The propagation of antibiotic resistance genes (ARGs) represents a global threat to both human health and food security. Assessment of ARG reservoirs and persistence is therefore critical for devising and evaluating strategies to mitigate ARG propagation. This study developed a novel, internal standard method to extract extracellular DNA (eDNA) and intracellular DNA (iDNA) from water and sediments, and applied it to determine the partitioning of ARGs in the Haihe River basin in China, which drains an area of intensive antibiotic use. The concentration of eDNA was higher than iDNA in sediment samples, likely due to the enhanced persistence of eDNA when associated with clay particles and organic matter. Concentrations of sul1, sul2, tetW, and tetT antibiotic resistance genes were significantly higher in sediment than in water, and were present at higher concentrations as eDNA than as iDNA in sediment. Whereas ARGs (frequently located on plasmid DNA) were detected for over 20 weeks, chromosomally encoded 16S rRNA genes were undetectable after 8 weeks, suggesting higher persistence of plasmid-borne ARGs in river sediment. Transformation of indigenous bacteria with added extracellular ARG (i.e., kanamycin resistance genes) was also observed. Therefore, this study shows that extracellular DNA in sediment is a major ARG reservoir that could facilitate antibiotic resistance propagation.

Yeast mhr1-1 was isolated as a defective mutation in mitochondrial DNA (mtDNA) recombination. About half of mhr1-1 cells lose mtDNA during growth at a higher temperature. Here, we show that mhr1-1 exhibits a defect in the partitioning of nascent mtDNA into buds and is a base-substitution mutation in MHR1 encoding a mitochondrial matrix protein. We found that the Mhr1 protein (Mhr1p) has activity to pair single-stranded DNA and homologous doublestranded DNA to form heteroduplex joints in vitro, and that mhr1-1 causes the loss of this activity, indicating its role in homologous mtDNA recombination. While the majority of the mtDNA in the mother cells consists of head-to-tail concatemers, more than half of the mtDNA in the buds exists as genome-sized monomers. The mhr1-1 Dcce1 double mutant cells do not maintain any mtDNA, indicating the strict dependence of mtDNA maintenance on recombination functions. These results suggest a mechanism for mtDNA inheritance similar to that operating in the replication and packaging of phage DNA.

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho–] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho–] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

Hypersuppressiveness, as observed inEukaryotic cells gain most of the energy required for cellular functions by oxidative respiration in mitochondria. These organelles contain hundreds to thousands of copies of a mitochondrial DNA (mtDNA) genome that encodes components essential for respiration and protein synthesis. Generally, mitochondrial alleles segregate during vegetative cell growth (vegetative segregation), and all copies of mtDNA within a cell or an individual generally have the same sequence (homoplasmy). In patients with mitochondrial myopathies, the progressive accumulation of mtDNA with large deletions leads to a heteroplasmic state in specific tissues (20; for review, see references 43 and 54). This phenomenon may be caused by the selective replication and/or segregation of mtDNA bearing the deletion; however, genetic analysis of mtDNA processes in mammals has been hampered by the strict maternal inheritance of mitochondria. The yeast Saccharomyces cerevisiae is a suitable model system because of its biparental mtDNA inheritance (14).The extremely biased inheritance of mtDNA with a large deletion is known in S. cerevisiae as "hypersuppressiveness. Two mechanisms for the initiation of mtDNA replication in yeast have been proposed: mitochondrial transcriptional RNA polymerase (Rpo41)-primed initiation and recombination-mediated initiation. Rpo41-primed DNA replication is similar to that observed in mammalian mitochondria. In support of this mechanism, each mtDNA replication origin shares sequence similarity with the heavy-strand replication origin of mammalian mtDNA, including the presence of a transcription promoter and three GC-rich clusters (2, 13). RNA synthesized by Rpo41 from mtDNA replication-origin promoters has been detected, and an endoribonuclease that cleaves the synthesized RNA at sites that correspond to regions of transition from RNA to DNA synthesis has been detected (3,49,53). The intact replication-origin promoter is required for hypersuppressiveness (36). However, this transcription-dependent process is not the sole mechanism for the initiation of mtDNA replication, since both the replication origin and RPO41 are dispensable for the maintenance of [rho Ϫ ] mtDNA (18,53). In addition, it has been reported that the selective inheritance of hypersuppressive [rho Ϫ ] mtDNA relative to nonhypersuppres-* Corresponding author. Mailing address: