Role of tol genes in cloacin DF13 susceptibility of Escherichia coli K-12 strains expressing the cloacin DF13-aerobactin receptor IutA

Abstract

IutA is the outer membrane protein receptor for ferric aerobactin and the bacteriocin cloacin DF13. Although the same receptor is shared, ferric aerobactin transport across the outer membrane in Escherichia coli is TonB dependent, whereas cloacin DF13 transport is not. We have recently observed that tolQ is required for cloacin DF13 susceptibility (J. A. Thomas and M. A. Valvano, FEMS Microbiol. Lett. 91:107-112, 1992). In this study, we demonstrate that the genes tolQ, toiR, and tolA, but not tolB, toWC, and ompF, are required for the internalization of cloacin DF13 and they are not involved in the transport of ferric aerobactin.IutA is an iron-regulated outer membrane protein that serves as a receptor for the hydroxamate siderophore ferric aerobactin (6, 16). In addition, IutA binds the bacteriocin cloacin DF13 (16,34) and presumably the bacteriophage B74K (24). Cloacin DF13 is produced by strains of Enterobacter cloacae and secreted as a protein complex comprising a 56-kDa activity protein and a 10-kDa immunity protein (8). Upon interaction with IutA, the immunity protein is released to the medium while cloacin is translocated across the outer membrane (15). Upon entry into the cell cytoplasm, cloacin can block protein synthesis by causing cleavage of the 16S ribosomal RNA (9, 22).Although both ferric aerobactin and cloacin DF13 interact with IutA primarily at the cell surface (34), the translocation of ferric aerobactin across the outer membrane is TonB dependent, whereas that of cloacin DF13 is not (5, 25). TonB, an inner membrane protein with a large periplasmic domain, interacts with various outer membrane receptors and is essential for the transport of ferrisiderophores, certain bacteriophages, the antibiotic albomycin, and group B colicins such as colicins B, G, D, H, and V (see reference 23 for a review). In contrast, group A colicins (colicins A, El, E2, E3, K, L, N, and S4) and filamentous bacteriophages fl, M13, and fd are TonB independent but they require the products of tol genes for translocation across the outer membrane (see reference 35 for a review). The Tol import system consists of various genes such as tolQ, toiR, tolA, tolB, and toiC whose products are located in different cell compartments; i.e., the inner membrane, periplasmic space, and outer membrane (3,26,27,35 Strains and plasmids used are described in Table 1. Purification of plasmid DNA and transformations were carried out as described elsewhere (30). Outer membrane protein preparations and immunoblot experiments were conducted by following reported procedures (19,31,32). Cloacin DF13 was purified by column chromatography with CM-Sephadex as previously described (8, 18), and its 50% killing activity was determined on susceptible cells (E. coli P9OC cells carrying pJHCV8) grown in M9 minimal medium containing 0.1% bovine serum albumin (9, 15). Cloacin sensitivity was evaluated by a plate killing assay in which 0.5 x 108 microorganisms grown overnight in M9 minimal medium were mixed with molten soft agarose containing the iron chela...

Biological and immunological comparisons of and porins

“…By analogy to the receptor complex OmpF-BtuB-LPS described for colicin A in E. coli [11,20], we propose that in addition to IutA, porin F is also involved in the cloacin DF13 receptor step in E. cloacae. This study also explains the apparent discrepancy concerning the porin requirement during cloacin uptake [16,21]. The observation that cloacin DF13, just as colicin A, required porins F or OmpF in the direct test, but could also use porins D or OmpC as a substitute after bypass treatment, supported the evidence that the porins participated, with To1 proteins [21], in the translocation of bacteriocin.…”

“…This study also explains the apparent discrepancy concerning the porin requirement during cloacin uptake [16,21]. The observation that cloacin DF13, just as colicin A, required porins F or OmpF in the direct test, but could also use porins D or OmpC as a substitute after bypass treatment, supported the evidence that the porins participated, with To1 proteins [21], in the translocation of bacteriocin. Taking into account the strategic role of specific OmpF domains during the colicin N uptake [6], the complete colicin N insensitivity observed here probably reflects the absence of these specific OmpF sites in E. cloacae porins F and D.…”

Surface expression of O‐specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein

“…All tol mutants were sensitive to 0.2% (w/v) sodium deoxycholate (Levengood‐Freyermuth et al ., 1993). Sensitivity to cloacin DF13 and colicin N were used as indicators of TolA function, and the assays were conducted as described previously (Thomas and Valvano, 1992; 1993).…”

Identification of an Essential Cleavage Site in ColE7 Required for Import and Killing of Cells

“…6C, red box). The Tol-dependent DF13, which binds to the receptor IutA (24), also shares high sequence homology with the E-group colicins in this linker region, and DF13 also has a lysine aligned with Arg 447 in ColE7. This implies that the cleavage observed in ColE7 at Arg 447 may be common to other Tol-dependent enzymatic colicins.…”