Cytochrome bd biosynthesis in Escherichia coli: the sequences of the cydC and cydD genes suggest that they encode the components of an ABC membrane transporter

Abstract

At least four genes are known to affect formation of the cytochrome bd-type terminal oxidase of Escherichia coli. In addition to the genes (cydA and cydB) encoding the two constituent subunits of this complex, a further two genes (cydC and cydD) map near 19 min on the E. coli chromosome. We report here the cloning of both genes on a 5.3 kb ClaI-HindIII restriction fragment, which, when used to transform either a cydC or cydD mutant, restored the ability of these mutants to grow on a selective medium containing azide and zinc ions and also restored the spectral signals associated with the cytochrome components of the oxidase complex. A subcloned 1.8 kb DdeI fragment similarly restored growth and cytochrome content of a cydD mutant, but not a cydC mutant. The complete nucleotide sequence of the ClaI-HindIII fragment reveals three open reading frames, one being trxB (19.3 min on the E. coli chromosome map, encoding thioredoxin reductase), confirming the mapping position of cydD previously established by P1-mediated transduction. Two ORFs identified by complementation experiments as cydD and cydC encode proteins with predicted molecular masses, respectively, of 65,103 and 62,946 Da. The hydropathy profile of each protein reveals an N-terminal hydrophobic domain and a C-terminal hydrophilic domain containing a putative nucleotide-binding site. The gene products probably constitute an ABC (ATP-binding cassette) family membrane transporter, the function of which is necessary for the formation of the cytochrome bd quinol oxidase. The CydDC system appears to be the first prokaryotic example of a heterodimeric ABC transport system in which each polypeptide contains both hydrophobic and ATP-binding domains.

The Escherichia coli ATP‐binding cassette (ABC) proteins

“…This family may also include YojI, which has a similar domain organization and a TMD closely related to the TMDs of the other members of the family, although the ABC domain of YojI is either of different origin or has diverged faster than the other members of the subfamily. No clear function for any of these proteins has been defined, although CydCD is required for biogenesis of cytochrome oxidase b (Poole et al, 1993). However, members of this family are the E. coli proteins most closely related to the mammalian multidrug resistance P-glycoprotein and the multidrug resistance protein of Lactococcus lactis, LmrA (van Veen et al, 1998).…”

A Bacterial Glutathione Transporter (Escherichia coli CydDC) Exports Reductant to the Periplasm

“…First, mutants defective in cydC or cydD encoding the polypeptides of the heterodimer, exhibit several defects of the periplasm, namely a hyperoxidized redox state and failure to assemble periplasmic cytochromes b and c (40,41). Second, cydDC mutants fail to assemble the cytochrome bd-type oxidase complex (25,41); in such mutants, the CydA and CydB subunits are detectable but the hemes are not incorporated into the subunits (42). Interestingly, both the hemes and the oxygen-reactive site (43) are thought to be close to the periplasmic side of the cytoplasmic membrane.…”

“…DTT was therefore first removed by solvent extraction with ethyl acetate, and the GSH was then oxidized to GSSG by exposure to air for 24 h. To determine whether the oxidation was complete, we employed DTNB, which undergoes oxidation in the presence of sulfhydryl groups. The transport assay conditions used to determine [ 35 (25) and periplasmic cytochromes c (41). Because GSH is a substrate of the CydDC transporter, it may be directly required in cytochrome bd assembly.…”

“…Recently, we demonstrated that an ABC-type transporter, CydDC, originally identified by its requirement for assembly of the cytochrome bd-type terminal oxidase of E. coli (23)(24)(25), is a transporter of cysteine outwards across the cytoplasmic membrane (26). Given the established roles of GSH in redox buffering in eukaryotic cells (7), we hypothesized that GSH is also a substrate of CydDC.…”

Characterization of CAF4 and CAF16 Reveals a Functional Connection between the CCR4-NOT Complex and a Subset of SRB Proteins of the RNA Polymerase II Holoenzyme

“…Deletion analysis indicated that an intact leucine-rich repeat (residues 345-470) of CCR4 was required for its interaction with both CAF4 and CAF16 (Table II, to encode a novel protein (yeast protein YKR036c) containing seven WD40 repeats in its C terminus (residues 320 -659). CAF16 when sequenced in its entirety was found to encode a protein (now designated YFL028c) that shares significant homology to the ABC ATPase family of proteins (31,32). ABC ATPases are principally found to play roles in transport across membranes and as membrane receptors (33).…”