ◽  
Inverse Pcr ◽  
E Coli ◽  
Chromosome Sequence ◽  
Tn10 Insertion ◽  
Insertion Sites ◽  
Recombination Hot Spots

ABSTRACT The chromosomal insertion sites of Tn10-containingEscherichia coli strains were amplified by inverse PCR, and the nucleotide sequences of the junctions were determined. In 95 strains analyzed, 88 unique Tn10 positions were determined and matched to the E. coli chromosome sequence. Two gaps in insertion site positions were noted, one including the terminus of DNA replication and another bounded by recombination hot spots RhsA and RhsB.

scholarly journals Greater Diversity of Shiga Toxin-Encoding Bacteriophage Insertion Sites among Escherichia coli O157:H7 Isolates from Cattle than in Those from Humans

2006 ◽  
Vol 73 (3) ◽  
pp. 671-679 ◽  
Author(s):  
Thomas E. Besser ◽  
Nurmohammad Shaikh ◽  
Nicholas J. Holt ◽  
Phillip I. Tarr ◽  
Michael E. Konkel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Diversity ◽  
Shiga Toxin ◽  
Insertion Site ◽  
Reservoir Host ◽  
Clinical Isolates ◽  
Escherichia Coli O157 ◽  
Human Pathogen ◽  
E Coli ◽  
Insertion Sites

ABSTRACT Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q 933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes.

scholarly journals Molecular surveillance of shiga toxigenic Escherichia coli in selected beef abattoirs in Osun State Nigeria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Femi Ayoade ◽  
Judith Oguzie ◽  
Philomena Eromon ◽  
Omolola E. Omotosho ◽  
Tosin Ogunbiyi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Block Design ◽  
Latex Agglutination ◽  
Pcr Analysis ◽  
Accession Number ◽  
E Coli ◽  
Representative Isolate ◽  
Randomized Block Design ◽  
Toxigenic Strains

AbstractShiga toxigenic strains of E. coli (STEC) known to be etiological agents for diarrhea were screened for their incidence/occurrence in selected abattoirs sources in Osogbo metropolis of Osun State, Nigeria using a randomized block design. Samples were plated directly on selective and differential media and E. coli isolates. Multiplex PCR analysis was used to screen for the presence of specific virulence factors. These were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Sequence analysis of PCR products was performed on a representative isolate showing the highest combination of virulence genes using the 16S gene for identification purposes only. Results showed that the average cfu/cm2 was significantly lower in the samples collected at Sekona-2 slaughter slab compared with those collected at Al-maleek batch abattoir and Sekona-1 slaughter slab in ascending order at P = 0.03. Moreover, the average cfu/cm2E. coli in samples collected from butchering knife was significantly lower when compared with that of the workers’ hand (P = 0.047) and slaughtering floor (P = 0.047) but not with the slaughter table (P = 0.98) and effluent water from the abattoir house (P = 0.39). These data suggest that the abattoir type may not be as important in the prevalence and spread of STEC as the hygiene practices of the workers. Sequence analysis of a representative isolate showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. This sequence was subsequently submitted to GenBank with accession number MW463885. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria. Although no case of E. coli O157 was found within the study area, percent occurrence of non-O157 STEC as high as 46.3% at some of the sampled sites is worrisome and requires regulatory interventions in ensuring hygienic practices at the abattoirs within the study area.

scholarly journals Bacteriophage Lambda: a Paradigm Revisited

2010 ◽  
Vol 84 (13) ◽  
pp. 6876-6879 ◽  
Author(s):  
Paul C. M. Fogg ◽  
Heather E. Allison ◽  
Jon R. Saunders ◽  
Alan J. McCarthy
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
High Frequency ◽  
Southern Hybridization ◽  
Integration Site ◽  
Bacteriophage Lambda ◽  
Rare Event ◽  
Low Frequency ◽  
E Coli ◽  
Very Low Frequency

ABSTRACT Bacteriophage lambda has an archetypal immunity system, which prevents the superinfection of its Escherichia coli lysogens. It is now known that superinfection can occur with toxigenic lambda-like phages at a high frequency, and here we demonstrate that the superinfection of a lambda lysogen can lead to the acquisition of additional lambda genomes, which was confirmed by Southern hybridization and quantitative PCR. As many as eight integration events were observed but at a very low frequency (6.4 × 10−4) and always as multiple insertions at the established primary integration site in E. coli. Sequence analysis of the complete immunity region demonstrated that these multiply infected lysogens were not immunity mutants. In conclusion, although lambda superinfection immunity can be confounded, it is a rare event.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica

1999 ◽  
Vol 181 (16) ◽  
pp. 4937-4948 ◽  
Author(s):  
Donna Perkins-Balding ◽  
Guy Duval-Valentin ◽  
Anna C. Glasgow
Keyword(s):  
Insertion Site ◽  
Extracellular Polysaccharide ◽  
Inverse Pcr ◽  
Target Sequence ◽  
Sequence Element ◽  
E Coli ◽  
Precise Excision ◽  
Circular Form ◽  
Pseudoalteromonas Atlantica ◽  
Circle Formation

ABSTRACT The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced intoEscherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 inE. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

scholarly journals Bacterial RecA Protein Promotes Adenoviral Recombination duringIn VitroInfection

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Jeong Yoon Lee ◽  
Ji Sun Lee ◽  
Emma C. Materne ◽  
Rahul Rajala ◽  
Ashrafali M. Ismail ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Human Adenovirus ◽  
Penton Base ◽  
Content Type ◽  
E Coli ◽  
Recombination Hot Spots ◽  
Loop 2

ABSTRACTAdenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of anEscherichia colilysate increased recombination; this was blocked in a RecA mutant strain,E. coliDH5α, or upon RecA depletion. Recombination increased in the presence ofE. colilysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiADsequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCEAdenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

scholarly journals DOMINANT MUTATORS IN ESCHERICHIA COLI

Genetics ◽  
1982 ◽  
Vol 100 (1) ◽  
pp. 7-18
Author(s):  
E C Cox ◽  
D L Horner
Keyword(s):  
Escherichia Coli ◽  
Genetic Analysis ◽  
Wild Type ◽  
E Coli ◽  
Tn10 Insertion ◽  
Three Factor

ABSTRACT In this paper we report on the isolation and genetic analysis of a series of strong mutators mapping at five minutes on the E. coli chromosome. These mutations are dominant and show no evidence of interaction in merodiploids. Cultures grown in broth medium exhibit mutant frequencies five to six orders of magnitude higher than mut+ strains. Cultures propagated in minimal salts media mutate at rates one to three orders higher than wild-type. Three-factor crosses have been used to order these mutators relative to metD, proA, and a Tn10 insertion near five minutes.

HIGH FREQUENCY OF GENETIC DUPLICATIONS IN THE dnaB REGION OF THE ESCHERICHIA COLI K12 CHROMOSOME

Genetics ◽  
1981 ◽  
Vol 98 (4) ◽  
pp. 677-689
Author(s):  
Robert A Sclafani ◽  
James A Wechsler
Keyword(s):  
Escherichia Coli ◽  
High Frequency ◽  
Cell Populations ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Recombination System ◽  
Tn10 Insertion ◽  
Insertion Mutants

ABSTRACT The region that includes the dnaB locus on the E. coli K12 chromosome was shown to be duplicated at high frequency in cell populations. The duplications were shown to be arranged in tandem and segregated at various frequencies. Segregation was dependent on the recA recombination system, but independent of recB,C. Though most of the data was obtained with dnaB::Tn10 insertion mutants, the duplications were shown to occur in the absence of Tn10.

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