CRISPR-Cas in Pseudomonas aeruginosa provides transient population-level immunity against high phage exposures.

The prokaryotic adaptive immune system, CRISPR-Cas (clustered regularly interspaced short palindromic repeats; CRISPR-associated), requires the acquisition of spacer sequences that target invading mobile genetic elements such as phages. Previous work has identified ecological variables that drive the evolution of CRISPR-based immunity of the model organism Pseudomonas aeruginosa PA14 against its phage DMS3vir, resulting in rapid phage extinction. However, it is unclear if and how stable such acquired immunity is within bacterial populations, and how this depends on the environment. Here, we examine the dynamics of CRISPR spacer acquisition and loss over a 30-day evolution experiment and identify conditions that tip the balance between long-term maintenance of immunity versus invasion of alternative resistance strategies that support phage persistence. Specifically, we find that both the initial phage dose and reinfection frequencies determine whether or not acquired CRISPR immunity is maintained in the long term, and whether or not phage can coexist with the bacteria. At the population genetics level, emergence and loss of CRISPR immunity are associated with high levels of spacer diversity that subsequently decline due to invasion of bacteria carrying pilus-associated mutations. Together, these results provide high resolution of the dynamics of CRISPR immunity acquisition and loss and demonstrate that the cumulative phage burden determines the effectiveness of CRISPR over ecologically relevant timeframes.

Evolution and conserved functionality of organ size and shape regulator PEAPOD.

Transcriptional regulator PEAPOD (PPD) and its binding partners comprise a complex that is conserved throughout many core eudicot plants with regard to protein domain sequence and the function of controlling organ size and shape. Orthologues of PPD also exist in the basal angiosperm Amborella trichopoda, various gymnosperm species, the lycophyte Selaginella moellendorffii and several monocot genera, although until now it was not known if these are functional sequences. Here we report constitutive expression of orthologues from species representing diverse taxa of plant phylogeny in the Arabidopsis Δppd mutant. PPD orthologues from S. moellendorffii, gymnosperm Picea abies, A. trichopoda, monocot Musa acuminata, and dicot Trifolium repens were able to complement the mutant and return it to the wild-type phenotype, demonstrating the conserved functionality of PPD throughout vascular plants. In addition, analysis of bryophyte genomes revealed potential PPD orthologues in model liverwort and moss species, suggesting a more primitive lineage for this conserved regulator. The Poaceae (grasses) lack the genes for the PPD module and the reason for loss of the complex from this economically significant family is unclear, given that grasses were the last of the flowering plants to evolve. Bioinformatic analyses identified putative PPD orthologues in close relatives of the Poaceae, indicating that the explanation for absence of PPD in the grasses may be more complex than previously considered. Understanding the mechanisms which led to loss of PPD from the grasses will provide insight into evolution of the Poaceae.

High viral abundance and low diversity are associated with increased CRISPR-Cas prevalence across microbial ecosystems.

CRISPR-Cas are adaptive immune systems that protect their hosts against viruses and other parasitic mobile genetic elements.1 Although widely distributed among prokaryotic taxa, CRISPR-Cas systems are not ubiquitous.2-4 Like most defense-system genes, CRISPR-Cas are frequently lost and gained, suggesting advantages are specific to particular environmental conditions.5 Selection from viruses is assumed to drive the acquisition and maintenance of these immune systems in nature, and both theory6-8 and experiments have identified phage density and diversity as key fitness determinants.9,10 However, these approaches lack the biological complexity inherent in nature. Here, we exploit metagenomic data from 324 samples across diverse ecosystems to analyze CRISPR abundance in natural environments. For each metagenome, we quantified viral abundance and diversity to test whether these contribute to CRISPR-Cas abundance across ecosystems. We find a strong positive association between CRISPR-Cas abundance and viral abundance. In addition, when controlling for differences in viral abundance, CRISPR-Cas systems are more abundant when viral diversity is low, suggesting that such adaptive immune systems may offer limited protection when required to target a diverse viral community. CRISPR-Cas abundance also differed among environments, with environmental classification explaining roughly a quarter of the variation in CRISPR-Cas relative abundance. The relationships between CRISPR-Cas abundance, viral abundance, and viral diversity are broadly consistent across environments, providing robust evidence from natural ecosystems that supports predictions of when CRISPR is beneficial. These results indicate that viral abundance and diversity are major ecological factors that drive the selection and maintenance of CRISPR-Cas in microbial ecosystems.

