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An antiviral gene is absent in DNA but expressed by rolling circle reverse transcription.

Bacteria defend against phage infection through a variety of antiphage defence systems 1 . Many defence systems were recently shown to deplete cellular nicotinamide adenine dinucleotide (NAD + ) in response to infection, by cleaving NAD + into ADP-ribose (ADPR) and nicotinamide 2-7 . It was demonstrated that NAD + depletion during infection deprives the phage of this essential molecule and impedes phage replication. Here we show that a substantial fraction of phages possess enzymatic pathways allowing reconstitution of NAD + from its degradation products in infected cells. We describe NAD + reconstitution pathway 1 (NARP1), a two-step pathway in which one enzyme phosphorylates ADPR to generate ADPR pyrophosphate (ADPR-PP), and the second enzyme conjugates ADPR-PP and nicotinamide to generate NAD + . Phages encoding NARP1 can overcome a diverse set of defence systems, including Thoeris, DSR1, DSR2, SIR2-HerA and SEFIR, all of which deplete NAD + as part of their defensive mechanism. Phylogenetic analyses show that NARP1 is primarily encoded on phage genomes, suggesting a phage-specific function in countering bacterial defences. A second pathway, NARP2, allows phages to overcome bacterial defences by building NAD + using metabolites different from ADPR-PP. Our findings reveal a unique immune evasion strategy in which viruses rebuild molecules depleted by defence systems, thus overcoming host immunity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems 1-7 . Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components 8-12 . In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD + (refs.  12-14 ). Although the structure of ThsA has been solved 15 , the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD + depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems 1-4 , but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation 1-5 . Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes 1,6,7 , but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion., (© 2023. The Author(s).)

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders., Competing Interests: Declaration of interests R.S. is a scientific cofounder and advisor of BiomX and Ecophage., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals 1-3 . In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function 1 . We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria., Competing Interests: Declaration of interests R.S. is a scientific cofounder and advisor of BiomX, Pantheon Bioscience, and Ecophage., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed., Competing Interests: Declaration of Interests R.S. is a scientific cofounder and consultant of BiomX Ltd., Pantheon Ltd., and EcoPhage., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a family of defence systems against bacteriophages (hereafter phages) that share ancestry with the cGAS-STING innate immune pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death. Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells. Here, we analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. We propose a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector function. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. We observed evidence of extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems. We expect that our classification and nomenclature scheme will guide future research in the developing CBASS field.

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP-AMP signalling during viral infection and anti-tumour immunity 1-5 . STING shares no structural homology with other known signalling proteins 6-9 , which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD + cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR-STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS-STING pathway in prokaryotic defence against bacteriophages.

Facing frequent phage challenges, bacteria have evolved numerous mechanisms to resist phage infection. A commonly used phage resistance strategy is abortive infection (Abi), in which the infected cell commits suicide before the phage can complete its replication cycle. Abi prevents the phage epidemic from spreading to nearby cells, thus protecting the bacterial colony. The Abi strategy is manifested by a plethora of mechanistically diverse defense systems that are abundant in bacterial genomes. In turn, phages have developed equally diverse mechanisms to overcome bacterial Abi. This review summarizes the current knowledge on bacterial defense via cell suicide. It describes the principles of Abi, details how these principles are implemented in a variety of natural defense systems, and discusses phage counter-defense mechanisms.

Viruses and their hosts are engaged in a constant arms race leading to the evolution of antiviral defence mechanisms. Recent studies have revealed that the immune arsenal of bacteria against bacteriophages is much more diverse than previously envisioned. These discoveries have led to seemingly contradictory observations: on one hand, individual microorganisms often encode multiple distinct defence systems, some of which are acquired by horizontal gene transfer, alluding to their fitness benefit. On the other hand, defence systems are frequently lost from prokaryotic genomes on short evolutionary time scales, suggesting that they impose a fitness cost. In this Perspective article, we present the 'pan-immune system' model in which we suggest that, although a single strain cannot carry all possible defence systems owing to their burden on fitness, it can employ horizontal gene transfer to access immune defence mechanisms encoded by closely related strains. Thus, the 'effective' immune system is not the one encoded by the genome of a single microorganism but rather by its pan-genome, comprising the sum of all immune systems available for a microorganism to horizontally acquire and use.

The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals 1,2 . The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response 3-5 . The production of cGAMP has also been detected in bacteria 6 , and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane 7 . However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages.

