Plant Transformation by Coinoculation with a Disarmed Agrobacterium tumefaciens Strain and an Escherichia coli Strain Carrying Mobilizable Transgenes

ABSTRACT Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.

Download Full-text

A Conductance Method for the Identification of Escherichia coli O157:H7 Using Bacteriophage AR1

Journal of Food Protection ◽

10.4315/0362-028x-65.1.12 ◽

2002 ◽

Vol 65 (1) ◽

pp. 12-17 ◽

Cited By ~ 20

Author(s):

TSUNG C. CHANG ◽

HWIA C. DING ◽

SHIOWWEN CHEN

Keyword(s):

Escherichia Coli ◽

Positive Reaction ◽

Present Method ◽

Culture Broth ◽

Coli Strain ◽

Bacterial Suspension ◽

Escherichia Coli O157 ◽

Bacterial Strains ◽

E Coli ◽

Conductance Method

The feasibility of using a specific phage (AR1) in conjunction with a conductance method for the identification of Escherichia coli O157:H7 was evaluated. The multiplication of strains of E. coli O157:H7 was inhibited by AR1; therefore, a time point (detection time, DT) at which an accelerating change in conductance in the culture broth was not obtained. Bacterial strains were subcultured on sorbitol-MacConkey agar and incubated at 35°C for 24 h, and the ability of the bacteria to ferment sorbitol was recorded. An aliquot of 0.5 ml of the bacterial suspension (107 CFU/ml) and 0.5 ml of the phage suspension (108 PFU/ml) were added to the conductance tube of a Malthus analyzer containing 5 ml of culture broth. The tubes were incubated at 35°C, and conductance changes in the tubes were continuously monitored at 6-min intervals for 24 h by the instrument. A positive reaction was defined as an E. coli strain that could not utilize sorbitol and caused no conductance change (i.e., no DT) within an incubation period of 24 h. Of the 41 strains of E. coli O157:H7 tested, all produced positive reactions. When a total of 155 strains of non-O157:H7 E. coli were tested, 14 did not have a DT within 24 h. However, among these 14 strains, 13 were sorbitol fermenters, and the remaining one was a nonfermenter. Therefore, by definition, only one strain produced a false-positive reaction. The sensitivity and specificity of the present method were 100% (41 of 41) and 99.4% (154 of 155), respectively. The present method incorporating conductimetric measurement and phage AR1 for the identification of E. coli O157:H7 was simple and capable of automation.

Download Full-text

Characterisation of Colistin -Resistant Enterobacterales and Acinetobacter Strains Carrying mcr Genes from Asian Aquaculture Products

Antibiotics ◽

10.3390/antibiotics10070838 ◽

2021 ◽

Vol 10 (7) ◽

pp. 838

Author(s):

Alžběta Kalová ◽

Tereza Gelbíčová ◽

Søren Overballe-Petersen ◽

Eva Litrup ◽

Renáta Karpíšková

Keyword(s):

Escherichia Coli ◽

Czech Republic ◽

Antimicrobial Resistance ◽

Coli Strain ◽

Bacterial Strains ◽

Asian Origin ◽

The Czech Republic ◽

E Coli ◽

Antimicrobial Resistance Genes ◽

Aquaculture Products

Aquaculture systems are widely recognised as hotspots for horizontal gene transfer, and the need for screening for bacteria carrying antimicrobial resistance genes in aquaculture systems is becoming more important. In this study, we characterised seventeen bacterial strains (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and A. nosocomialis) resistant to colistin originating from retailed aquaculture products imported from Vietnam to the Czech Republic. The mcr-1.1 gene was found located on plasmid types IncHI2, IncI2, and IncX4, as well as on the rarely described plasmid types IncFIB-FIC and IncFIB(K), phage-like plasmid p0111, and on the chromosome of E. coli. One E. coli strain carried the mcr-3.5 gene on IncFII(pCoo) plasmid in addition to the mcr-1.1 gene located on IncHI2 plasmid. K. pneumoniae was found to carry the mcr-1.1 and mcr-8.2 genes on IncFIA(HI1) plasmid. The mcr-4.3 gene was found on similar untypeable plasmids of A. baumannii and A. nosocomialis strains, pointing to the possible interspecies transfer of plasmids carrying the mcr-4 gene. Our results highlight that some aquaculture products of Asian origin can represent an important source of variable plasmids carrying mcr genes. The results showed an involvement of phages in the incorporation of the mcr-1 gene into plasmids or the chromosome in E. coli strains from aquaculture. The detection of E. coli with the mcr-1 gene in the chromosome points to the risks associated with the stabilisation of the mcr genes in the bacterial chromosome.

