Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli

Abstract

The tonA gene codes for an outer membrane protein, a receptor of phage T5, the TonA protein. Strains harboring pLG513, a multicopy plasmid in which the tonA gene has been cloned, overproduced TonA protein, which appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell envelope proteins as a 78,000-molecular-weight protein. Identical results have been observed by Plastow et al. (FEBS Lett. 131:262-264, 1981) with plasmid pLC19-19, in which the tonA gene has also been cloned. The activity of the TonA protein, measured by its capacity to inactivate phage T5, increased by five- to sixfold in purified envelopes of cells harboring pLG513 compared with cells lacking the plasmid. Solubilization of the cytoplasmic membrane by Triton-Mg2+ treatment did not increase this activity. However, partial solubilization of outer membrane proteins by Triton-EDTA unmasked further T5 receptor activity, resulting in a final increase of around 50-fold, a value more consistent with the expected gene dosage effect. Treatment of whole cells by trypsin in conditions in which trypsin is allowed to enter the outer membrane revealed that part of the overproduced T5 receptors were embedded in the outer membrane and masked by a trypsin-sensitive protein. In addition, no T5 receptor activity was detected in either the periplasmic space or the cytoplasm. These results suggest that all of the overproduced TonA molecules were synthesized in an active form and integrated in the outer membrane, but only a small fraction could be reached or recognized by phage T5 in vivo.

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli

“…However, phage adsorption and vitamin B12 transport in whole cells were only amplified three to sevenfold. A similar apparent crypticity of phage receptors has been reported for the case of T5 binding to cells with the clonedfluA (tonA) gene (22). It remains to be seen whether this phenomenon reflects improper insertion of the receptors to the membrane or their interaction (e.g., aggregation) such that only a few receptors are active or exposed at the surface.…”

Studies on the gene for the multivalent vitamin B12 receptor of Escherichia coli

“…However, despite a 50-fold increase in the concentration of the btuB protein in vivo, and despite the fact that the majority of these molecules were apparently in the outer membrane, there is only about a four-fold increase in the number of functional vitamin B12 receptors when btuB is present on a multicopy vector. Similar observations have been made of other E. coli receptors (e.g., TonB [20]). Thus, although the outer membrane can accommodate a larger number of receptor molecules (with a resultant decrease in the levels of the major outer membrane proteins), only a small proportion of these molecules are active.…”

Molecular cloning of the ferrichrome-iron receptor of Escherichia coli K-12

“…For pBR322, the plasmid copy number has been shown to be 20 to 50 (14). Menichi and Buu (27) recently determined that in intact bacteria, only 3% of the TonA (=FhuA) protein which was made by an overproducing strain was available to act as receptor for phage T5. Phage binding activity could be unmasked by partial solubilization of outer membrane proteins by Triton-EDTA so as to aohieve a 50-fold increase in T5 receptor activity.…”