The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis, which is characterized by rampant intraspecies recombination, rather than that of Rickettsia, where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A-and B-group strains that infect D. simulans. These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.horizontal transfer ͉ recombination ͉ ankyrin repeat gene ͉ genome evolution ͉ insect symbiosis Wolbachia pipientis are intracellular ␣-proteobacteria of the order Rickettsiales that infect insects as well as isopods, spiders, scorpions, mites, and filarial nematodes (1, 2). These bacteria represent a single species, with strains classified into supergroups, of which the most abundant are supergroups A and B.

Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6͞orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates. In S. acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.T he prokaryotes are divided into two main lineages, the Bacteria and Archaea domains (1). Strikingly, proteins involved in replication, transcription, translation, and recombination in archaea are closely related to the corresponding eukaryotic proteins, whereas the bacterial information machinery is considerably less similar (2).The mode of chromosome replication is a fundamental distinguishing feature between bacteria and eukaryotes. Whereas bacteria replicate their chromosome(s) bidirectionally from a single replication origin, eukaryotic chromosomes contain multiple start sites for DNA synthesis. Through the use of multiple origins, the chromosome replication stage (S phase) of a eukaryotic cell cycle may be similar in length to the corresponding stage (C period) of a bacterium containing a considerably smaller genome.Sulfolobus acidocaldarius and Sulfolobus solfataricus belong to the Crenarchaeota phylum within the Archaea domain. The organisms are hyperthermophilic acidophiles that display optimal growth at Ϸ80°C and pH 3 and were originally isolated from geothermal hot springs (3, 4). We have initiated studies of the cell cycle in the two species, with the main focus on chromosome replication, genome segregation, and cell division (5-9).We decided to investigate the chromosome replication characteristics of S. acidocaldarius and S. solfataricus by marker frequency (MF) analysis, by using whole-genome DNA microarrays developed in our laboratory. The MF technique is based on the fact that the copy number of a chromosomal DNA marker located close to a replication origin, on average, will be higher than that of a marker located near a terminus in a replicating cell population (see description of the principle of MF analysis in supporting information, which is published on the PNAS web site). The approach has been successfully used in combination with microarrays to study replication c...

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.