Download Full-text

Construction of Disarmed Ti Plasmids Transferable between Escherichia coli and Agrobacterium Species

Applied and Environmental Microbiology ◽

10.1128/aem.01856-08 ◽

2009 ◽

Vol 75 (7) ◽

pp. 1845-1851 ◽

Cited By ~ 7

Author(s):

Kazuya Kiyokawa ◽

Shinji Yamamoto ◽

Kei Sakuma ◽

Katsuyuki Tanaka ◽

Kazuki Moriguchi ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Plant Transformation ◽

Ti Plasmid ◽

Broad Host Range ◽

Bacterial Strains ◽

E Coli ◽

Ti Plasmids ◽

The Right ◽

Border Sequence

ABSTRACT Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1λpir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

Download Full-text

Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

Pathogens ◽

10.3390/pathogens10050549 ◽

2021 ◽

Vol 10 (5) ◽

pp. 549

Author(s):

Julia Ittensohn ◽

Jacqueline Hemberger ◽

Hannah Griffiths ◽

Maren Keller ◽

Simone Albrecht ◽

...

Keyword(s):

Escherichia Coli ◽

Protein C ◽

Interleukin 1 ◽

Coli Strain ◽

Uropathogenic Escherichia Coli ◽

Reporter Construct ◽

Regulation Of Expression ◽

E Coli ◽

Strain Cft073 ◽

Myeloid Differentiation Factor

The uropathogenic Escherichia coli strain CFT073 causes kidney abscesses in mice Toll/interleukin-1 receptor domain-containing protein C (TcpC) dependently and the corresponding gene is present in around 40% of E. coli isolates of pyelonephritis patients. It impairs the Toll-like receptor (TLR) signaling chain and the NACHT leucin-rich repeat PYD protein 3 inflammasome (NLRP3) by binding to TLR4 and myeloid differentiation factor 88 as well as to NLRP3 and caspase-1, respectively. Overexpression of the tcpC gene stopped replication of CFT073. Overexpression of several tcpC-truncation constructs revealed a transmembrane region, while its TIR domain induced filamentous bacteria. Based on these observations, we hypothesized that tcpC expression is presumably tightly controlled. We tested two putative promoters designated P1 and P2 located at 5′ of the gene c2397 and 5′ of the tcpC gene (c2398), respectively, which may form an operon. High pH and increasing glucose concentrations stimulated a P2 reporter construct that was considerably stronger than a P1 reporter construct, while increasing FeSO4 concentrations suppressed their activity. Human urine activated P2, demonstrating that tcpC might be induced in the urinary tract of infected patients. We conclude that P2, consisting of a 240 bp region 5′ of the tcpC gene, represents the major regulator of tcpC expression.

Download Full-text

Enterohemorrhagic Escherichia coli Colony Check Assay for the Identification of Serogroups O26, O45, O103, O111, O121, O145, and O157 Colonies Isolated on Plating Media

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-555 ◽

2014 ◽

Vol 77 (7) ◽

pp. 1212-1218 ◽

Cited By ~ 1

Author(s):

BURTON BLAIS ◽

MYLÈNE DESCHÊNES ◽

GEORGE HUSZCZYNSKI ◽

MARTINE GAUTHIER

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

High Throughput Screening ◽

Enterohemorrhagic Escherichia Coli ◽

Test Results ◽

Bacterial Strains ◽

Enrichment Broth ◽

Substrate Solution ◽

E Coli ◽

Assay Performance

A simple immunoenzymatic enterohemorrhagic Escherichia coli (EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coli serogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coli isolates including several nontarget Shiga toxin–producing E. coli serogroups (all unreactive), and 33 non–E. coli strains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

Download Full-text

Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli during Initial Contact with Cattle Colonic Explants

Microorganisms ◽

10.3390/microorganisms8111662 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1662

Author(s):

