NorM, a putative efflux pump of Vibrio cholerae, is a member of the multidrug and toxic compound extrusion family of transporters. We demonstrate that NorM confers resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Inactivation of norM rendered V. cholerae hypersensitive towards these fluoroquinolones. Multiple sequence alignment of members of its family identified several regions of high sequence conservation. The topology of NorM was determined using ␤-lactamase and chloramphenicol acetyltransferase fusions. Multidrug efflux pumps extrude a variety of structurally unrelated drugs from cells (3,23,30), and pumps such as AcrAB have been associated with the intrinsic reduced susceptibility of organisms such as Proteus mirabilis to certain drugs (26,27,37). A family of multidrug efflux proteins has been identified which utilize the electrochemical potential of Na ϩ transport across membranes as the driving force (8,21,24,25,34) and which show sequence similarity. These transporters constitute the multidrug and toxic compound extrusion (MATE) family, which contains more than 30 proteins present in all three kingdoms (7), including NorM proteins from Vibrio parahaemolyticus (21), Neisseria gonorrhoeae, Neisseria meningitidis (27), and Brucella melitensis (4); YdhE, a NorM homologue in Escherichia coli (21); VmrA from V. parahaemolyticus (8); VcmB, VcmD, VcmH, VcmN, VcmA, and VcrM of non-O1 Vibrio cholerae (2, 24); PmpM of Pseudomonas aeruginosa (11); and BexA of Bacteroides thetaiotaomicron (20).Recently, quinolone resistance was reported for clinical isolates of V. cholerae from Calcutta, India (10). In our laboratory, we have demonstrated that an efflux pump-dependent mechanism imparts fluoroquinolone (FQ) resistance to clinical isolates of V. cholerae (1), making it important to understand the mechanism of action of FQ-specific pumps.Chromosome I of V. cholerae encodes a putative counterpart of NorM (12) that has a high level of sequence similarity (Fig. 1) to the NorM protein of V. parahaemolyticus. In this report, we demonstrate that disruption of the norM gene of V. cholerae confers sensitivity towards fluoroquinolones, making it likely that NorM is one of the FQ resistance-determining efflux pumps of this organism. Otsuka et al. (29) have only recently demonstrated that three conserved acidic residues in the predicted transmembrane region of NorM of V. parahaemolyticus are involved in the Na ϩ -dependent drug transport process. However, no detailed information is available on the topology of this transporter or the role of conserved residues in the periplasmic and cytoplasmic loops of NorM in FQ efflux. With this in view, we have carried out a detailed mutational and topological analysis of V. cholerae NorM, with the particular aim of identifying residues crucial for imparting FQ resistance. We focused on residues conserved in the MATE family of transporters, particularly acidic residues, and determined the effects of mutating these residues on the norfloxacin (NOR) sensitivity of the resulting ...

We conclude that the modified Hb in cigarette smokers may further enhance the oxidative insult within the cell.

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Linezolid, one of the reserve antibiotic of oxazolidinone class has wide range of antimicrobial activity. Here we have conducted a fundamental study concerning the dynamics of its interaction with bovine serum albumin (BSA), and the post binding modification of the later by employing different spectroscopic (absorption, fluorescence and circular dichroism (CD) spectroscopy) and molecular docking tools. Gradual quenching of the tryptophan (Trp) fluorescence upon addition of linezolid to BSA confirms their interaction. Analysis of fluorescence quenching at different temperature indicates that the interaction is made by static complex formation and the BSA has one binding site for the drug. The negative Gibbs energy change (ΔG), and positive values of enthalpy change (ΔH) and entropy change (ΔS) strongly suggest that it is an entropy driven spontaneous and endothermic reaction. The reaction involves hydrophobic pocket of the protein, which is further stabilized by hydrogen bonding and electrostatic interactions as evidenced from 8-anilino-1-napthalene sulfonic acid, sucrose and NaCl binding studies. These findings also support the molecular docking study using AutoDock 4.2. The influence of this interaction on the secondary structure of the protein is negligible as evidenced by CD spectroscopy. So, from these findings, we conclude that linezolid interacts with BSA in 1:1 ratio through hydrophobic, hydrogen bonding and ionic interactions, and this may not affect the secondary structure of the protein.