Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.     

Distinct roles of peroxynitrite and hydroxyl radical in triggering stunned myocardium-like impairment of cardiac myocytes in vitro

Myocardial stunning is characterized by the impairment of excitation-contraction coupling via a decrease in myofilament Ca²⁺ responsiveness, thought to be triggered by hydroxyl radicals (·OH) generated upon reperfusion. Since peroxynitrite is also expected to be produced during reperfusion, we examined whether it can induce a stunned myocardium-like impairment of cardiac myocytes. Its effect on cultured cardiac myocytes was compared with that of hydrogen peroxide (H₂O₂), ·OH source. Infusion of peroxynitrite (0.2 mM) induced a decrease in cell motion and a complete arrest in diastole at 2.9 ± 0.3 min, which coincided with an elevation in [Ca²⁺]_i. Arrest induced by infusion of H²O² (10 mM) was not associated with an increase in [Ca²⁺]_i. The ATP content was unaffected by peroxynitrite (control, 34.3 ± 3.4: + peroxynitrite, 32.9 ± 3.5 nmol/mg protein) and the cells remained viable. Sulfhydryl (SH) content was decreased by peroxynitrite, but not by H₂O₂. The membrane fluidity (a measure of peroxidation of the membrane lipids) was not affected by peroxynitrite, but was decreased by H₂O₂. Onset time of arrest was unaffected by deferoxamine (0.2 mM), but was delayed by DTT (10 mM) (from 2.9 ± 0.3 to 19.2 ± 1.6 min). Nitrotyrosine content was unchanged by peroxynitrite, and its augmentation with Fe³⁺/EDTA (1 mM) was not associated with a shortened onset time of arrest. The function of the Na⁺/Ca²⁺ exchanger was impaired by peroxynitrite, but not by H₂O₂. Peroxynitrite and H₂O₂ each induce arrest, but only the former increases [Ca²⁺]_i. One of the mechanisms of the increase in [Ca²⁺]_i is Na⁺/Ca²⁺ exchanger dysfunction. The impairments were induced through SH oxidation by peroxynitrite, but through lipid peroxidation by H₂O₂. Myocardial stunning may be induced by both species in concert.     

High-temperature-induced transposition of insertion elements in burkholderia multivorans ATCC 17616

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42 degrees C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.