Plasmid DNA Binds to the Core Oligosaccharide Domain of LPS Molecules of E. coli Cell Surface in the CaCl 2 -Mediated Transformation Process Subrata Panja, Pulakesh Aich, Bimal Jana, and Tarakdas Basu* Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741 235, West Bengal, India Received May 12, 2008; Revised Manuscript Received July 15, 2008 In the standard procedure for artificial transformation of E. coli by plasmid DNA, cellular competence for DNA uptake is developed by suspending the cells in ice-cold CaCl 2 (50-100 mM). It is believed that CaCl 2 helps DNA adsorption to the lipopolysaccharide (LPS) molecules on E. coli cell surface; however, the binding mechanism is mostly obscure. In this report, we present our findings of an in-depth study on in vitro interaction between plasmid DNA and E. coli LPS, using different techniques like absorption and circular dichroism spectroscopy, isothermal titration calorimetry, electron and atomic force microscopy, and so on. The results suggest that the Ca(II) ions, forming coordination complexes with the phosphates of DNA and LPS, facilitate the binding between them. The binding interaction appears to be cooperative, reversible, exothermic, and enthalpy-driven in nature. Binding of LPS causes a partial transition of DNA from B- to A-form. Finer study with the hydrolyzed products of LPS shows that only the core oligosaccharide domain of LPS is responsible for the interaction with DNA. Moreover, the biological significance of this interaction becomes evident from the observation that E. coli cells, from which the LPS have been leached out considerably, show higher efficiency of transformation, when transformed with plasmid-LPS complex rather than plasmid DNA alone. Introduction The outer leaflet of the outer membrane of E. coli predomi- nantly consists of certain proteins and a class of macroamphi- phile, the LPS, whereas the inner leaflet is composed of phos- pholipids and proteins. One bacterial cell contains approximately 3.5 × 10 6 LPS molecules occupying an area of 4.9 µm 2 . As the surface of an E. coli cell amounts to 6.7 µm 2 , it appears that three-quarters of bacterial surface consist of LPS, the remaining area being filled by proteins. 1 LPS has three co- valently linked domains: (1) the lipid A moiety, a glucosamine- based phospholipid (instead of the classical glycero-phospho- lipids of membrane), serves as the hydrophobic membrane anchor of LPS, (2) the core region, a nonrepeating oligosac- charide decorated with several phosphate-containing substitu- ents, and (3) the O-antigen, a distal repeating oligosaccharide. 2,3 This communication mainly deals with the in vitro interaction between E. coli LPS and plasmid DNA. The biological importance of this study lies at the step of DNA adsorption on cell surface in the artificial transformation process of Gram- negative bacteria like E. coli. In gene technology, transformation is an important basic technique, which involves binding of DNA to the cell surface followed by its uptake across the wall-mem- brane complex into the cytoplasm. E. coli cells can be made competent for DNA uptake by suspending cells in ice-cold CaCl 2 (50-100 mM) and then subjecting them to a brief heat shock at 42 °C for 90 s. 4,5 However, the exact mechanism of this widely used CaCl 2 -mediated artificial transformation pro- cedure is still unknown. It is just believed that the CaCl 2 helps DNA adsorption to the competent cell surface and the heat- shock step facilitates penetration of the adsorbed DNA into the cell cytosol. In the process of the investigation of the hidden mechanism of transformation, our previous findings suggest (1) the initial step of DNA adsorption on the cell surface, perhaps, involves the binding of DNA to the LPS molecules via the divalent cation Ca(II) 6-8 and (2) the brief heat shock step facilitates entry of adsorbed DNA into the cell cytoplasm (a) by releasing lipids and thereby forming pores on the outer membrane 9 and (b) by depolarizing the inner membrane and thereby decreasing the potential barrier for movement of negatively charged DNA molecules into cell interior. 10 The results of the present in-depth study with plasmid pUC19 DNA and E. coli LPS reveal that the calcium ions, forming coordina- tion complexes with the phosphates of DNA and LPS, facilitate DNA-LPS binding and the interaction is cooperative, reversible, exothermic and enthalpy-driven in nature; moreover, the “core oligosaccharide” (COS) part of LPS is the site, which binds with DNA. The biological significance of this in vitro interaction becomes evident from the fact that the E. coli cells, from which the LPS molecules are leached out, can be transformed more by DNA-LPS complex than by DNA alone. Materials and Methods Bacterial Strain and Plasmid. The E. coli XL1-Blue, 11 a genetically engineered highly transformable strain and the plasmid pUC19, 12 a cloning vector were used for this study. Fine Chemicals and Biochemicals. Different fine- and biochemicals, used in this study, were of molecular biology grade and were procured from different companies like Sigma-Aldrich (U.S.A.), Pharmacia- Amersham (Sweden), and Sisco Research Laboratories (India). Isolation and Purification of Plasmid DNA. Plasmid DNA was isolated from the transformed strain of E. coli (XL1-Blue + pUC19) by the method of alkaline lysis with SDS and purified by the method of polyethylene glycol. 13,14 The purity of plasmid DNA was checked by measuring its (absorbance) 260nm /(absorbance) 280nm ; the ratio attained the value of about 1.90 in our case. Moreover, the absence of any LPS in the purified DNA sample was checked by the KDO test [the colorimetric estimation of unique sugar 2-keto-3-deoxyoctulosonate * To whom correspondence should be addressed. Tel.: +91 (033) 25828750. Fax: +91 (033) 25828282. E-mail: tarakdb@yahoo.com. Biomacromolecules 2008, 9, 2501–2509 2501 10.1021/bm8005215 CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008