The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.

The Na + -translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrixassisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide. ß

Na + -translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS^PAGE of the purified complex, NqrB and NqrC subunits were found to give yellowĝ reen fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. NTerminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress. z 2000 Federation of European Biochemical Societies.

K+-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake. A 41 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E. coli trkH (V. alginolyticus trkH) and a new gene, o d l , whose function is not clear. Products of V, alginolyticus trkA and od9 were detected in E. coli minicells. tr&A and trkH from V. alginolyticus restored growth at low K+ concentrations of an E. coli AtrkA and an E. coli AtrkG AtrkH strain, respectively, suggesting that these V , alginolyticus genes can functionally replace their E. coli counterparts. In addition, a plasmid containing V. alginolyticus trkAH permitted growth of an E. coli AsapABCDF (AtrkE) strain at low K+ concentrations. This effect was mainly due to V. alginolyticus trkH and was enhanced by trkA from this organism. Measurements of net K+-uptake rates indicated that the presence of these genes in E. coli renders the Trk systems independent of products from the E. coli sapABCDF (trkE) operon.

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