Identification of the gate regions in the primary structure of the secretin pIV.

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.

In vitro characterization of a bacterial manganese uptake regulator of the fur superfamily.

Fur proteins generally act as negative transcriptional regulators by binding to target regulatory sequences (fur boxes) in the promoter regions of iron-responsive genes. Recently, Rhizobium leguminosarum was reported to contain a protein (Mur(Rl)) of Fur-like sequence, which, under manganese-replete conditions in its native background, repressed transcription of an ABC-type Mn(II) transporter by binding to two nonpalindromic mur boxes in its promoter region. Mur(Rl) displays apparently unusual regulatory flexibility in that it can also repress iron-responsive genes in Escherichia coli under iron-replete conditions. In this study, we quantify the affinities for binding a number of first-row transition-metal cations by Mur(Rl) and demonstrate that, in a fashion similar to E. coli Fur, Mur(Rl) binds Mn(II), Fe(II), Zn(II), and Co(II) with similar micromolar-order dissociation constants. In contrast to the vast majority of Fur proteins, however, Mur(Rl) lacks any high-affinity structural Zn(II) sites. Furthermore, we show that holoMur(Rl) binds as one and two homodimers to both mur and fur boxes in a concentration-dependent fashion in the presence of not only Mn(II) and Fe(II) but also Zn(II) and Co(II). We have developed an analytical method for determination of the individual dissociation constants and find that the DNA-binding affinities are essentially independent of the metal co-effector. These results complement those obtained in vivo by other authors and suggest that the Fur-like protein of R. leguminosarum, a competent ferric uptake regulator in E. coli, is insufficiently discriminating in its metal-binding characteristics to function as a regulator of iron homeostasis in its native background.