scholarly journals High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

2014 ◽  
Vol 80 (13) ◽  
pp. 3826-3834 ◽  
Author(s):  
Junjie Yang ◽  
Bingbing Sun ◽  
He Huang ◽  
Yu Jiang ◽  
Liuyang Diao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Modification ◽  
Target Gene ◽  
Donor Plasmid ◽  
Content Type ◽  
Recognition Sites ◽  
Genetic Modifications ◽  
Pcr Targeting ◽  
Λ Red ◽  
Red Recombination

ABSTRACTGenetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of theEscherichia colichromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used forcadAknockout,gdhAknock-in, seamless deletion ofpepD, site-directed mutagenesis of the essentialmetKgene, and replacement ofmetKwith theRickettsiaS-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

scholarly journals UV Light Induces IS10 Transposition in Escherichia coli

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1173-1181
Author(s):  
Zehava Eichenbaum ◽  
Zvi Livneh
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Target Gene ◽  
Uv Light ◽  
Insertion Site ◽  
Donor Plasmid ◽  
Donor And Acceptor ◽  
Low Copy Number ◽  
Uv Induction ◽  
Target Plasmid

Abstract A new mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid was used to investigate the effect of UV light on intermolecular transposition of IS10. Inactivation of the target gene by IS10 insertion was detected by the expression of the tet gene from the phage 434 PR promoter, followed by Southern blot analysis of plasmids isolated from TetR colonies. UV irradiation of cells harboring the target plasmid and a donor plasmid carrying an IS10 element led to an increase of up to 28-fold in IS10 transposition. Each UV-induced transposition of IS10 was accompanied by fusion of the donor and acceptor plasmid into a cointegrate structure, due to coupled homologous recombination at the insertion site, similar to the situation in spontaneous IS10 transposition. UV radiation also induced transposition of IS10 from the chromosome to the target plasmid, leading almost exclusively to the integration of the target plasmid into the chromosome. UV induction of IS10 transposition did not depend on the umuC and uvrA gene product, but it was not observed in lexA3 and ΔrecA strains, indicating that the SOS stress response is involved in regulating UV-induced transposition. IS10 transposition, known to increase the fitness of Escherichia coli, may have been recruited under the SOS response to assist in increasing cell survival under hostile environmental conditions. To our knowledge, this is the first report on the induction of transposition by a DNA-damaging agent and the SOS stress response in bacteria.

scholarly journals Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V. Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Target Gene ◽  
Target Genes ◽  
Rna Seq ◽  
Content Type ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

scholarly journals Highly Efficient Method for Introducing Successive Multiple Scarless Gene Deletions and Markerless Gene Insertions into the Yersinia pestis Chromosome

2008 ◽  
Vol 74 (13) ◽  
pp. 4241-4245 ◽  
Author(s):  
Wei Sun ◽  
Shifeng Wang ◽  
Roy Curtiss
Keyword(s):  
Yersinia Pestis ◽  
Efficient Method ◽  
Genetic Modification ◽  
Gene Deletion ◽  
Gene Deletions ◽  
Live Attenuated Vaccines ◽  
Highly Efficient ◽  
Λ Red ◽  
Red Recombination

ABSTRACT An efficient two-step recombination method for markerless gene deletion and insertion that can be used for repetitive genetic modification in Yersinia pestis was developed. The method combines λ Red recombination and counterselective screening (sacB gene) and can be used for genetic modification of Y. pestis to construct live attenuated vaccines.

scholarly journals Performance of the 47-Kilodalton Membrane Protein versus DNA Polymerase I Genes for Detection of Treponema pallidum by PCR in Ulcers

2014 ◽  
Vol 53 (3) ◽  
pp. 976-980 ◽  
Author(s):  
Angèle Gayet-Ageron ◽  
Frédéric Laurent ◽  
Jacques Schrenzel ◽  
Béatrice Charton ◽  
Gisela Jimenez-Getaz ◽  
...  
Keyword(s):  
Target Gene ◽  
Treponema Pallidum ◽  
Secondary Syphilis ◽  
Dna Polymerase I ◽  
Perfect Agreement ◽  
Content Type ◽  
Sexually Transmitted ◽  
Polymerase I ◽  
Pcr Targeting ◽  
Protein Versus

Treponema pallidumPCR (Tp-PCR) is a direct diagnostic method for primary and secondary syphilis, but there is no recommendation regarding the best choice of target gene. In this study, we sequentially tested 272 specimens from patients with sexually transmitted ulcers usingTp-PCR targeting thetpp47and thenpolAgenes. The two methods showed similar accuracies and an almost-perfect agreement.

