Archaeal Taxa Research Articles

Archaeal Lineages within the Human Microbiome: Absent, Rare or Elusive?

Archaea are well-recognized components of the human microbiome. However, they appear to be drastically underrepresented compared to the high diversity of bacterial taxa which can be found on various human anatomic sites, such as the gastrointestinal environment, the oral cavity and the skin. As our “microbial” view of the human body, including the methodological concepts used to describe them, has been traditionally biased towards bacteria, the question arises whether our current knowledge reflects the actual ratio of archaea versus bacteria or whether we have failed so far to unravel the full diversity of human-associated archaea. This review article hypothesizes that distinct archaeal lineages within humans exist, which still await our detection. First, previously unrecognized taxa might be quite common but they have eluded conventional detection methods. Two recent prime examples are described that demonstrate that this might be the case for specific archaeal lineages. Second, some archaeal taxa might be overlooked because they are rare and/or in low abundance. Evidence for this exists for a broad range of phylogenetic lineages, however we currently do not know whether these sporadically appearing organisms are mere transients or important members of the so called “rare biosphere” with probably basic ecosystem functions. Lastly, evidence exists that different human populations harbor different archaeal taxa and/or the abundance and activity of shared archaeal taxa may differ and thus their impact on the overall microbiome. This research line is rather unexplored and warrants further investigation. While not recapitulating exhaustively all studies on archaeal diversity in humans, this review highlights pertinent recent findings that show that the choice of appropriate methodological approaches and the consideration of different human populations may lead to the detection of archaeal lineages previously not associated with humans. This in turn will help understand variations found in the overall microbiomes from different individuals and ultimately may lead to the emergence of novel concepts/mechanisms impacting human health.

Contrasting spatial patterns and ecological attributes of soil bacterial and archaeal taxa across a landscape.

Even though recent studies have clarified the influence and hierarchy of environmental filters on bacterial community structure, those constraining bacterial populations variations remain unclear. In consequence, our ability to understand to ecological attributes of soil bacteria and to predict microbial community response to environmental stress is therefore limited. Here, we characterized the bacterial community composition and the various bacterial taxonomic groups constituting the community across an agricultural landscape of 12 km2, by using a 215 × 215 m systematic grid representing 278 sites to precisely decipher their spatial distribution and drivers at this scale. The bacterial and Archaeal community composition was characterized by applying 16S rRNA gene pyrosequencing directly to soil DNA from samples. Geostatistics tools were used to reveal the heterogeneous distribution of bacterial composition at this scale. Soil physical parameters and land management explained a significant amount of variation, suggesting that environmental selection is the major process shaping bacterial composition. All taxa systematically displayed also a heterogeneous and particular distribution patterns. Different relative influences of soil characteristics, land use and space were observed, depending on the taxa, implying that selection and spatial processes might be differentially but not exclusively involved for each bacterial phylum. Soil pH was a major factor determining the distribution of most of the bacterial taxa and especially the most important factor explaining the spatial patterns of α-Proteobacteria and Planctomycetes. Soil texture, organic carbon content and quality were more specific to a few number of taxa (e.g., β-Proteobacteria and Chlorobi). Land management also influenced the distribution of bacterial taxa across the landscape and revealed different type of response to cropping intensity (positive, negative, neutral or hump-backed relationships) according to phyla. Altogether, this study provided valuable clues about the ecological behavior of soil bacterial and archaeal taxa at an agricultural landscape scale and could be useful for developing sustainable strategies of land management.

