Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8-+Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8-->Pro and Val-10-*Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.

SummaryMany strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5Ј region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.

Spectroscopic analysis of membranes isolated from Mycobacterium smegmatis, along with analysis of its genome, indicates that the cytochrome c branch of its respiratory pathway consists of a modified bc 1 complex that contains two cytochromes c in its c 1 subunit, similar to other acid-fast bacteria, and an aa 3-type cytochrome c oxidase. A functional association of the cytochrome bcc and aa 3 complexes was indicated by the findings that levels of detergent sufficient to completely disrupt isolated membranes failed to inhibit quinol-driven O2 reduction, but known inhibitors of the bc 1 complex did inhibit quinol-driven O2 reduction. The gene for subunit II of the aa 3-type oxidase indicates the presence of additional charged residues in a predicted extramembrane domain, which could mediate an intercomplex association. However, high concentrations of monovalent salts had no effect on O2 reduction, suggesting that ionic interactions between extramembrane domains do not play the major role in stabilizing the bcc–aa 3 interaction. Divalent cations did inhibit electron transfer, likely by distorting the electron-transfer interface between cytochrome c 1 and subunit II. Soluble cytochrome c cannot donate electrons to the aa 3-type oxidase, even though key cytochrome c-binding residues are conserved, probably because the additional residues of subunit II prevent the binding of soluble cytochrome c. The results indicate that hydrophobic interactions are the primary forces maintaining the bcc–aa 3 interaction, but ionic interactions may assist in aligning the two complexes for efficient electron transfer.