scholarly journals Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

2015 ◽  
Vol 81 (16) ◽  
pp. 5650-5659 ◽  
Author(s):  
Shan Goh ◽  
Angela Hohmeier ◽  
Timothy C. Stone ◽  
Victoria Offord ◽  
Francisco Sarabia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Target Gene ◽  
Essential Gene ◽  
Transcript Level ◽  
Essential Genes ◽  
Content Type ◽  
Reading Frame ◽  
Knockout Mutants ◽  
Bacterial Genes ◽  
Relationship Of

ABSTRACTEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, theEscherichia coliribF-ileS-lspA-fkpB-ispHoperon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlappingileS-lspAgenes. We found upstream and downstream polar silencing effects when eitherileSorlspAwas silenced, indicating coupled expression. Weighted MTL50values (means and standard deviations) ofribF,ileS, andlspAwere 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P= 0.71 by weighted one-way analysis of variance). The gene requirement forispHcould not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated thatileS-lspAexpression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

scholarly journals Novel Aggregative Adherence Fimbria Variant of Enteroaggregative Escherichia coli

2015 ◽  
Vol 83 (4) ◽  
pp. 1396-1405 ◽  
Author(s):  
Rie Jønsson ◽  
Carsten Struve ◽  
Nadia Boisen ◽  
Ramona Valentina Mateiu ◽  
Araceli E. Santiago ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Encoding ◽  
Content Type ◽  
Fimbrial Subunit ◽  
Eaec Strain ◽  
Aggregative Adherence ◽  
In The Beginning ◽  
Important Variant ◽  
Pcr Targeting

EnteroaggregativeEscherichia coli(EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with traveler's diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termedagg5A. The sequence of theagg5DCBAgene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of theagg3Agene. The completeagg5DCBAgene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherentE. coliHB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found theagg5Agene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant.

scholarly journals Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants

2016 ◽  
Vol 82 (18) ◽  
pp. 5505-5518 ◽  
Author(s):  
Shuai Zhi ◽  
Graham Banting ◽  
Qiaozhi Li ◽  
Thomas A. Edge ◽  
Edward Topp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wastewater Treatment ◽  
Intergenic Region ◽  
Municipal Wastewater ◽  
Treated Wastewater ◽  
Content Type ◽  
E Coli ◽  
Biomarker Pattern ◽  
Biomarker Analysis ◽  
Pcr Targeting

ABSTRACTEscherichia colihas been proposed to have two habitats—the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains ofE. colihave evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the survivingE. colistrains were found to contain a genetic insertion element (IS30) located within theuspC-flhDCintergenic region. The positional location of the IS30element was not observed across a library of 845E. coliisolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animalE. coliisolates (n= 1,177). Phylogenetics clustered the IS30element-containing wastewaterE. coliisolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only thefimHmarker. Our data suggest that wastewater contains a naturalized resident population ofE. coli. We developed an endpoint PCR targeting the IS30element within theuspC-flhDCintergenic region, and all raw sewage samples (n= 21) were positive for this marker. Conversely, the prevalence of this marker inE. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater.IMPORTANCEThe results of this study demonstrate that some strains ofE. coliappear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of usingE. colias a microbial indicator of wastewater treatment performance, suggesting that theE. colistrains present in human and animal feces may be very different from those found in treated wastewater.

scholarly journals Acquisition of a High Diversity of Bacteria during the Hajj Pilgrimage, Including Acinetobacter baumannii withblaOXA-72and Escherichia coli withblaNDM-5Carbapenemase Genes

2016 ◽  
Vol 60 (10) ◽  
pp. 5942-5948 ◽  
Author(s):  
Thongpan Leangapichart ◽  
Philippe Gautret ◽  
Karolina Griffiths ◽  
Khadidja Belhouchat ◽  
Ziad Memish ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Rectal Swab ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Pharyngeal Swab ◽  
Content Type ◽  
Carbapenemase Gene ◽  
Before And After ◽  
Pcr Targeting

ABSTRACTPilgrims returning from the Hajj (pilgrimage to Mecca) can be carriers of multidrug-resistant bacteria (MDR). Pharyngeal and rectal swab samples were collected from 98 pilgrims before and after they traveled to the Hajj in 2014 to investigate the acquisition of MDR bacteria. The bacterial diversity in pharyngeal swab samples was assessed by culture with selective media. There was a significantly higher diversity of bacteria in samples collected after the return from the Hajj than in those collected before (P= 0.0008). Surprisingly,Acinetobacter baumanniistrains were isolated from 16 pharyngeal swab samples (1 sample taken during the Hajj and 15 samples taken upon return) and 26 post-Hajj rectal swab samples, while none were isolated from samples taken before the Hajj. Testing of all samples by real-time PCR targetingblaOXA-51gave positive results for only 1% of samples taken during the Hajj, 21/90 (23.3%) pharyngeal swab samples taken post-Hajj, and 35/90 (38.9%) rectal swab samples taken post-Hajj. One strain ofA. baumanniiisolated from the pharynx was resistant to imipenem and harbored ablaOXA-72carbapenemase gene. Multilocus sequence typing analysis of 43A. baumanniiisolates revealed a huge diversity of 35 sequence types (STs), among which 18 were novel STs reported for the first time in this study. Moreover, we also found oneEscherichia coliisolate, collected from a rectal swab sample from a pilgrim taken after the Hajj, which harboredblaNDM-5,blaCTX-M-15,blaTEM-1, andaadA2(ST2659 and ST181). In conclusion, pilgrims are at a potential risk of acquiring and transmitting MDRAcinetobacterspp. and carbapenemase-producing Gram-negative bacteria during the Hajj season.

