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.

Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa

Plasmid ◽  
1987 ◽  
Vol 17 (3) ◽  
pp. 222-232 ◽  
Author(s):  
Michael Pinkney ◽  
Bimal D.M. Theophilus ◽  
Simon R. Warne ◽  
William C.A. Tacon ◽  
Christopher M. Thomas
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Broad Host Range ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors

Gene ◽  
1983 ◽  
Vol 26 (2-3) ◽  
pp. 273-282 ◽  
Author(s):  
Miroslawa M. Bagdasarian ◽  
Egon Amann ◽  
Rudolf Lurz ◽  
Beate Rückert ◽  
Michael Bagdasarian
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Expression Vectors ◽  
Broad Host Range ◽  
Tac Promoter

scholarly journals Natural Transformation of Plasmid DNA in Escherichia coli in Sterilized Soil Column

1970 ◽  
Vol 25 (1) ◽  
pp. 49-52
Author(s):  
M Mahabub-Uz-Zaman ◽  
Zia Uddin Ahmed
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Natural Transformation ◽  
Soil Column ◽  
Limiting Factor ◽  
Broad Host Range ◽  
E Coli ◽  
Competent Cells ◽  
Broad Host Range Plasmid ◽  
Plasmid Puc18

The present study was carried out to assess transformability of natural and laboratory strains of Escherichia coli by plasmid DNA under different transformation conditions in sterilized soil column. Transformation experiments were carried out in laboratory conditions and in sterile soil columns with CaCl2-treated competent cells and non-competent cells at log phase and stationary phase of growth using the broad host range plasmid pUC18. In soil column experiments, transformants were obtained after CaCl2 induced competence in both E. coli K12 DH5α and strain BM09 in the frequency of 10-8 to 10-9. In natural transformation assays, transformants appeared only in log phase cells of strain DH5α at a lower frequency (5.0 x 10-9), and in CaCl2-competent BM09 cells, but not in fresh cells. Thus the major limiting factor for natural transformation in environmental E. coli in soil column is probably the absence of a competent state. The significance of this finding has been discussed with respect to generally observed lower antibiotic resistance in environmental E. coli isolates from aquatic sources. Keywords: Natural transformation; Plasmid DNA; Escherichia coli; Competent stateDOI: http://dx.doi.org/10.3329/bjm.v25i1.4856 Bangladesh J Microbiol, Volume 25, Number 1, June 2008, pp 49-52

Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida

2012 ◽  
Vol 161 (2) ◽  
pp. 71-79 ◽  
Author(s):  
Sonja Christina Troeschel ◽  
Stephan Thies ◽  
Olga Link ◽  
Catherine Isabell Real ◽  
Katja Knops ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Broad Host Range ◽  
Shuttle Vectors ◽  
Expression In Escherichia Coli

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 Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

1998 ◽  
Vol 180 (22) ◽  
pp. 6023-6030 ◽  
Author(s):  
Carla L. Easter ◽  
Helmut Schwab ◽  
Donald R. Helinski
Keyword(s):  
Host Range ◽  
Plasmid Stability ◽  
Broad Host Range ◽  
E Coli ◽  
Broad Host Range Plasmid ◽  
Resolution System ◽  
Stable Maintenance ◽  
Plasmid Stabilization ◽  
Plasmid Rk2

ABSTRACT The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons,parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, andparCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or bothparB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss ofparCBA activity. For P. aeruginosaPAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosaPAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parAand res alone were sufficient for stabilization. Thecer resolvase system of plasmid ColE1 and theloxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all threeE. coli strains. These observations indicate that theparA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of theparC and parB genes but that in at least oneE. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or thecer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.

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 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.

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