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Calcium Chloride Made E. coli Competent for Uptake of Extraneous DNA Through Overproduction of OmpC Protein

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Abstract

In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100 mM CaCl2. Analysis of the whole protein profile of CaCl2-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40 % lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl2-shock. From these results it can be suggested that in the process of CaCl2-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins.

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Abbreviations

E. coli :

Escherichia coli

MALDI-MS:

Matrix assisted laser desorption ionization-mass spectroscopy

OmpA/C:

Outer membrane protein A/C

Da/kDa:

Dalton/kilo Dalton

MOPS:

3-(N-morpholino)propanesulfonic acid

OD:

Optical density

SDS:

Sodium dodecyl sulphate

DTT:

Dithiothreitol

TOF:

Time of flight

GPS:

Global Protein Server

IEF:

Isoelectric focusing

Wt :

Wild type

μCi:

Micro Currie

ng:

Nano gram

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Acknowledgment

We acknowledge Coli Genetic Stock Centre, Yale University, USA for sending us the strains JW4103 and BW25113. We are grateful to Dr. J. H. Lakey of The University of Newcastle for his kind gift of the strains RAM 363 and RAM 105, Dr. Sekhar C. Mande of Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India for his generous gift of the plasmid pKY206, Dr. Rukhsana Chowdhury and Mr. Sandeep Chakraborty of IICB, Kolkata for analyzing samples in their proteomics facility. We are indebted to the Department of Science and Technology, Govt. of India for the financial assistance (Project No. SR/SO/BB-51/2006) and also for its ‘FIST Programme – 2001-2011’ & PURSE Programme – 2012-15, going on in our department/university to provide different instrumental and infrastructural support.

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  1. Pulakesh Aich

    Present address: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741 235, West Bengal, India

    Pulakesh Aich, Monobesh Patra, Arijit Kumar Chatterjee, Sourav Singha Roy & Tarakdas Basu

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  1. Pulakesh Aich
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  2. Monobesh Patra
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Correspondence to Tarakdas Basu.

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Aich, P., Patra, M., Chatterjee, A.K. et al. Calcium Chloride Made E. coli Competent for Uptake of Extraneous DNA Through Overproduction of OmpC Protein. Protein J 31, 366–373 (2012). https://doi.org/10.1007/s10930-012-9411-z

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