Small RNAs (sRNAs) are central regulators of gene expression in bacteria, controlling target genes posttranscriptionally by base pairing with their mRNAs. sRNAs are involved in many cellular processes and have unique regulatory characteristics. In this review, we discuss the properties of regulation by sRNAs and how it differs from and combines with transcriptional regulation. We describe the global characteristics of the sRNA-target networks in bacteria using graph-theoretic approaches and review the local integration of sRNAs in mixed regulatory circuits, including feed-forward loops and their combinations, feedback loops, and circuits made of an sRNA and another regulator, both derived from the same transcript. Finally, we discuss the competition effects in posttranscriptional regulatory networks that may arise over shared targets, shared regulators, and shared resources and how they may lead to signal propagation across the network.

Antibiotic resistance genes are commonly regulated by sophisticated mechanisms that activate gene expression in response to antibiotic exposure. Growing evidence suggest that cis-acting non-coding RNAs play a major role in regulating the expression of many resistance genes, specifically those which counteract the effects of translation-inhibiting antibiotics. These ncRNAs reside in the 5'UTR of the regulated gene, and sense the presence of the antibiotics by recruiting translating ribosomes onto short upstream open reading frames (uORFs) embedded in the ncRNA. In the presence of translation-inhibiting antibiotics ribosomes arrest over the uORF, altering the RNA structure of the regulator and switching the expression of the resistance gene to 'ON'. The specificity of these riboregulators is tuned to sense-specific classes of antibiotics based on the length and composition of the respective uORF. Here we review recent work describing new types of antibiotic-sensing RNA-based regulators and elucidating the molecular mechanisms by which they function to control antibiotic resistance in bacteria., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A common strategy for regulation of gene expression in bacteria is conditional transcription termination. This strategy is frequently employed by 5'UTR cis-acting RNA elements (riboregulators), including riboswitches and attenuators. Such riboregulators can assume two mutually exclusive RNA structures, one of which forms a transcriptional terminator and results in premature termination, and the other forms an antiterminator that allows read-through into the coding sequence to produce a full-length mRNA. We developed a machine-learning based approach, which, given a 5'UTR of a gene, predicts whether it can form the two alternative structures typical to riboregulators employing conditional termination. Using a large positive training set of riboregulators derived from 89 human microbiome bacteria, we show high specificity and sensitivity for our classifier. We further show that our approach allows the discovery of previously unidentified riboregulators, as exemplified by the detection of new LeuA leaders and T-boxes in Streptococci Finally, we developed PASIFIC (www.weizmann.ac.il/molgen/Sorek/PASIFIC/), an online web-server that, given a user-provided 5'UTR sequence, predicts whether this sequence can adopt two alternative structures conforming with the conditional termination paradigm. This webserver is expected to assist in the identification of new riboswitches and attenuators in the bacterial pan-genome., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. Laboratory experiments, however, often study evolution in well-mixed systems. Here, we introduce an experimental device, the microbial evolution and growth arena (MEGA)-plate, in which bacteria spread and evolved on a large antibiotic landscape (120 × 60 centimeters) that allowed visual observation of mutation and selection in a migrating bacterial front. While resistance increased consistently, multiple coexisting lineages diversified both phenotypically and genotypically. Analyzing mutants at and behind the propagating front, we found that evolution is not always led by the most resistant mutants; highly resistant mutants may be trapped behind more sensitive lineages. The MEGA-plate provides a versatile platform for studying microbial adaption and directly visualizing evolutionary dynamics., (Copyright © 2016, American Association for the Advancement of Science.)

Whole-transcriptome sequencing studies from recent years revealed an unexpected complexity in transcriptomes of bacteria and archaea, including abundant non-coding RNAs, cis-antisense transcription and regulatory untranslated regions (UTRs). Understanding the functional relevance of the plethora of non-coding RNAs in a given organism is challenging, especially since some of these RNAs were attributed to 'transcriptional noise'. To allow the search for conserved transcriptomic elements we produced comparative transcriptome maps for multiple species across the microbial tree of life. These transcriptome maps are detailed in annotations, comparable by gene families, and BLAST-searchable by user provided sequences. Our transcriptome collection includes 18 model organisms spanning 10 phyla/subphyla of bacteria and archaea that were sequenced using standardized RNA-seq methods. The utility of the comparative approach, as implemented in our web server, is demonstrated by highlighting genes with exceptionally long 5'UTRs across species, which correspond to many known riboswitches and further suggest novel putative regulatory elements. Our study provides a standardized reference transcriptome to major clinically and environmentally important microbial phyla. The viewer is available at http://exploration.weizmann.ac.il/TCOL, setting a framework for comparative studies of the microbial non-coding genome., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak-to-trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes., (Copyright © 2015, American Association for the Advancement of Science.)

