OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.

[1] Dehydration embrittlement of metamorphosed oceanic crust and mantle in the subducting slab may be responsible for the occurrence of intermediate-depth earthquakes. We explore the possibility that this hypothesis can explain the morphology of the double seismic zones observed in northeast Japan, southwest Japan, northeast Taiwan, northern Chile, Cape Mendocino, and eastern Aleutians. We calculate transient temperature structures of slabs based on geologically estimated subduction histories of these regions. We then determine dehydration loci of metamorphosed oceanic crust and serpentinized mantle using experimentally derived phase diagrams. The depth range of the dehydration loci of metamorphosed oceanic crust and serpentine is dependent on slab age. The dehydration loci of serpentine produce a double-layered structure. Because the upper dehydration loci of serpentine are mostly located in the wedge mantle above the slab, we regard the upper plane seismicity representing dehydration embrittlement in the oceanic crust, and we fix the slab geometry so that the upper plane seismicity is just below the upper surface of the slab. We find that the lower plane seismicity is located at the lower dehydration loci of serpentine, which indicates that the morphology of the double seismic zones is consistent with the dehydration embrittlement.INDEX TERMS: 7218 Seismology: Lithosphere and upper mantle; 7209 Seismology: Earthquake dynamics and mechanics; 7220 Seismology: Oceanic crust; KEYWORDS: double seismic zone, dehydration embrittlement, serpentine, oceanic crust, subducting plate Citation: Yamasaki, T., and T. Seno, Double seismic zone and dehydration embrittlement of the subducting slab,