scholarly journals Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

2010 ◽  
Vol 192 (24) ◽  
pp. 6418-6427 ◽  
Author(s):  
Lionel Ferrières ◽  
Gaëlle Hémery ◽  
Toan Nham ◽  
Anne-Marie Guérout ◽  
Didier Mazel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Random Mutagenesis ◽  
Broad Host Range ◽  
Donor Strain ◽  
Bacteriophage Mu ◽  
Genetic Determinants ◽  
Sensitive Species ◽  
E Coli ◽  
Original Construction

ABSTRACT Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.

scholarly journals LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7

2020 ◽  
Vol 94 (12) ◽  
Author(s):  
Peipei Chen ◽  
Huzhi Sun ◽  
Huiying Ren ◽  
Wenhua Liu ◽  
Guimei Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Inner Core ◽  
Phage Infection ◽  
Broad Host Range ◽  
Content Type ◽  
E Coli ◽  
Phage Adsorption ◽  
Specific Receptors ◽  
K 12

ABSTRACT Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions. IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

scholarly journals A broad-host-range event detector: expanding and quantifying performance between Escherichia coli and Pseudomonas species

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Nymul Khan ◽  
Enoch Yeung ◽  
Yuliya Farris ◽  
Sarah J Fansler ◽  
Hans C Bernstein
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Memory Device ◽  
Broad Host Range ◽  
Complex Environments ◽  
New Host ◽  
E Coli ◽  
Pseudomonas Fluorescens Sbw25 ◽  
The Common ◽  
New Applications

Abstract Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads—Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

scholarly journals Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

2018 ◽  
Author(s):  
Vanesa Amarelle ◽  
Ananda Sanches-Medeiros ◽  
Rafael Silva-Rocha ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Broad Host Range ◽  
Functional Metagenomics ◽  
Alternative Hosts ◽  
E Coli ◽  
Broad Host Range Plasmid

AbstractAs the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

scholarly journals Inhibition of Antimicrobial-Resistant Escherichia coli Using a Broad Host Range Phage Cocktail Targeting Various Bacterial Phylogenetic Groups

2021 ◽  
Vol 12 ◽  
Author(s):  
Jinshil Kim ◽  
Haejoon Park ◽  
Sangryeol Ryu ◽  
Byeonghwa Jeon
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Host Range ◽  
Microscopic Analysis ◽  
Broad Host Range ◽  
Phylogenetic Groups ◽  
Electron Microscopic ◽  
E Coli ◽  
Phage Cocktail ◽  
Transmission Electron Microscopic Analysis

Antimicrobial-resistant (AMR) commensal Escherichia coli is a major reservoir that disseminates antimicrobial resistance to humans through the consumption of contaminated foods, such as retail poultry products. This study aimed to control AMR E. coli on retail chicken using a broad host range phage cocktail. Five phages (JEP1, 4, 6, 7, and 8) were isolated and used to construct a phage cocktail after testing infectivity on 67 AMR E. coli strains isolated from retail chicken. Transmission electron microscopic analysis revealed that the five phages belong to the Myoviridae family. The phage genomes had various sizes ranging from 39 to 170 kb and did not possess any genes associated with antimicrobial resistance and virulence. Interestingly, each phage exhibited different levels of infection against AMR E. coli strains depending on the bacterial phylogenetic group. A phage cocktail consisting of the five phages was able to infect AMR E. coli in various phylogenetic groups and inhibited 91.0% (61/67) of AMR E. coli strains used in this study. Furthermore, the phage cocktail was effective in inhibiting E. coli on chicken at refrigeration temperatures. The treatment of artificially contaminated raw chicken skin with the phage cocktail rapidly reduced the viable counts of AMR E. coli by approximately 3 log units within 3 h, and the reduction was maintained throughout the experiment without developing resistance to phage infection. These results suggest that phages can be used as a biocontrol agent to inhibit AMR commensal E. coli on raw chicken.

Detection of Escherichia coli with Fluorescent Labeled Phages That Have a Broad Host Range to E. coli in Sewage Water

2008 ◽  
Vol 24 (2) ◽  
pp. 481-486 ◽  
Author(s):  
M. Namura ◽  
T. Hijikata ◽  
K. Miyanaga ◽  
Y. Tanji
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Sewage Water ◽  
Broad Host Range ◽  
E Coli

scholarly journals Diversity and Local Coadaptation of Escherichia coli and Coliphages From Small Ruminants

2020 ◽  
Vol 11 ◽  
Author(s):  
Felipe Molina ◽  
Alfredo Simancas ◽  
Rafael Tabla ◽  
Antonia Gómez ◽  
Isidro Roa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Raw Milk ◽  
Coliform Bacteria ◽  
Cell Protein ◽  
Broad Host Range ◽  
High Positive Correlation ◽  
E Coli ◽  
Raw Milk Cheese

