Microbiomes are vast communities of microbes and viruses that populate all natural ecosystems. Viruses have been considered the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared to other environments. Here we investigate the origin, evolution, and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboratory, we obtained DNA sequences of crAssphage from over one-third of the world's countries, and showed that its phylogeography is locally clustered within countries, cities, and individuals. We also found colinear crAssphage-like genomes in both Old-World and New-World primates, challenging genomic mosaicism and suggesting that the association of crAssphage with primates may be millions of years old. We conclude that crAssphage is a benign globetrotter virus that may have co-evolved with the human lineage and an integral part of the normal human gut virome.

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67 phox and fulllength Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47 phox . We subjected chimera p67 phox -(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67 phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67 phox and Rac1, in the p67 phox -(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67 phox . This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67 phox to the membrane; the other is to induce an "activating" conformational change in p67 phox . . production, is probably the consequence of a conformational change in gp91 phox , caused by the interaction of cytochrome b 559 with one or several of the cytosolic oxidase components (8). Establishing contact with cytochrome b 559 requires the translocation to the plasma membrane of p47 phox , p67 phox , and Rac, a process known as oxidase assembly (reviewed in Refs. 9 and 10). Stimulus-elicited activation of the oxidase in intact cells can be mimicked in a cell-free system, in which phagocyte membranes or purified and relipidated cytochrome b 559 are mixed with the cytosolic components p47 phox , p67 phox , and Rac, in the presence of an anionic amphiphile, serving as an activator (11-15). The amphiphile acts by causing a conformational change in p47 phox (16) and, possibly, also by the induction of structural changes in cytochrome b 559 (17).The impairment of O 2 . production in the p47 phox -deficient form of chronic granulomatous disease shows that p47 phox is * This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 428/01, the Roberts-Guthman Chair in Immunopharmacology (to E. P.), and National Institutes of Health Grant GM46372 (to C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefor...

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA conferring sensitivity in a dominant fashion to two antibiotics, streptomycin and nalidixic acid, respectively. Unique selective pressure is generated to enrich for bacteria that harbor the phages carrying the sensitizing constructs. This selection pressure is based on a toxic compound, tellurite, and therefore does not forfeit any antibiotic for the sensitization procedure. We further demonstrate a possible way of reducing undesirable recombination events by synthesizing dominant sensitive genes with major barriers to homologous recombination. Such synthesis does not significantly reduce the gene's sensitization ability. Unlike conventional bacteriophage therapy, the system does not rely on the phage's ability to kill pathogens in the infected host, but instead, on its ability to deliver genetic constructs into the bacteria and thus render them sensitive to antibiotics prior to host infection. We believe that transfer of the sensitizing cassette by the constructed phage will significantly enrich for antibiotic-treatable pathogens on hospital surfaces. Broad usage of the proposed system, in contrast to antibiotics and phage therapy, will potentially change the nature of nosocomial infections toward being more susceptible to antibiotics rather than more resistant.

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.host takeover | bacterial division | bacteriophage biology T he abundance of bacteriophages and their importance to microbial evolution, and consequently to major ecological issues, provide an incentive to study their biology. Bacteriophage T7 and its host, Escherichia coli, provide a model for systematically studying host-virus interactions. The genetics of both the phage and its host have been studied extensively, and the putative functions or tentative physiological roles of over half of the 56 genes of phage T7 and the 4,453 genes of E. coli have been identified (1-3). T7 is a virulent phage which, upon infection of its host E. coli, produces over 100 progeny phage per host in less than 25 min. It is an obligatory lytic phage, meaning that a successful phage growth cycle always results in lysis of the host. The genome of T7 is a 39,937-bp double-stranded DNA molecule (4). Despite extensive knowledge of phage T7, the mechanism by which it takes over the host molecular machinery remains obscure. Specific functions have been attributed to over