Genetic evaluation of ESBL-producing Escherichia coli urinary isolates in Otago, New Zealand.

OBJECTIVES: The incidence of infections with ESBL-producing Escherichia coli (ESBL-Ec) in New Zealand is increasing. ESBL-Ec most commonly cause urinary tract infections and are seen in both community and hospitalized patients. The reason for the increasing incidence of ESBL-Ec infections is unknown. METHODS: In this study, 65 urinary ESBL-Ec isolates from the Otago region in 2015 were fully genetically characterized to understand the mechanisms of transmission. The ESBL gene, E. coli STs, plasmid types and genetic context (e.g. insertion sequences) of ESBL genes were determined by a combination of whole genome and plasmid sequencing. The phylogenetic relationships of the isolates were compared with ESBL-Ec isolates sequenced as part of the 2016 nationwide survey. RESULTS: Significant diversity of E. coli strains, plasmids, and the genetic context of ESBL genes was seen. However, there was evidence of common mobile genetic elements in unrelated ESBL-Ec. CONCLUSIONS: Multiple introductions of ESBL resistance genes or resistant bacterial strains with limited horizontal transmission of mobile genetic elements accounts for the increased incidence of ESBL-Ec in this low prevalence area. Future studies should investigate modes of transmission of ESBL-Ec in the Otago region.

Soil Nitrogen Treatment Alters Microbiome Networks Across Farm Niches.

Agriculture is fundamental for food production, and microbiomes support agriculture through multiple essential ecosystem services. Despite the importance of individual (i.e., niche specific) agricultural microbiomes, microbiome interactions across niches are not well-understood. To observe the linkages between nearby agricultural microbiomes, multiple approaches (16S, 18S, and ITS) were used to inspect a broad coverage of niche microbiomes. Here we examined agricultural microbiome responses to 3 different nitrogen treatments (0, 150, and 300 kg/ha/yr) in soil and tracked linked responses in other neighbouring farm niches (rumen, faecal, white clover leaf, white clover root, rye grass leaf, and rye grass root). Nitrogen treatment had little impact on microbiome structure or composition across niches, but drastically reduced the microbiome network connectivity in soil. Networks of 16S microbiomes were the most sensitive to nitrogen treatment across amplicons, where ITS microbiome networks were the least responsive. Nitrogen enrichment in soil altered soil and the neighbouring microbiome networks, supporting our hypotheses that nitrogen treatment in soil altered microbiomes in soil and in nearby niches. This suggested that agricultural microbiomes across farm niches are ecologically interactive. Therefore, knock-on effects on neighbouring niches should be considered when management is applied to a single agricultural niche.

Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity.

CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.

Sharing a ß-Glucan Meal: Transcriptomic Eavesdropping on a Bacteroides ovatus-Subdoligranulum variabile-Hungatella hathewayi Consortium.

Whole-transcriptome analysis was used to investigate the molecular interplay between three bacterial species that are members of the human gut microbiota. Bacteroides ovatus, Subdoligranulum variabile, and Hungatella hathewayi formed associations in cocultures fed barley ß-glucan, a constituent of dietary fiber. B. ovatus depolymerized ß-glucan and released, but did not utilize, 3-O-ß-cellobiosyl-d-glucose (DP3) and 3-O-ß-cellotriosyl-d-glucose (DP4). These oligosaccharides provided growth substrates for S. variabile and H. hathewayi with a preference for DP4 in the case of the latter species. There was increased transcription of a B. ovatus mixed-linkage-ß-glucan utilization locus, as well as carbohydrate transporters in S. variabile and H. hathewayi when in batch coculture. Increased transcription of the ß-glucan utilization locus did not occur in continuous culture. Evidence for interactions relating to provision of cobalamin, alterations to signaling, and modulation of the "stringent response" (an adaptation to nutrient deprivation) were detected. Overall, we established a bacterial consortium based on barley ß-glucan in vitro, which can be used to investigate aspects of the functional blueprint of the human gut microbiota.IMPORTANCE The microbial community, mostly composed of bacterial species, residing in the human gut degrades and ferments polysaccharides derived from plants (dietary fiber) that would not otherwise be digested. In this way, the collective metabolic actions of community members extract additional energy from the human diet. While the variety of bacteria present in the microbial community is well known, the formation of bacterial consortia, and the consequent interactions that result in the digestion of dietary polysaccharides, has not been studied extensively. The importance of our work was the establishment, under laboratory conditions, of a consortium of gut bacteria that formed around a dietary constituent commonly present in cereals. This enabled the metabolic interplay between the bacterial species to be studied. This kind of knowledge is required to construct an interactive, metabolic blueprint of the microbial community that inhabits the human gut.

