INTRODUCTION .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 DOMAIN ORGANIZATION: The Typical ABC Transporter . . . . . . . . . . . . . . . . . 73 THE TRANSMEMBRANE DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 The "Two-Times-Six" Helix Paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Sequence Similarities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 THE ATP-BINDING DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 PERIPLASMIC-BINDING PROTEINS ... . 84 SUBSTRATE SPECIFICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 THE ROLE OF ATP : Coupling Energy to Transport . . . . . . . . . . . . . . . . . . . " . . . . . 88 COVALENT MODIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 CELLULAR FUNCTIONS OF ABC TRANSPORTERS . . . . . . . . . . . . . . . . . . . . . . . 9 1 Nutrient Uptake . . . . . . . . . . . . ....... . 9 1 Protein Export ....... . .... . . . . . . . . . . . . ... . ......... . ...... . ... . .. 93 Intracellular Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Regulation of ABC Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Regulation by ABC Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Drug and Antibiotic Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Channel Functions: CFTR and P-glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 MECHANISMS OF SOLUTE TRANSLOCATION . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Structure of the Transmembrane Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Channels and Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 I Energy Coupling andlor Gating . 102 CONCLUDING REMARKS . 103

ABC Transporters: From Microorganisms to Man

Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.

Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

Some species of bacteria have low-permeability membrane barriers and are thereby "intrinsically" resistant to many antibiotics; they are selected out in the multitude of antibiotics present in the hospital environment and thus cause many hospital-acquired infections. Some strains of originally antibiotic-susceptible species may also acquire resistance through decreases in the permeability of membrane barriers. Another mechanism for preventing access of drugs to targets is the membrane-associated energy-driven efflux, which plays a major role in drug resistance, especially in combination with the permeation barrier. Recent results indicate the existence of bacterial efflux systems of extremely broad substrate specificity, in many ways reminiscent of the multidrug resistance pump of mammalian cells. One such system seems to play a major role in the intrinsic resistance of Pseudomonas aeruginosa, a common opportunistic pathogen. As the pharmaceutical industry succeeds in producing agents that can overcome specific mechanisms of bacterial resistance, less specific resistance mechanisms such as permeability barriers and multidrug active efflux may become increasingly significant in the clinical setting.

https://science.sciencemag.org/content/sci/264/5157/382.full.pdf

Prevention of drug access to bacterial targets: permeability barriers and active efflux

Antibiotics--compounds that are literally 'against life'--are typically antibacterial drugs, interfering with some structure or process that is essential to bacterial growth or survival without harm to the eukaryotic host harbouring the infecting bacteria. We live in an era when antibiotic resistance has spread at an alarming rate and when dire predictions concerning the lack of effective antibacterial drugs occur with increasing frequency. In this context it is apposite to ask a few simple questions about these life-saving molecules. What are antibiotics? Where do they come from? How do they work? Why do they stop being effective? How do we find new antibiotics? And can we slow down the development of antibiotic-resistant superbugs?

Molecular mechanisms that confer antibacterial drug resistance

The discovery, commercialization, and routine administration of antimicrobial compounds to treat infections revolutionized modern medicine and changed the therapeutic paradigm. Indeed, antibiotics have become one of the most important medical interventions needed for the development of complex medical approaches such as cutting-edge surgical procedures, solid organ transplantation, and management of patients with cancer, among others. Unfortunately, the marked increase in antimicrobial resistance among common bacterial pathogens is now threatening this therapeutic accomplishment, jeopardizing the successful outcomes of critically ill patients. In fact, the World Health Organization has named antibiotic resistance as one of the three most important public health threats of the 21st century (
1
).

Mechanisms of Antibiotic Resistance

Summary

A Staphylococcus epidermidis plasmid conferring inducible resistance to 14-membered ring macrolides and type B streptogramins has been analysed and the DNA sequence of the gene responsible for resistance determined. A single open reading frame of 1.464kbp, preceded by a complex control region containing a promoter and two ribosomal binding sites, was identified. The deduced sequence of the 488-amino-acid protein (MsrA) revealed the presence of two ATP-binding motifs homologous to those of a family of transport-related proteins from Gram-negative bacteria and eukaryotic cells, including the P-glycoprotein responsible for multidrug resistance. In MsrA, but not these other proteins, the two potential ATP-binding domains are separated by a Q-linker of exceptional length. Q-linkers comprise a class of flexible inter-domain fusion junctions that are typically rich in glutamine and other hydrophilic amino acids and have a characteristic spacing of hydrophobic amino acids, as found in the MsrA sequence. Unlike the other transport-related proteins, which act in concert with one or more hydrophobic membrane proteins, MsrA appears to function independently when cloned in a heterologous host (Staphylococcus aureus RN4220). MsrA might, therefore, interact with and confer antibiotic specificity upon other transmembrane efflux complexes of staphylococcal cells. The active efflux of [14C]-erythromycin from cells of S. aureus RN4220 containing msrA has been demonstrated.

Inducible erythromycin resistance in staphlyococci is encoded by a member of the ATP‐binding transport super‐gene family

The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII-ORF4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium-dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII-ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway-specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2005.04817.x

Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor

Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei.

Summary

The arginine-dependent repressor-activator from Bacillus subtilis, AhrC, has been overexpressed in Escherichia coli and purified to homogeneity. AhrC, expressed in E. coli, is able to repress a Bacillus promoter (argCp), which lies upstream of the argC gene. The purified protein is a hexamer with a subunit molecular mass of 16.7 kDa. Its ability to recognize DNA has been examined in vitro using argCp in both DNase I and hydroxyl radical protection assays. AhrC binds at two distinct sites within the argCp fragment. One site, argCO1, with the highest affinity for protein, is located within the 5′ promoter sequences, whilst the other, argCO2, is within the coding region of argC. The data are consistent with the binding of a single hexamer of AhrC to argCO1 via four of its subunits, possibly allowing the remaining two subunits to bind at argCO2in vivo forming a repression loop similar to those observed for the E coli Lac repressor.

Simon Baumberg

Papers

Inducible erythromycin resistance in staphlyococci is encoded by a member of the ATP‐binding transport super‐gene family

Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor

Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei.

Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis

Identification of a chromosomally encoded abc-transport system with which the staphylococcal erythromycin exporter msra may interact