BackgroundThe genome of Helicobacter pylori, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian H. pylori genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains.ResultsA phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including oipA, hopMN, babABC, sabAB and vacA-2) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (tipα), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (miaA, tilS), a DNA recombinase/exonuclease that recognizes genome identity (addA), and DNA/RNA hybrid nucleases (rnhAB). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of cagA, vacA, homC (outer membrane protein), sotB (sugar transport), and a translation fidelity factor (miaA). Large divergence was seen in genes related to antibiotics: frxA (metronidazole resistance), def (peptide deformylase, drug target), and ftsA (actin-like, drug target).ConclusionsThese results demonstrate dramatic genome evolution within a species, especially in likely host interaction genes. The East Asian strains appear to differ greatly from the European strains in electron transfer and redox reactions. These findings also suggest a model of adaptive evolution through proteome diversification and selection through modulation of translational fidelity. The results define H. pylori East Asian lineages and provide essential information for understanding their pathogenesis and designing drugs and therapies that target them.

The RecF pathway of Escherichia coli is important for recombinational repair of DNA breaks and gaps. Here we reconstitute in vitro a seven-protein reaction that recapitulates early steps of dsDNA break repair using purified RecA, RecF, RecO, RecR, RecQ, RecJ, and SSB proteins, components of the RecF system. Their combined action results in processing of linear dsDNA and its homologous pairing with supercoiled DNA. RecA, RecO, RecR, and RecJ are essential for joint molecule formation, whereas SSB and RecF are stimulatory. This reconstituted system reveals an unexpected essential function for RecJ exonuclease: the capability to resect duplex DNA. RecQ helicase stimulates this processing, but also disrupts joint molecules. RecO and RecR have two indispensable functions: They mediate exchange of RecA for SSB to form the RecA nucleoprotein filament, and act with RecF to load RecA onto the SSB-ssDNA complex at processed ssDNA-dsDNA junctions. The RecF pathway has many parallels with recombinational repair in eukaryotes.[Keywords: DSB repair; Rad52 group; RecA; RecF; RecQ; recombination] Supplemental material is available at http://www.genesdev.org.

In Escherichia coli, chi (5'-GCTGGTGG-3') is a recombination hotspot recognized by the RecBCD enzyme. Recognition of chi reduces both nuclease activity and translocation speed of RecBCD and activates RecA-loading ability. RecBCD has two motor subunits, RecB and RecD, which act simultaneously but independently. A longstanding hypothesis to explain the changes elicited by chi interaction has been "ejection" of the RecD motor from the holoenzyme at chi. To test this proposal, we visualized individual RecBCD molecules labeled via RecD with a fluorescent nanoparticle. We could directly see these labeled, single molecules of RecBCD moving at up to 1835 bp/s (approximately 0.6 microm/s). Those enzymes translocated to chi, paused, and continued at reduced velocity, without loss of RecD. We conclude that chi interaction induces a conformational change, resulting from binding of chi to RecC, and not from RecD ejection. This change is responsible for alteration of RecBCD function that persists for the duration of DNA translocation.

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/ Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.A type II restriction enzyme, such as R.EcoRI, will make a double-stranded break at a specific sequence on DNA (49). A cognate modification enzyme (M.EcoRI) can methylate the same sequence and protect it from restriction cleavage. The genes involved are tightly linked and form a type II restrictionmodification (RM) gene complex. Type II RM gene complexes will attack unmodified foreign DNA such as bacteriophage DNA but not the modified DNA of the cells where they reside. They have been considered to function as bacterial tools against invasion by foreign DNA.We found that elimination of type II RM gene complexes from bacterial cells by a competing genetic element inhibits cell growth (43,44). Our experiments suggested the following course of events after a cell has lost a type II RM gene complex. In the descendants of the cell that have lost the type II RM gene complex, the number of molecules of the modification enzyme will decrease with each cell division. Eventually, the capacity of the enzyme to modify the many sites needed to protect the newly replicated chromosomes from the remaining pool of restriction enzyme will become inadequate. Chromosomal DNA will then be cleaved at the unmodified sites, and the cells will be killed. This is reminiscent of postsegregational cell killing mechanisms, which have been shown to contribute to the stable maintenance of plasmids (11,12,21). Indeed, linkage of several type II RM gene complexes stabilizes plasmids (29,32,43...