Global dispersion and local diversification of the methane seep microbiome

Methane seeps are widespread seafloor ecosystems shaped by the emission of gas from seabed reservoirs. The microorganisms inhabiting methane seeps transform the chemical energy in methane to products that sustain rich benthic communities around the gas leaks. Despite the biogeochemical relevance of microbial methane removal at seeps, the global diversity and dispersion of seep microbiota remain unknown. Here we determined the microbial diversity and community structure of 23 globally distributed methane seeps and compared these to the microbial communities of 54 other seafloor ecosystems, including sulfate-methane transition zones, hydrothermal vents, coastal sediments, and deep-sea surface and subsurface sediments. We found that methane seep communities show moderate levels of microbial richness compared with other seafloor ecosystems and harbor distinct bacterial and archaeal taxa with cosmopolitan distribution and key biogeochemical functions. The high relative sequence abundance of ANME (anaerobic methanotrophic archaea), as well as aerobic Methylococcales, sulfate-reducing Desulfobacterales, and sulfide-oxidizing Thiotrichales, matches the most favorable microbial metabolisms at methane seeps in terms of substrate supply and distinguishes the seep microbiome from other seafloor microbiomes. The key functional taxa varied in relative sequence abundance between different seeps due to the environmental factors, sediment depth and seafloor temperature. The degree of endemism of the methane seep microbiome suggests a high local diversification in these heterogeneous but long-lived ecosystems. Our results indicate that the seep microbiome is structured according to metacommunity processes and that few cosmopolitan microbial taxa mediate the bulk of methane oxidation, with global relevance to methane emission in the ocean.

Microbial community successional patterns in beach sands impacted by the Deepwater Horizon oil spill.

Although petroleum hydrocarbons discharged from the Deepwater Horizon (DWH) blowout were shown to have a pronounced impact on indigenous microbial communities in the Gulf of Mexico, effects on nearshore or coastal ecosystems remain understudied. This study investigated the successional patterns of functional and taxonomic diversity for over 1 year after the DWH oil was deposited on Pensacola Beach sands (FL, USA), using metagenomic and 16S rRNA gene amplicon techniques. Gamma- and Alphaproteobacteria were enriched in oiled sediments, in corroboration of previous studies. In contrast to previous studies, we observed an increase in the functional diversity of the community in response to oil contamination and a functional transition from generalist populations within 4 months after oil came ashore to specialists a year later, when oil was undetectable. At the latter time point, a typical beach community had reestablished that showed little to no evidence of oil hydrocarbon degradation potential, was enriched in archaeal taxa known to be sensitive to xenobiotics, but differed significantly from the community before the oil spill. Further, a clear succession pattern was observed, where early responders to oil contamination, likely degrading aliphatic hydrocarbons, were replaced after 3 months by populations capable of aromatic hydrocarbon decomposition. Collectively, our results advance the understanding of how natural benthic microbial communities respond to crude oil perturbation, supporting the specialization-disturbance hypothesis; that is, the expectation that disturbance favors generalists, while providing (microbial) indicator species and genes for the chemical evolution of oil hydrocarbons during degradation and weathering.

Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans.

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions.

Soil pH determines microbial diversity and composition in the park grass experiment.

The Park Grass experiment (PGE) in the UK has been ongoing since 1856. Its purpose is to study the response of biological communities to the long-term treatments and associated changes in soil parameters, particularly soil pH. In this study, soil samples were collected across pH gradient (pH 3.6-7) and a range of fertilizers (nitrogen as ammonium sulfate, nitrogen as sodium nitrate, phosphorous) to evaluate the effects nutrients have on soil parameters and microbial community structure. Illumina 16S ribosomal RNA (rRNA) amplicon sequencing was used to determine the relative abundances and diversity of bacterial and archaeal taxa. Relationships between treatments, measured soil parameters, and microbial communities were evaluated. Clostridium, Bacteroides, Bradyrhizobium, Mycobacterium, Ruminococcus, Paenibacillus, and Rhodoplanes were the most abundant genera found at the PGE. The main soil parameter that determined microbial composition, diversity, and biomass in the PGE soil was pH. The most probable mechanism of the pH impact on microbial community may include mediation of nutrient availability in the soil. Addition of nitrogen to the PGE plots as ammonium sulfate decreases soil pH through increased nitrification, which causes buildup of soil carbon, and hence increases C/N ratio. Plant species richness and plant productivity did not reveal significant relationships with microbial diversity; however, plant species richness was positively correlated with soil microbial biomass. Plants responded to the nitrogen treatments with an increase in productivity and a decrease in the species richness.

