Study of the Interaction between Bacteriophage T4 asiA andEscherichia coliς⁷⁰, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity toE. coliCells by Coexpression of a Truncated ς⁷⁰Fragment

Abstract

The interaction of T4 phage-encoded anti-sigma factor, asiA, andEscherichia coli ς70 was studied by using the yeast two-hybrid system. Truncation of ς70 to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased β-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated ς70 fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated ς70 and asiA could be expressed in large quantities and purified.

A single in vivo‐selected point mutation in the active center of Toxoplasma gondii ferredoxin‐NADP+ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

“…Along those lines, placing a negative charge at this position in TgFNR (S267E) does not lead to a repulsion of Fd in vivo, as might be expected (data not shown). Together with other reports [26–28], these data emphasize the advantage of the two‐hybrid approach in this context.…”

Isomerase-Independent Chaperone Function of Cyclophilin Ensures Aggregation Prevention of Adenosine Kinase Both in vitro and under in vivo Conditions

“…To investigate whether LdCyP or its N-terminal truncated version, like in vitro , also prevents aggregate formation under in vivo conditions, we designed vectors that simultaneously expressed both AdK and LdCyP or its truncated version in E. coli . Several workers in the past have used a similar coexpression strategy to study protein−protein interactions in vivo ( − ). However, the strategy that we adapted had several distinct advantages over others.…”

Dimerization properties of TraM, the antiactivator that modulates TraR‐mediated quorum‐dependent expression of the Ti plasmid tra genes

“…The antiactivator targets the C-terminal region including the helix-turn-helix domain of TraR, and inhibits the interaction between TraR and its cognate DNA recognition site Swiderska et al ., 2001). In this regard, TraM functions like other antiactivators including NifL of Klebsiella pneumonia (Barrett et al ., 2001) and MecA of Bacillus subtilis (Kong and Dubnau, 1994), as well as antisigma factors such as AsiA of bacteriophage T4 (Sharma et al ., 1999) and FlgM of Salmonella typhimurium (Chadsey and Hughes, 2001). The traM gene codes for an 11.2 kDa protein containing 102 amino acids, the C-terminal region of which contains a strongly hydrophobic domain.…”