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Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5′ end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis , E. faecium , E. casseliflavus , and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards. [ABSTRACT FROM AUTHOR]

Nitrogenase is an oxygen‐vulnerable metalloenzyme that catalyzes nitrogen fixation. It largely remains unknown how nitrogenase coexists with oxygenic photosynthesis in nonheterocystous cyanobacteria, since there have been no appropriate model cyanobacteria so far. Here, we demonstrate in vivo transposon tagging in the nonheterocystous cyanobacterium Leptolyngbya boryana as a forward genetics approach. By conjugative transfer, a mini‐Tn5‐derived vector, pKUT‐Tn5‐Sm/Sp, was transferred from Escherichia coli to L. boryana cells. Of 1839 streptomycin‐resistant colonies, we isolated three mutants showing aberrant diazotrophic growth. Genome resequencing identified the insertion sites of the transposon in the mutants. This in vivo transposon tagging mutagenesis of L. boryana provides a promising system to investigate molecular mechanisms to resolve the Oxygen Paradox between nitrogen fixation and oxygenic photosynthesis in cyanobacteria. [ABSTRACT FROM AUTHOR]