58 Upstream of cusCFBA is a promoter region with high sequence similarity to other known copper and silver resistance genes in Gram-negative bacteria. Transcribed in the opposite direction, cusR and cusS encode a two component regulatory system. The cusA gene is preceded by cusB, encoding the MFP, cusF, encoding the small periplasmic metallochaperone, and cusC, encoding the OMF protein. coli CusA was later found to be a putative monovalent cation metal transporter, 19, 54 which is closely related to SilA of Salmonella typhimurium, 55- 57 The chromosomally encoded cus locus consists of two back-to-back operons, cusCFBA and cusRS. The first HME-RND protein identified was the cadmium and zinc specified CzcA divalent cation efflux pump. While the CusCFBA efflux system has been found to have high specificity for Cu + and Ag +, in contrast to the broad specificity of HAE-RND proteins, both HAE- and HME-RND family pumps are believed to be key components of antimicrobial resistance in Gram-negative pathogens. In addition, the proposed transport mechanisms of the pump will be discussed. This review will focus on the known structural information for the CusCFBA efflux system, much of which has been solved in our lab. Among all RND family efflux complexes, CusCFBA is one of only three with structural information for all components, including MexAB-OprM 43- 46 and AcrAB-TolC. The inner membrane copper and silver ion transporter CusA operates in conjunction with the MFP CusB, 38 OMF CusC, 39, 40 and periplasmic metallochaperone protein CusF 41, 42 to form one of only two known tetrapartite efflux systems. 19, 32, 33 The heavy metal efflux RND (HME-RND) subfamily 10, 34 contains only CusA. The hydrophobic and amphiphilic efflux RND (HAE-RND) protein subfamily 10 includes the multidrug efflux pumps AcrB, 15- 25 AcrD, 19, 26 AcrF, 19, 27, 28 MdtB, 19, 29- 31 MdtC, 19, 29- 31 and YhiV. coli, there are seven known RND proteins that can be divided into two subfamilies. They are exemplified by the RND superfamily, members of which are essential to the multidrug resistance observed in many pathogens. 13 Composed of an inner membrane transporter, a periplasmic membrane fusion protein (MFP), and an outer membrane factor (OMF) channel protein, tripartite efflux pumps represent the most powerful antibiotic efflux machinery available to bacteria. 12 These organisms are capable of coordinating multiprotein structures that bridge both the inner and outer membranes to expel toxic compounds directly from the cell. pneumoniae, Pseudomonas aeruginosa, Streptomyces, Salmonella, and Legionella spp., have been found to contain transporters of all five families. Gram-negative bacteria, including pathogenic E. 6 (i) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily 7 (TC 3.A.1) (ii) the major facilitator superfamily (MFS) 8 (TC 2.A.1) (iii) the small multidrug resistance (SMR) family 9 (TC 2.A.7) (iv) the resistance-nodulation-cell division (RND) superfamily 10 (TC 2.A.6) and (v) the multidrug and toxic compound extrusion (MATE) family 11 (TC 2.A.66). 4 Based on sequence similarity, function, and energy source these pumps can be divided into five families: 5. 2, 3 Antibiotic resistance systems of this type have been found in every bacterial species tested. One of the major means by which bacteria exhibit antibiotic resistance is the expression of multidrug efflux pumps, which detoxify diverse antimicrobials by directly exporting them from the cell. 1 The complete failure of both gonorrhea and tuberculosis treatments, due to resistance to all available drugs, is already a reality. Recently, the World Health Organization reported that more than 50% of infections by the common pathogens Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were resistant to routine antibacterial drugs, in many settings. However, it is widely accepted that the increased use of these drugs has resulted in bacteria with developed resistance to such treatments, representing a major public health crisis. Antibiotics have been used to treat infections for over a century.