Bacilus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.Mammalian cells selected in culture for resistance to various lipophilic cytotoxic drugs often demonstrate resistance not only to the selective agent but also to a large group of apparently unrelated toxic compounds. This phenomenon, called multidrug resistance (MDR), is based on active efflux of drugs from the cells, performed by a membrane ATPase pump, P-glycoprotein (reviews in refs. 1 and 2). In MDR cells, the mdrl gene coding for this protein is frequently amplified (1). Overexpression of the P-glycoprotein gene is believed to be responsible for clinical drug resistance in many tumors.The most intriguing question in the area of MDR is the mechanism of extremely broad chemical specificity of P-glycoprotein. Its substrates have almost nothing in common, except that most of them bear positive electric charge and all of them are moderately hydrophobic. Some substances, such as reserpine and verapamil, are potent inhibitors of P-glycoprotein activity, apparently by a competition mechanism (3, 4). P-glycoprotein belongs to a large family of membrane ATPase pumps both of eukaryotic and of prokaryotic origin (2). It is, however, the only member of this family with a proven ability to transport multiple drugs. Even close homologues ofP-glycoprotein-the mammalian mdr2 gene product (5) and the yeast STE6 protein (6)-do not share this property. Here we describe MDR in Gram-positive bacteria and characterize the gene of a bacterial multidrug transporter.s MATERIALS AND METHODS Bacteria and Plasmids. Bacillus subtilis BD170 (trpC2, thr-S) and BD224 (trpC2, thr-S, recE4), Escherichia coli JM103, B. subtilis plasmids pCB20 (7) and pUB110, E. coli plasmids pUC19 and pBluescript KS(+) (Stratagene), and shuttle plasmid pMK3 (8) were used in this work.Selection of Resistant Bacteria and Sensitivity Assay. BD170 bacteria were selected with rhodamine 6G in the liquid antibiotic medium 3 (Difco) supplemented with 0.4% glucose.Bacteria were grown at 370C with the drug at 0.5, 1, 2, 3, and 4,g/ml, consecutively. The cultures were diluted 1:100 with fresh drug-containing medium every 1-2 days. The drug concentration was in...

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Proliferation of bacterial pathogens in blood represents one of the most dangerous stages of infection. Growth in blood serum depends on the ability of a pathogen to adjust metabolism to match the availability of nutrients. Although certain nutrients are scarce in blood and need to be de novo synthesized by proliferating bacteria, it is unclear which metabolic pathways are critical for bacterial growth in blood. In this study, we identified metabolic functions that are essential specifically for bacterial growth in the bloodstream. We used two principally different but complementing techniques to comprehensively identify genes that are required for the growth of Escherichia coli in human serum. A microarray-based and a dye-based mutant screening approach were independently used to screen a library of 3,985 single-gene deletion mutants in all non-essential genes of E. coli (Keio collection). A majority of the mutants identified consistently by both approaches carried a deletion of a gene involved in either the purine or pyrimidine nucleotide biosynthetic pathway and showed a 20- to 1,000-fold drop in viable cell counts as compared to wild-type E. coli after 24 h of growth in human serum. This suggests that the scarcity of nucleotide precursors, but not other nutrients, is the key limitation for bacterial growth in serum. Inactivation of nucleotide biosynthesis genes in another Gram-negative pathogen, Salmonella enterica, and in the Gram-positive pathogen Bacillus anthracis, prevented their growth in human serum. The growth of the mutants could be rescued by genetic complementation or by addition of appropriate nucleotide bases to human serum. Furthermore, the virulence of the B. anthracis purE mutant, defective in purine biosynthesis, was dramatically attenuated in a murine model of bacteremia. Our data indicate that de novo nucleotide biosynthesis represents the single most critical metabolic function for bacterial growth in blood and reveal the corresponding enzymes as putative antibiotic targets for the treatment of bloodstream infections.

Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins.

Bacterial populations contain persisters, cells which survive exposure to bactericidal antibiotics and other lethal factors. Persisters do not have a genetic resistance mechanism, and their means to tolerate killing remain unknown. In exponentially growing populations of Escherichia coli the frequency of persister formation usually is 10 ؊7 to 10 ؊5 . It has been shown that cells overexpressing either of the toxic proteins HipA and RelE, both members of the bacterial toxin-antitoxin (TA) modules, have the ability to form more persisters, suggesting a specific role for these toxins in the mechanism of persistence. However, here we show that cells expressing proteins that are unrelated to TA modules but which become toxic when ectopically expressed, chaperone DnaJ and protein PmrC of Salmonella enterica, also form 100-to 1,000-fold more persisters. Thus, persistence is linked not only to toxicity caused by expression of HipA or dedicated toxins but also to expression of other unrelated proteins.