The immunity proteins coded by the usp gene operon have

The immunity proteins coded by the usp gene operon have AZD6244 price a characteristic two-histidine region which appears to enable the inactivation of the Usp DNase activity [10]. However, Usp-encoding strains that do not have all three orfU immunity protein genes have been described. All three immunity proteins are thus not essential for the protection of the Usp producers, although Usp is lethal when it is expressed alone in E. coli. It has been postulated that none of the three proteins is exclusively required for Usp protein synthesis [6]. As protection of the Usp-producing bacterial

cell might be provided by a mechanism that is different from that of the colicins, we have investigated the E. coli Usp-associated immunity protein Imu3, previously designated

OrfU3. Our study indicates that Imu3 has protective non specific DNA-binding abilities that could have possible biotechnological potential. Results and discussion Isolation of Immunity protein 3 (Imu3) with Ni-NTA affinity chromatography provided protein fractions with appropriate purity; (Figure  1A). DNA binding ability was not affected by the presence or absence of the his-tag, as both precipitated linear DNA (Additional file 1: Figure S1). The theoretical and actual mass (11.497 kDa) of the purified Imu3 differed by 1.5 Da (measured by ESI + and Q-Tof; Waters-Micromass, United Kingdom, data not shown), indicating that Imu3 is not post-translationally modified. Parret and DeMot [5] previously Ergoloid described an approximately 45% sequence identity of the C-terminal region of the Usp protein with known nuclease colicins, such as colicins E7 and E9. Although it has been shown that colicin E7 and its immunity

protein form a high-affinity complex [11], we were not able to confirm the formation of a high affinity complex between Usp and any of the three smaller proteins encoded downstream of the usp gene (data not shown) which were previously proposed to protect the Usp-producing cell against its endonucleolytic activity [5]. Nevertheless, our results showed that Imu3 protects isolated DNA from digestion by the nuclease colicin E7, indicating a nonspecific protection mechanism that is distinct from that of the colicin immunity proteins (Figure  2). Figure 1 Purified Imu3 protein. (A) SDS PAGE gel of Imu3 isolated using Ni-NTA agarose affinity chromatography, M: PageRuler Prestained Protein Ladder (Fermentas). (B) Superimposed chromatograms of Imu3 protein monomers (darker line) (HPLC, size-exlusion) with absorption values at 280 nm normalised. LexA protein self-cleavage products were used as standards (lighter line). Figure 2 Imu3 protection against colicin E7 DNase activity.

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