Zachary R. Stromberg ◽

Rick E. Masonbrink ◽

Melha Mellata

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Bacterial Colonization ◽

Coli Strain ◽

Fold Change ◽

Initial Contact ◽

Lon Protease ◽

E Coli ◽

Log2 Fold Change ◽

Colonization Factors

Foodborne pathogens are a public health threat globally. Shiga toxin-producing Escherichia coli (STEC), particularly O26, O111, and O157 STEC, are often associated with foodborne illness in humans. To create effective preharvest interventions, it is critical to understand which factors STEC strains use to colonize the gastrointestinal tract of cattle, which serves as the reservoir for these pathogens. Several colonization factors are known, but little is understood about initial STEC colonization factors. Our objective was to identify these factors via contrasting gene expression between nonpathogenic E. coli and STEC. Colonic explants were inoculated with nonpathogenic E. coli strain MG1655 or STEC strains (O26, O111, or O157), bacterial colonization levels were determined, and RNA was isolated and sequenced. STEC strains adhered to colonic explants at numerically but not significantly higher levels compared to MG1655. After incubation with colonic explants, flagellin (fliC) was upregulated (log2 fold-change = 4.0, p < 0.0001) in O157 STEC, and collectively, Lon protease (lon) was upregulated (log2 fold-change = 3.6, p = 0.0009) in STEC strains compared to MG1655. These results demonstrate that H7 flagellum and Lon protease may play roles in early colonization and could be potential targets to reduce colonization in cattle.

Download Full-text

In VivoTransmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness

Applied and Environmental Microbiology ◽

10.1128/aem.04193-14 ◽

2015 ◽

Vol 81 (10) ◽

pp. 3561-3570 ◽

Cited By ~ 20

Author(s):

Timothy J. Johnson ◽

Randall S. Singer ◽

Richard E. Isaacson ◽

Jessica L. Danzeisen ◽

Kevin Lang ◽

...

Keyword(s):

Escherichia Coli ◽

High Dose ◽

Broad Host Range ◽

Antibiotic Administration ◽

Dose Administration ◽

Content Type ◽

E Coli ◽

The Impact ◽

Selection Of

ABSTRACTIncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensalEscherichia colihost. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containingE. colifrom pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containingE. coliin pig feces (P< 0.001) and increased movement of the IncA/C plasmid to other indigenousE. colihosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other thanE. coli.In vitrocompetition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage inE. coliandSalmonella.In vitrotransfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracyclinein vitrostrongly selected for IncA/C plasmid-containingE. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.

Download Full-text

An Improved Binary Vector and Escherichia coli Strain for Agrobacterium tumefaciens-Mediated Plant Transformation

G3 Genes|Genome|Genetics ◽

10.1534/g3.116.029405 ◽

2016 ◽

Vol 6 (7) ◽

pp. 2195-2201 ◽

Cited By ~ 3

Author(s):

Michael R. Watson ◽

Yu-fei Lin ◽

Elizabeth Hollwey ◽

Rachel E. Dodds ◽

Peter Meyer ◽

...

Keyword(s):

Escherichia Coli ◽

Agrobacterium Tumefaciens ◽

Plant Transformation ◽

Binary Vector ◽

Coli Strain

Download Full-text

Developing a Model to Study Commensal Translocation during IBD

Inquiry@Queen's Undergraduate Research Conference Proceedings ◽

10.24908/iqurcp.9963 ◽

2018 ◽

Author(s):

Kathy Yu

Keyword(s):

Bacterial Translocation ◽

Imaging Techniques ◽

Coli Strain ◽

The Body ◽

Conjugation System ◽

E Coli ◽

Normal Microbiota ◽

Bowel Diseases ◽

Inflammatory Bowel ◽

Future Work

Inflammatory bowel diseases (IBD) is severe inflammation of the gastrointestinal tract. This can lead to a breakdown of mucosal barriers, causing dissemination of commensal bacteria throughout the body. To better understand bacterial translocation during IBD, aim to develop a fluorescent microbiota in mice that we can interrogate using live imaging techniques. Our preliminary experiments depleted commensals using broad-spectrum antibiotics, and replaced these microbiota with a fluorescent E. coli strain. The length of time that E.coli stays in the mice gut were monitored. We show that E. coli can persist in the ‘germ-free’ mouse gut for at least 21 days; control mice lose all added E. coli by 8-14 days. The establishment of the E. coli colony suggests this could be a reasonable model to study bacterial translocation. We are currently going to treat the colonized mice with DSS to induce colitis, and then to study translocation of E. coli by intravital microscopy. Considering E. coli is only a fraction of the normal microbiota and perhaps not a relevant model, future work aims at making a fluorescent microbiota consisting of multiple endogenous murine microbes. This will entail the use of a bacterial conjugation system capable of ubiquitously transforming many microbial species.

Download Full-text

Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli

Synthetic Biology ◽

10.1093/synbio/ysaa018 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Ryo Yoshida ◽

Hisashi Hemmi

Keyword(s):

Escherichia Coli ◽

Biosynthetic Pathway ◽

Membrane Lipids ◽

Extreme Environments ◽

Halophilic Archaea ◽

Coli Strain ◽

Glycerol Moiety ◽

Hyperthermophilic Archaeon ◽

E Coli ◽

Aeropyrum Pernix

Abstract Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.

Download Full-text