A prospective study of bloodstream infections among febrile adolescents and adults attending Yangon General Hospital, Yangon, Myanmar.

Data on causes of community-onset bloodstream infection in Myanmar are scarce. We aimed to identify etiological agents of bloodstream infections and patterns of antimicrobial resistance among febrile adolescents and adults attending Yangon General Hospital (YGH), Yangon, Myanmar. We recruited patients ≥12 years old with fever ≥38°C who attended YGH from 5 October 2015 through 4 October 2016. A standardized clinical history and physical examination was performed. Provisional diagnoses and vital status at discharge was recorded. Blood was collected for culture, bloodstream isolates were identified, and antimicrobial susceptibility testing was performed. Using whole-genome sequencing, we identified antimicrobial resistance mechanisms of Enterobacteriaceae and sequence types of Enterobacteriaceae and Streptococcus agalactiae. Among 947 participants, 90 (9.5%) had bloodstream infections (BSI) of which 82 (91.1%) were of community-onset. Of 91 pathogens isolated from 90 positive blood cultures, we identified 43 (47.3%) Salmonella enterica including 33 (76.7%) serovar Typhi and 10 (23.3%) serovar Paratyphi A; 20 (22.0%) Escherichia coli; 7 (7.7%) Klebsiella pneumoniae; 6 (6.6%), Staphylococcus aureus; 4 (4.4%) yeasts; and 1 (1.1%) each of Burkholderia pseudomallei and Streptococcus agalactiae. Of 70 Enterobacteriaceae, 62 (88.6%) were fluoroquinolone-resistant. Among 27 E. coli and K. pneumoniae, 18 (66.6%) were extended-spectrum beta-lactamase (ESBL)-producers, and 1 (3.7%) each were AmpC beta-lactamase- and carbapenemase-producers. Fluoroquinolone resistance was associated predominantly with mutations in the quinolone resistance-determining region. blaCTX-M-15 expression was common among ESBL-producers. Methicillin-resistant S. aureus was not detected. Fluoroquinolone-resistant, but not multiple drug-resistant, typhoidal S. enterica was the leading cause of community-onset BSI at a tertiary hospital in Yangon, Myanmar. Fluoroquinolone and extended-spectrum cephalosporin resistance was common among other Enterobactericeae. Our findings inform empiric management of severe febrile illness in Yangon and indicate that measures to prevent and control enteric fever are warranted. We suggest ongoing monitoring and efforts to mitigate antimicrobial resistance among community-onset pathogens.

Exploitation of the Cooperative Behaviors of Anti-CRISPR Phages.

Bacteriophages encoding anti-CRISPR proteins (Acrs) must cooperate to overcome phage resistance mediated by the bacterial immune system CRISPR-Cas, where the first phage blocks CRISPR-Cas immunity in order to allow a second Acr phage to successfully replicate. However, in nature, bacteria are frequently not pre-immunized, and phage populations are often not clonal, exhibiting variations in Acr presence and strength. We explored how interactions between Acr phages and initially sensitive bacteria evolve, both in the presence and absence of competing phages lacking Acrs. We find that Acr phages benefit "Acr-negative" phages by limiting the evolution of CRISPR-based resistance and helping Acr-negative phages to replicate on resistant host sub-populations. These benefits depend on the strength of CRISPR-Cas inhibitors and result in strong Acrs providing smaller fitness advantages than weaker ones when Acr phages compete with Acr-negative phages. These results indicate that different Acr types shape the evolutionary dynamics and social interactions of phage populations in natural communities.