Microbial community structure and functioning in marine sediments associated with diffuse hydrothermal venting assessed by integrated meta‐omics

Deep-sea hydrothermal vents are unique environments on Earth, as they host chemosynthetic ecosystems fuelled by geochemical energy with chemolithoautotrophic microorganisms at the basis of the food webs. Whereas discrete high-temperature venting systems have been studied extensively, the microbiotas associated with low-temperature diffuse venting are not well understood. We analysed the structure and functioning of microbial communities in two diffuse venting sediments from the Jan Mayen vent fields in the Norwegian-Greenland Sea, applying an integrated 'omics' approach combining metatranscriptomics, metaproteomics and metagenomics. Polymerase chain reaction-independent three-domain community profiling showed that the two sediments hosted highly similar communities dominated by Epsilonproteobacteria, Deltaproteobacteria and Gammaproteobacteria, besides ciliates, nematodes and various archaeal taxa. Active metabolic pathways were identified through transcripts and peptides, with genes of sulphur and methane oxidation, and carbon fixation pathways highly expressed, in addition to genes of aerobic and anaerobic (nitrate and sulphate) respiratory chains. High expression of chemotaxis and flagella genes reflected a lifestyle in a dynamic habitat rich in physico-chemical gradients. The major metabolic pathways could be assigned to distinct taxonomic groups, thus enabling hypotheses about the function of the different prokaryotic and eukaryotic taxa. This study advances our understanding of the functioning of microbial communities in diffuse hydrothermal venting sediments.

Cell Size Distributions of Soil Bacterial and Archaeal Taxa

Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes in situ, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 μm to 0.2 μm) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 μm in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including Acidobacteria, Gemmatimonadetes, Crenarchaeota, Verrucomicrobia, and Elusimicrobia). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized.

Transcriptional response of bathypelagic marine bacterioplankton to the Deepwater Horizon oil spill

The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean between April and July 2010, stimulating microbial blooms of petroleum-degrading bacteria. To understand the metabolic response of marine microorganisms, we sequenced ≈ 66 million community transcripts that revealed the identity of metabolically active microbes and their roles in petroleum consumption. Reads were assigned to reference genes from ≈ 2700 bacterial and archaeal taxa, but most assignments (39%) were to just six genomes representing predominantly methane- and petroleum-degrading Gammaproteobacteria. Specific pathways for the degradation of alkanes, aromatic compounds and methane emerged from the metatranscriptomes, with some transcripts assigned to methane monooxygenases representing highly divergent homologs that may degrade either methane or short alkanes. The microbial community in the plume was less taxonomically and functionally diverse than the unexposed community below the plume; this was due primarily to decreased species evenness resulting from Gammaproteobacteria blooms. Surprisingly, a number of taxa (related to SAR11, Nitrosopumilus and Bacteroides, among others) contributed equal numbers of transcripts per liter in both the unexposed and plume samples, suggesting that some groups were unaffected by the petroleum inputs and blooms of degrader taxa, and may be important for re-establishing the pre-spill microbial community structure.

Stability along with Extreme Variability in Core Genome Evolution

The shape of the distribution of evolutionary distances between orthologous genes in pairs of closely related genomes is universal throughout the entire range of cellular life forms. The near invariance of this distribution across billions of years of evolution can be accounted for by the Universal Pace Maker (UPM) model of genome evolution that yields a significantly better fit to the phylogenetic data than the Molecular Clock (MC) model. Unlike the MC, the UPM model does not assume constant gene-specific evolutionary rates but rather postulates that, in each evolving lineage, the evolutionary rates of all genes change (approximately) in unison although the pacemakers of different lineages are not necessarily synchronized. Here, we dissect the nearly constant evolutionary rate distribution by comparing the genome-wide relative rates of evolution of individual genes in pairs or triplets of closely related genomes from diverse bacterial and archaeal taxa. We show that, although the gene-specific relative rate is an important feature of genome evolution that explains more than half of the variance of the evolutionary distances, the ranges of relative rate variability are extremely broad even for universal genes. Because of this high variance, the gene-specific rate is a poor predictor of the conservation rank for any gene in any particular lineage.