Bacteriophages are highly specific predators that drive bacterial diversity through coevolution while striking tradeoffs among preserving host populations for long-term exploitation and increasing their virulence, structural stability, or host range. Escherichia coli and other coliform bacteria present in the microbiota of milk and during early ripening of raw milk cheeses have been linked to the production of gas, manifested by the appearance of eyes, and the development of off-flavors; thus, they might cause early blowing and cheese spoilage. Here, we report the characterization of coliphages isolated from manure from small ruminant farms and E. coli strains isolated from goat and sheep raw milk cheese. Additionally, the virulence and host range of locally isolated and laboratory collection phages were determined by comparing the susceptibility of E. coli strains from different sources. In agreement with the high genetic diversity found within the species E. coli, clustering analysis of whole-cell protein revealed a total of 13 distinct profiles but none of the raw milk cheese isolates showed inhibition of growth by reference or water-isolated coliphages. Conversely, 10 newly isolated phages had a broad host range (i.e., able to lyse ≥50% of bacterial hosts tested), thus exhibiting utility for biocontrol and only one cheese-isolated E. coli strain was resistant to all the phages. Whereas there was a high positive correlation between bacterial susceptibility range and lysis intensity, the phages virulence decreased as range increased until reaching a plateau. These results suggest local gene-for-gene coevolution between hosts and phages with selective tradeoffs for both resistance and competitive ability of the bacteria and host-range extension and virulence of the phage populations. Hence, different phage cocktail formulations might be required when devising long-term and short-term biocontrol strategies.

scholarly journals PROPERTIES OF A BACTERIOPHAGE DERIVED FROM ESCHERICHIA COLI K235

1963 ◽  
Vol 117 (2) ◽  
pp. 285-302 ◽  
Author(s):  
Margeris A. Jesaitis ◽  
John J. Hutton
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Host Bacterium ◽  
Temperate Bacteriophage ◽  
Genetic Determinants ◽  
E Coli ◽  
Immunological Specificity

A temperate bacteriophage was isolated from the colicinogenic strain of Escherichia coli K235 and characterized. This phage, termed PK, is related to P2 virus morphologically, serologically, and, possibly, genetically and it bears no relationship to the T-even phages. It was also demonstrated that PK virus and colicine K differ both in their host range and in their immunological specificity, and that PK prophage does not induce the colicinogenesis in its host bacterium. It was concluded that the formation of colicine K. and PK phage in E. coli K235 are controlled by different genetic determinants.

scholarly journals Complete Genome Sequence of Broad-Host-Range Shiga Toxin-Converting Bacteriophage SH2026Stx1, Isolated from Escherichia coli O157:H7

2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Mingrui Duan ◽  
Samuel S. Hunter ◽  
Scott A. Minnich ◽  
Matthew W. Fagnan ◽  
Daniel D. New ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Genome Sequence ◽  
Shiga Toxin ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Broad Host Range ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
E Coli

ABSTRACT The Shiga toxin-encoding phage SH2026Stx1 was isolated from Escherichia coli O157:H7 strain 2026. SH2026Stx1 and its detoxified derivative can infect a broad range of E. coli strains, including commensal, enteropathogenic, and enteroaggregative strains. We report here the complete genome sequence of phage SH2026Stx1 and its important features.

scholarly journals A broad-host-range event detector: expanding and quantifying performance across bacterial species

2018 ◽  
Author(s):  
Nymul Khan ◽  
Enoch Yeung ◽  
Yuliya Farris ◽  
Sarah J. Fansler ◽  
Hans C. Bernstein
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Bacterial Species ◽  
Memory Device ◽  
Bench Mark ◽  
Broad Host Range ◽  
Complex Environments ◽  
New Host ◽  
E Coli ◽  
New Applications

ABSTRACTModern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains ofEscherichia coli. It is clear that new applications – such as chemical sensing and event logging in complex environments – will benefit from new host chassis. This study quantitatively compared how a chemical event logger performed across multiple microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads –Pseudomonas fluorescensSBW25 andPseudomonas putidaDSM 291. Quantitative comparisons were made between these two non-traditional hosts and two bench-markEscherichia colichassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and down-stream recombinant region and via data-driven kinetic models. ThePseudomonadsproved to be acceptable chassis for the operation of this event logger, which outperformed the commonE. coliDH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

scholarly journals Potential Pathogenicity and Host Range of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates from Healthy Poultry

2011 ◽  
Vol 77 (16) ◽  
pp. 5830-5833 ◽  
Author(s):  
Valeria Bortolaia ◽  
Jesper Larsen ◽  
Peter Damborg ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Virulence Genes ◽  
Broad Host Range ◽  
Content Type ◽  
E Coli ◽  
Type Complex ◽  
Extended Spectrum ◽  
Zoonotic Risk ◽  
Potential Pathogenicity

ABSTRACTThirty of 33 epidemiologically unrelated extended-spectrum β-lactamase (ESBL)-producingEscherichia coliisolates from healthy poultry lacked the virulence genes commonly associated with human-pathogenic strains. The main zoonotic risk is associated with the broad host range of avianE. colibelonging to sequence type complex 10 and of IncN and IncI1 plasmids carryingblaCTX-MorblaSHV.

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