Roles for λ Orf and Escherichia coli RecO, RecR and RecF in λ Recombination

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 357-369
Author(s):  
James A Sawitzke ◽  
Franklin W Stahl
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Gene Product ◽  
Strand Break ◽  
Bacteriophage Λ ◽  
Recf Pathway ◽  
The Right ◽  
Mutant Cells ◽  
Λ Red ◽  
Red Recombination

Bacteriophage λ lacking its Red recombination functions requires either its own gene product, Orf, or the product of Escherichia coli's recO, recR and recF genes (RecORF) for efficient recombination in recBC sbcB sbcC mutant cells (the RecF pathway). Phage crosses under conditions of a partial block to DNA replication have revealed the following: (1) In the presence of Orf, RecF pathway recombination is similar to λ Red recombination; (2) Orf is necessary for focusing recombination toward the right end of the chromosome as λ is conventionally drawn; (3) RecORF-mediated RecF pathway recombination is not focused toward the right end of the chromosome, which may indicate that RecORF travels along the DNA; (4) both Orf- and RecORF-mediated RecF pathway recombination are stimulated by DNA replication; and (5) low level recombination in the simultaneous absence of Orf and RecORF may occur by a break-copy mechanism that is not initiated by a double strand break. Models for the roles of Orf and RecO, RecR and RecF in recombination are presented.

scholarly journals The emerging importance of Shiga toxin-producing Escherichia coli other than serogroup O157 in England

2021 ◽  
Vol 70 (7) ◽  
Author(s):  
Bhavita Vishram ◽  
Claire Jenkins ◽  
David R. Greig ◽  
Gauri Godbole ◽  
Kevin Carroll ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Severe Disease ◽  
Descriptive Analysis ◽  
Fold Increase ◽  
Surveillance Data ◽  
Bloody Diarrhoea ◽  
Content Type ◽  
Emerging Risk ◽  
Pcr Targeting

Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe disease and large outbreaks. In England, the incidence and clinical significance of STEC serogroups other than O157 (non-O157) is unknown due to a testing bias for detection of STEC O157. Since 2013, the implementation of PCR to detect all STEC serogroups by an increasing number of diagnostic laboratories has led to an increase in the detection of non-O157 STEC. Hypothesis/Gap statement. Due to a bias in testing methodologies to select for STEC serogroup O157 in frontline diagnostic laboratories in most countries, very little surveillance data have been previously generated on non-O157 STEC. Aim. Five years (2014–2018) of STEC national surveillance data were extracted and descriptive analysis undertaken to assess disease severity of non-O157 STEC strains. Methods. Data from 1 January 2014 to 31 December 2018 were extracted from the National Enhanced Surveillance System for STEC and analysed. Results. The implementation of Gastrointestinal Polymerase Chain Reaction (GI-PCR) has resulted in a four-fold increase in the detection of non-O157 STEC cases between 2014 and 2018. There were 2579 cases infected with 97 different non-O157 serogroups. The gender distribution was similar amongst STEC O157 and non-O157 STEC cases with 57 and 56 % of cases being female respectively, but a significantly higher proportion of cases (P <0.001) under 5 years of age was observed among STEC O157 (22 %) cases compared to non-O157 STEC (14 %). The most common non-O157 serogroups were O26 (16 %), O146 (11 %), O91 (10 %), O128 (7 %), O103 (5 %) and O117 (3 %). Overall, rates of bloody diarrhoea were highest in O26 (44 %) and O103 (48 %) cases and lowest in STEC O117 cases (17 %). Strains harbouring Shiga toxin stx1a caused the highest proportion of diarrhoea (93 %) and caused the same level of bloody diarrhoea as stx2a (39 %). However, stx2a caused the highest proportion of vomiting (46 %), hospitalisation (49 %) and considerably more HUS (29 %) than other stx profiles. Conclusion. The implementation of PCR targeting stx at diagnostic laboratories has shown that non-O157 STEC, most notably STEC O26, are an emerging risk to public health.

Sign in / Sign up

Export Citation Format

Share Document