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system of bacteria and archaea constitutes a mechanism of acquired adaptive immunity against phages, which is based on genome-encoded markers of previously infecting phage sequences ("spacers"). As a repository of phage sequences, these spacers make the system particularly suitable for elucidating phage-bacteria interactions in metagenomic studies. Recent metagenomic analyses of CRISPRs associated with the human microbiome intriguingly revealed conserved "memory spacers" shared by bacteria in multiple unrelated, geographically separated individuals. Here, we discuss possible avenues for explaining this phenomenon by integrating insights from CRISPR biology and phage-bacteria ecology, with a special focus on the human gut. We further explore the growing body of evidence for the role of CRISPR/Cas in regulating the interplay between bacteria and lysogenic phages, which may be intimately related to the presence of memory spacers and sheds new light on the multifaceted biological and ecological modes of action of CRISPR/Cas.

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.

The bacterial community in the human gut has crucial health roles both in metabolic functions and in protection against pathogens. Phages, which are known to significantly affect microbial community composition in many ecological niches, have the potential to impact the gut microbiota, yet thorough characterization of this relationship remains elusive. We have reconstructed the content of the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals and used it to identify a catalog of 991 phages targeted by CRISPR across all individuals. Our results show that 78% of these phages are shared among two or more individuals. Moreover, a significant fraction of phages found in our study are shown to exist in fecal samples previously derived from American and Japanese individuals, identifying a common reservoir of phages frequently associated with the human gut microbiome. We further inferred the bacterial hosts for more than 130 such phages, enabling a detailed analysis of phage-bacteria interactions across the 124 individuals by correlating patterns of phage abundance with bacterial abundance and resistance. A subset of phages demonstrated preferred association with host genomes as lysogenized prophages, with highly increased abundance in specific individuals. Overall, our results imply that phage-bacterial attack-resistance interactions occur within the human gut microbiome, possibly affecting microbiota composition and human health. Our finding of global sharing of gut phages is surprising in light of the extreme genetic diversity of phages found in other ecological niches.

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.

Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. Here, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. The commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.

The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

Arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats that are separated by similarly sized non-repetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism. This Progress discusses the structure and function of this newly recognized antiviral mechanism.

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Coral microbiology is an emerging field, driven largely by a desire to understand, and ultimately prevent, the worldwide destruction of coral reefs. The mucus layer, skeleton and tissues of healthy corals all contain large populations of eukaryotic algae, bacteria and archaea. These microorganisms confer benefits to their host by various mechanisms, including photosynthesis, nitrogen fixation, the provision of nutrients and infection prevention. Conversely, in conditions of environmental stress, certain microorganisms cause coral bleaching and other diseases. Recent research indicates that corals can develop resistance to specific pathogens and adapt to higher environmental temperatures. To explain these findings the coral probiotic hypothesis proposes the occurrence of a dynamic relationship between symbiotic microorganisms and corals that selects for the coral holobiont that is best suited for the prevailing environmental conditions. Generalization of the coral probiotic hypothesis has led us to propose the hologenome theory of evolution.

Background: Clustered regularly interspaced short palindromic repeats (CRISPRs) are a novel class of direct repeats, separated by unique spacer sequences of similar length, that are present in approximately 40% of bacterial and most archaeal genomes analyzed to date. More than 40 gene families, called CRISPR-associated sequences (CASs), appear in conjunction with these repeats and are thought to be involved in the propagation and functioning of CRISPRs. It has been recently shown that CRISPR provides acquired resistance against viruses in prokaryotes., Results: Here we analyze CRISPR repeats identified in 195 microbial genomes and show that they can be organized into multiple clusters based on sequence similarity. Some of the clusters present stable, highly conserved RNA secondary structures, while others lack detectable structures. Stable secondary structures exhibit multiple compensatory base changes in the stem region, indicating evolutionary and functional conservation., Conclusion: We show that the repeat-based classification corresponds to, and expands upon, a previously reported CAS gene-based classification, including specific relationships between CRISPR and CAS subtypes.