Molecular mechanisms of antimicrobial resistance and phylogenetic relationships of Salmonella enterica isolates from febrile patients in Yangon, Myanmar.

BACKGROUND: Enteric fever is common in southeast Asia. However, there is little information on the circulating Salmonella enterica strains causing enteric fever in Myanmar. METHODS: We performed antimicrobial susceptibility testing and whole genome sequencing on S. enterica bloodstream isolates from febrile patients aged ≥12 y attending two hospitals in Yangon, Myanmar, from 5 October 2015 through 4 October 2016. We identified the serovar of S. enterica, determined antimicrobial susceptibility and the molecular mechanisms of resistance. We analysed phylogenetic relationships among Myanmar S. enterica isolates and those with isolates from neighbouring countries. RESULTS: Of 73 S. enterica isolated, 39 (53%) were serovar Typhi and 34 (47%) were Paratyphi A. All isolates were susceptible to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole but resistant to ciprofloxacin. We identified mutations in chromosomal genes gyrA, gyrB and parC as responsible for fluoroquinolone resistance. All S. enterica Typhi isolates were of 4.3.1 subclade (formerly known as H58) and formed two closely related genotypic clusters; both clusters were most closely related to isolates from India from 2012. All S. enterica Paratyphi A were lineage C, clade C4 and were closely related. CONCLUSION: Our study describes currently circulating S. enterica serovars in Myanmar, the genetic basis of their antimicrobial resistance and provides a genotypic framework for epidemiologic study.

Bifidobacterium bifidum ATCC 15696 and Bifidobacterium breve 24b Metabolic Interaction Based on 2'-O-Fucosyl-Lactose Studied in Steady-State Cultures in a Freter-Style Chemostat.

Infants fed breast milk harbor a gut microbiota in which bifidobacteria are generally predominant. The metabolic interactions of bifidobacterial species need investigation because they may offer insight into the colonization of the gut in early life. Bifidobacterium bifidum ATCC 15696 hydrolyzes 2'-O-fucosyl-lactose (2FL; a major fucosylated human milk oligosaccharide) but does not use fucose released into the culture medium. However, fucose is a growth substrate for Bifidobacterium breve 24b, and both strains utilize lactose for growth. The provision of fucose and lactose by B. bifidum (the donor) allowing the growth of B. breve (the beneficiary) conforms to the concept of syntrophy, but both strains will compete for lactose to multiply. To determine the metabolic impact of this syntrophic/competitive relationship on the donor, the transcriptomes of B. bifidum were determined and compared in steady-state monoculture and coculture using transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR). B. bifidum genes upregulated in coculture included those encoding alpha-l-fucosidase and carbohydrate transporters and those involved in energy production and conversion. B. bifidum abundance was the same in coculture as in monoculture, but B. breve dominated the coculture numerically. Cocultures during steady-state growth in 2FL medium produced mostly acetate with little lactate (acetate:lactate molar ratio, 8:1) compared to that in monobatch cultures containing lactose (2:1), which reflected the maintenance of steady-state cells in log-phase growth. Darwinian competition is an implicit feature of bacterial communities, but syntrophy is a phenomenon putatively based on cooperation. Our results suggest that the regulation of syntrophy, in addition to competition, may shape bacterial communities.IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the infant bowel) using in vitro experimentation with bacterial cultures maintained under controlled growth and environmental conditions. We studied the growth of bifidobacteria whose nutrition centered on the hydrolysis of a human milk oligosaccharide. The results revealed responses relating to metabolism occurring in a Bifidobacterium bifidum strain when it provided nutrients that allowed the growth of Bifidobacterium breve, and so discovered biochemical features of these bifidobacteria in relation to metabolic interaction in the shared environment. These kinds of experiments are essential in developing concepts of bifidobacterial ecology that relate to the development of the gut microbiota in early life.

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.

F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

Phylogenetic and functional potential links pH and N2O emissions in pasture soils.

Denitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir &nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.

Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR-Cas system.

CRISPR-Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR-Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR-Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3'-5' translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition ('targeted acquisition') is a major contributor to adaptation in type I-F CRISPR-Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.

CRISPRDetect: A flexible algorithm to define CRISPR arrays.

BACKGROUND: CRISPR (clustered regularly interspaced short palindromic repeats) RNAs provide the specificity for noncoding RNA-guided adaptive immune defence systems in prokaryotes. CRISPR arrays consist of repeat sequences separated by specific spacer sequences. CRISPR arrays have previously been identified in a large proportion of prokaryotic genomes. However, currently available detection algorithms do not utilise recently discovered features regarding CRISPR loci. RESULTS: We have developed a new approach to automatically detect, predict and interactively refine CRISPR arrays. It is available as a web program and command line from bioanalysis.otago.ac.nz/CRISPRDetect. CRISPRDetect discovers putative arrays, extends the array by detecting additional variant repeats, corrects the direction of arrays, refines the repeat/spacer boundaries, and annotates different types of sequence variations (e.g. insertion/deletion) in near identical repeats. Due to these features, CRISPRDetect has significant advantages when compared to existing identification tools. As well as further support for small medium and large repeats, CRISPRDetect identified a class of arrays with 'extra-large' repeats in bacteria (repeats 44-50 nt). The CRISPRDetect output is integrated with other analysis tools. Notably, the predicted spacers can be directly utilised by CRISPRTarget to predict targets. CONCLUSION: CRISPRDetect enables more accurate detection of arrays and spacers and its gff output is suitable for inclusion in genome annotation pipelines and visualisation. It has been used to analyse all complete bacterial and archaeal reference genomes.

H2 metabolism is widespread and diverse among human colonic microbes.

Microbial molecular hydrogen (H2) cycling is central to metabolic homeostasis and microbial composition in the human gastrointestinal tract. Molecular H2 is produced as an endproduct of carbohydrate fermentation and is reoxidised primarily by sulfate-reduction, acetogenesis, and methanogenesis. However, the enzymatic basis for these processes is incompletely understood and the hydrogenases responsible have not been investigated. In this work, we surveyed the genomic and metagenomic distribution of hydrogenase-encoding genes in the human colon to infer dominant mechanisms of H2 cycling. The data demonstrate that 70% of gastrointestinal microbial species listed in the Human Microbiome Project encode the genetic capacity to metabolise H2. A wide variety of anaerobically-adapted hydrogenases were present, with [FeFe]-hydrogenases predominant. We subsequently analyzed the hydrogenase gene content of stools from 20 healthy human subjects. The hydrogenase gene content of all samples was overwhelmingly dominated by fermentative and electron-bifurcating [FeFe]-hydrogenases emerging from the Bacteroidetes and Firmicutes. This study supports that H2 metabolism in the human gut is driven by fermentative H2 production and interspecies H2 transfer. However, it suggests that electron-bifurcation rather than respiration is the dominant mechanism of H2 reoxidation in the human colon, generating reduced ferredoxin to sustain carbon-fixation (e.g. acetogenesis) and respiration (via the Rnf complex). This work provides the first comprehensive bioinformatic insight into the mechanisms of H2 metabolism in the human colon.

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival.

Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H2) is a niche process. To gain a broader insight into the importance of microbial H2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H2. The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H2-based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H2-sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content (pO2) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H2 metabolism.

Computational Detection of CRISPR/crRNA Targets.

The CRISPR-Cas systems in bacteria and archaea provide protection by targeting foreign nucleic acids. The sequence of the "spacers" within CRISPR arrays specifically determines the targets in invader genomes. These spacers provide the short specific RNA nucleotide sequences within the guide crRNAs. In addition to complementarity in the spacer-target (protospacer) interaction, short flanking protospacer adjacent motifs (PAMs), or mismatching flanks have a discriminatory role in accurate target detection. Here, we describe a bioinformatic method, called CRISPRTarget, to use the sequence of a CRISPR array (e.g., predicted via CRISPRDetect/CRISPRDirection) to identify the foreign nucleic acids it targets.