Foreign signal peptides can constitute a barrier to functional expression of periplasmic proteins in Haemophilus influenzae

Abstract

To study the periplasmic branch of iron (ferric ion) uptake systems in Gram-negative bacteria, genetic reconstitution experiments were initiated inHaemophilus influenzaeinvolving exchange of the periplasmic iron-binding protein. The expression of many of the heterologous periplasmic ferric-binding proteins (FbpAs) was quite limited. Transformation experiments with thefbpAgene fromNeisseria gonorrhoeaeyielded two colony sizes with different phenotypic characteristics. The small colonies contained the intactN. gonorrhoeae fbpAgene and were deficient in utilization of transferrin iron. The large colonies contained hybridH. influenzae/N. gonorrhoeae fbpAgenes, were proficient in transferrin iron utilization and had enhanced levels of expression of FbpA. These hybrid genes included several that encoded the matureN. gonorrhoeaeFbpA with theH. influenzaesignal peptide. To more fully evaluate the effect of foreign signal peptides, a series of hybrid genes were prepared that exchanged the signal peptides fromH. influenzaeFbpA,N. gonorrhoeaeFbpA and the TEM-1β-lactamase. The presence of theH. influenzaeleader was required for functional expression of FbpAs and was shown to dramatically increase the level ofβ-lactamase activity.