scholarly journals ArdC protein overpasses the recipient hsdRMS restriction system broadening conjugation host range

2019 ◽  
Author(s):  
Lorena González-Montes ◽  
Irene del Campo ◽  
Fernando de la Cruz ◽  
Gabriel Moncalian
Keyword(s):  
Host Range ◽  
Recipient Cell ◽  
Broad Host Range ◽  
Rna Seq ◽  
Ssdna Binding ◽  
E Coli ◽  
Broad Host Range Plasmid ◽  
Donor Cells ◽  
Metalloprotease Activity ◽  
Restriction Modification

AbstractPlasmids, when transferred by conjugation, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida, but not from E. coli to E. coli. Surprisingly, expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. Thus, ArdC has antirestriction activity against HsdRMS system, and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and in the presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity is not needed for antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggers an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies.

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 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.

Transformation of Acidiphilium by electroporation and conjugation

1992 ◽  
Vol 38 (5) ◽  
pp. 387-393 ◽  
Author(s):  
Anne W. Glenn ◽  
Frank F. Roberto ◽  
Thomas E. Ward
Keyword(s):  
Host Range ◽  
Transformation Efficiency ◽  
Agar Medium ◽  
Recipient Cell ◽  
Coli Strain ◽  
Broad Host Range ◽  
E Coli ◽  
Acidophilic Bacteria ◽  
Nutrient Agar Medium ◽  
Broad Host Range Plasmids

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigaed. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 °C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 °C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10–15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 μg/mL. Transformation efficiencies in these experiments ranged up to 104 transformants/μg DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10−5–10−9 per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain. Key words: acidophilic bacteria, endogenous plasmids, broad-host-range plasmids.

Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control

1985 ◽  
pp. 173-188 ◽  
Author(s):  
Richard J. Meyer ◽  
Lung-Shen Lin ◽  
Kyunghoon Kim ◽  
Michael A. Brasch
Keyword(s):  
Host Range ◽  
Copy Number ◽  
Broad Host Range ◽  
Copy Number Control ◽  
Broad Host Range Plasmid

scholarly journals Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 27-36 ◽  
Author(s):  
A Greener ◽  
S M Lehman ◽  
D R Helinski
Keyword(s):  
Host Range ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Firefly Luciferase ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Broad Host Range Plasmid ◽  
Transcriptional Start Sites ◽  
Plasmid Rk2

Abstract A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

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