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.

scholarly journals Plasmid Evolution and Interaction between the Plasmid Addiction Stability Systems of Two Related Broad-Host-Range IncQ-Like Plasmids

2004 ◽  
Vol 186 (7) ◽  
pp. 2123-2133 ◽  
Author(s):  
Shelly M. Deane ◽  
Douglas E. Rawlings
Keyword(s):  
Host Range ◽  
Plasmid Stability ◽  
Amino Acid Identity ◽  
The Other ◽  
Host Cells ◽  
Broad Host Range ◽  
Plasmid Curing ◽  
Plasmid Evolution ◽  
E Coli ◽  
Broad Host Range Plasmids

ABSTRACT Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmids are likely to encounter each other. We investigated the relative efficiencies of the two stability systems and whether they had evolved apart sufficiently for each pas to stabilize a plasmid in the presence of the other. The three-component pTF-FC2 pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells (±92% and ±60% after 100 generations, respectively). The PasA antidote of each pas was unable to neutralize the PasB toxin of the other plasmid. The pas proteins of each plasmid autoregulated their own expression as well as that of the pas of the other plasmid. The pas of pTF-FC2 was more effective at repressing the pas operon of pTC-F14 than the pas of pTC-F14 was able to repress itself or the pas of pTF-FC2. This increased efficiency was not due to the PasC of pTF-FC2. The effect of this stronger repression was that pTF-FC2 displaced pTC-F14 when the two plasmids were coresident in the same E. coli host cell. Plasmid curing resulted in the arrest of cell growth but did not cause cell death, and plasmid stability was not influenced by the E. coli mazEF genes.

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 Evolution of IncA/CblaCMY-2-Carrying Plasmids by Acquisition of theblaNDM-1Carbapenemase Gene

2011 ◽  
Vol 56 (2) ◽  
pp. 783-786 ◽  
Author(s):  
Alessandra Carattoli ◽  
Laura Villa ◽  
Laurent Poirel ◽  
Rémy A. Bonnin ◽  
Patrice Nordmann
Keyword(s):  
Host Range ◽  
Large Scale ◽  
The United States ◽  
Broad Host Range ◽  
New Delhi ◽  
Yersinia Ruckeri ◽  
Content Type ◽  
Photobacterium Damselae ◽  
Environmental Bacteria ◽  
Broad Host Range Plasmids

ABSTRACTTheblaNDM-1gene has been reported to be often located on broad-host-range plasmids of the IncA/C type in clinical but also environmental bacteria recovered from the New Delhi, India, area. IncA/C-type plasmids are the main vehicles for the spread of the cephalosporinase geneblaCMY-2, frequently identified in the United States, Canada, and Europe. In this study, we completed the sequence of IncA/C plasmid pNDM-KN carrying theblaNDM-1gene, recovered from aKlebsiella pneumoniaeisolate from Kenya. This sequence was compared with those of three IncA/C-type reference plasmids fromEscherichia coli,Yersinia ruckeri, andPhotobacterium damselae. Comparative analysis showed that theblaNDM-1gene was located on a widely diffused plasmid scaffold known to be responsible for the spread ofblaCMY-2-like genes and consequently for resistance to broad-spectrum cephalosporins. Considering that IncA/C plasmids possess a broad host range, this scaffold might support a large-scale diffusion of theblaNDM-1gene among Gram-negative rods.

Transformation of Brucella Species with Suicide and Broad Host-Range Plasmids

2003 ◽  
pp. 143-148 ◽  
Author(s):  
John R. McQuiston ◽  
Gerhardt G. Schurig ◽  
Nammalwar Sriranganathan ◽  
Stephen M. Boyle
Keyword(s):  
Host Range ◽  
Broad Host Range ◽  
Brucella Species ◽  
Broad Host Range Plasmids

Plasmids captured in C. metallidurans CH34: defining the PromA family of broad-host-range plasmids

2009 ◽  
Vol 96 (2) ◽  
pp. 193-204 ◽  
Author(s):  
Géraldine A. Van der Auwera ◽  
Jaroslaw E. Król ◽  
Haruo Suzuki ◽  
Brian Foster ◽  
Rob Van Houdt ◽  
...  
Keyword(s):  
Host Range ◽  
Broad Host Range ◽  
Broad Host Range Plasmids
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