The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB 2 A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane ␣-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes.Multiple drug resistant bacteria poses a threat to man's fight against infectious diseases. Some multiple drug resistance systems may detoxify their substrates by transport across the complete cell wall of Gram-negative bacteria, across cytoplasmic membrane, periplasm, and outer membrane. These assumed transenvelope transporters are composed of a pump protein that energizes the transport, in addition to a membrane fusion and an outer membrane-associated protein (1, 2). The pump protein may be an ATP-binding cassette transporter (3, 4), a transporter of the major facilitator superfamily (5), or a resistance-nodulation-cell division (RND) 1 protein (4, 6, 7). The archetype of the RND permease superfamily family is CzcA from the Gram-negative bacterium Ralstonia sp. CH34 (formerly Alcaligenes eutrophus strain CH34) (8 -12).This bacterium contains at least seven heavy metal resistance determinants, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (163 kilobase pairs) and pMOL30 (238 kb) (8, 13-16). One of them, the czc-determinant of plasmid pMOL30, mediates inducible resistance to millimolar concentrations of Co 2ϩ , Zn 2ϩ , and Cd 2ϩ in strain CH34 (8, 17). The products of the genes czcA, czcB, and czcC form a membrane-bound protein complex catalyzing an energy-dependent efflux of these three metal cations (9, 11), probably across the complete envelope. The mechanism of action of CzcCB 2 A is that of a proton/cation antiporter, and the K m values of the efflux system for the substrate heavy metal cations are also in the millimolar range (10). Although indirect evidence led to the assumption that CzcA is the central cation/proton antiporter of the CzcCB 2 A complex (10), this has not been shown directly. This paper demonstrates that CzcA is a cation/proton antiporter, and develops the model of CzcA as a two-channel pump based on topology studies and the function of CzcA mutant proteins. This model sheds some light on other RND proteins involved in multiple drug resistance of bacteria or with previously unknown functions in mammals. EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Growt...

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The Drosophila Spätzle protein, involved in the embryonic development of the dorsal-ventral axis and in the adult immune response, is expressed as a proprotein and is activated by the serine proteinases Easter or Spätzle-processing enzyme. Proteolytic cleavage generates a 106-amino acid COOH-terminal fragment, C106, homologous to the mature form of nerve growth factor NGF, a cystine knot protein. Through alternative splicing, the Spätzle gene encodes for several isoforms that (with one exception, the "propeptide isoform") share C106 but differ in the prosequence. Three isoforms have been expressed recombinantly in Escherichia coli strains. The propeptide isoform could be expressed in soluble form and is unstructured according to CD and NMR measurements. Dimeric full-length Spätzle isoforms have been refolded from insoluble inclusion bodies and are able to rescue Spätzle-deficient embryos. Although the two full-length isoforms exhibit similar far-UV CD spectra, large differences in tryptophan fluorescence quenching by the respective pro-parts are observed. Both full-length isoforms exhibited highly cooperative folding transitions. Proteolytic digestion using trypsin resulted in C106, whose unfolding exhibits lower thermodynamic stability and cooperativity compared with the full-length proteins. The structure of C106 reveals a T-shaped dimer with significant differences to NGF and a deep internal cavity. Substantial ␤-sheet formation is observed between the two monomers, whereas a long loop containing the single tryptophan residue is disordered in the crystals. Our results suggest that the propeptides stabilize the tertiary structure of the "mature" Spätzle cystine knot.The Spätzle protein is the precursor of a nerve growth factorlike ligand in Drosophila melanogaster (1). It defines the dorsalventral axis in Drosophila embryos and acts in the initiation of immune response to fungal and bacterial infection in adult flies (2). Sequence homology to coagulogen and human nerve growth factor (NGF), 2 together with the spacing of cysteine residues, suggests a cystine knot motif, in which two disulfide bridges form a ring through which a third disulfide bridge is threaded (3).Extracellular binding of mature Spätzle to its receptor Toll is thought to lead to receptor dimerization and autophosphorylation of the cytoplasmic Toll/interleukin-1 receptor domains (4, 5). Although Toll is distributed uniformly within the embryonic perivittelline membrane, a concentration gradient of Spätzle results in dorsal-ventral asymmetry. The extracellular domain of the transmembrane receptor Toll consists of two leucine-rich repeat domains that bind the ligand. The cytoplasmic domain of Toll shares sequence similarity with the vertebrate Toll-like receptors, such as interleukin-1 receptor (6, 7), also involved in innate immunity.Activation of Spätzle proceeds via one of two extracellular proteolytic cascades. In the developmental pathway, Spätzle is activated by Easter, generating a 12-kDa COOH-terminal fragment, C106, that is capable of a...

Chromate resistance in Ralstonia metallidurans CH34 is based on chromate efflux catalyzed by ChrA efflux pumps. The bacterium harbors two chromate resistance determinants, the previously known chr(1) on plasmid pMOL28 (genes chrI, chrB(1), chrA(1), chrC, chrE, chrF(1)) and chr(2) on the chromosome (genes chrB(2), chrA(2), chrF(2)). Deletion of the genes chrI, chrC, chrA(2), chrB(2) and chrF(2) influenced chromate resistance and transcription from a chrBp(1) ::lacZ fusion. Deletion of the plasmid-encoded gene chrB(1) did not change chromate resistance or chrBp(1) regulation. Northern hybridization and primer-extension experiments were used to study transcription of the plasmid-encoded chr(1) determinant. Transcription of chrB(1), chrA(1) and chrC was induced by chromate. The presence of sulfate influenced transcription positively. The chrBp(1), chrAp(1) and chrCppromoters showed some similarity to heat-shock promoters. Transcription of the gene rpoH encoding a putative heat-shock sigma factor was also induced by chromate, but rpoH was not essential for chromate resistance. The ChrC protein was purified as a homotetramer and exerted superoxide dismutase activity. Thus, possible regulators for chromate resistance (ChrI, ChrB(1), ChrB(2), ChrF(1), and ChrF(2)) and an additional detoxification system (ChrC) were newly identified as parts of chromate resistance in R. metallidurans.

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