Comparative genomics of defense systems in archaea and bacteria

Our knowledge of prokaryotic defense systems has vastly expanded as the result of comparative genomic analysis, followed by experimental validation. This expansion is both quantitative, including the discovery of diverse new examples of known types of defense systems, such as restriction-modification or toxin-antitoxin systems, and qualitative, including the discovery of fundamentally new defense mechanisms, such as the CRISPR-Cas immunity system. Large-scale statistical analysis reveals that the distribution of different defense systems in bacterial and archaeal taxa is non-uniform, with four groups of organisms distinguishable with respect to the overall abundance and the balance between specific types of defense systems. The genes encoding defense system components in bacterial and archaea typically cluster in defense islands. In addition to genes encoding known defense systems, these islands contain numerous uncharacterized genes, which are candidates for new types of defense systems. The tight association of the genes encoding immunity systems and dormancy- or cell death-inducing defense systems in prokaryotic genomes suggests that these two major types of defense are functionally coupled, providing for effective protection at the population level.

Archaeal Assemblages Inhabiting Temperate Mixed Forest Soil Fluctuate in Taxon Composition and Spatial Distribution over Time

This study explored the persistence and spatial distribution of a diverse Archaeal assemblage inhabiting a temperate mixed forest ecosystem. Persistence under native conditions was measured from 2001 to 2010, 2011, and 2012 by comparison of 16S rRNA gene clone libraries. The Archaeal assemblages at each of these time points were found to be significantly different (AMOVA, P < 0.01), and the nature of this difference was dependent on taxonomic rank. For example, the cosmopolitan genus g_Ca. Nitrososphaera (I.1b) was detected at all time points, but within this taxon the abundance of s_SCA1145, s_SCA1170, and s_Ca. N. gargensis fluctuated over time. In addition, spatial heterogeneity (patchiness) was measured at these time points using 1D TRFLP-SSCP fingerprinting to screen soil samples covering multiple spatial scales. This included soil collected from small volumes of 3 cubic centimeters to larger scales—over a surface area of 50 m2, plots located 1.3 km apart, and a separate locality 23 km away. The spatial distribution of Archaea in these samples changed over time, and while g_Ca. Nitrososphaera (I.1b) was dominant over larger scales, patches were found at smaller scales that were dominated by other taxa. This study measured the degree of change for Archaeal taxon composition and patchiness over time in temperate mixed forest soil.

Structure and diversity of bacterial, eukaryotic and archaeal communities in glacial cryoconite holes from the Arctic and the Antarctic

The cryosphere presents some of the most challenging conditions for life on earth. Nevertheless, (micro)biota survive in a range of niches in glacial systems, including water-filled depressions on glacial surfaces termed cryoconite holes (centimetre to metre in diameter and up to 0.5 m deep) that contain dark granular material (cryoconite). In this study, the structure of bacterial and eukaryotic cryoconite communities from ten different locations in the Arctic and Antarctica was compared using T-RFLP analysis of rRNA genes. Community structure varied with geography, with greatest differences seen between communities from the Arctic and the Antarctic. DNA sequencing of rRNA genes revealed considerable diversity, with individual cryoconite hole communities containing between six and eight bacterial phyla and five and eight eukaryotic 'first-rank' taxa and including both bacterial and eukaryotic photoautotrophs. Bacterial Firmicutes and Deltaproteobacteria and Epsilonproteobacteria, eukaryotic Rhizaria, Haptophyta, Choanomonada and Centroheliozoa, and archaea were identified for the first time in cryoconite ecosystems. Archaea were only found within Antarctic locations, with the majority of sequences (77%) related to members of the Thaumarchaeota. In conclusion, this research has revealed that Antarctic and Arctic cryoconite holes harbour geographically distinct highly diverse communities and has identified hitherto unknown bacterial, eukaryotic and archaeal taxa, therein.

Ecological structuring of bacterial and archaeal taxa in surface ocean waters

The Global Ocean Sampling (GOS) expedition is currently the largest and geographically most comprehensive metagenomic dataset, including samples from the Atlantic, Pacific, and Indian Oceans. This study makes use of the wide range of environmental conditions and habitats encompassed within the GOS sites in order to investigate the ecological structuring of bacterial and archaeal taxon ranks. Community structures based on taxonomically classified 16S ribosomal RNA (rRNA) gene fragments at phylum, class, order, family, and genus rank levels were examined using multivariate statistical analysis, and the results were inspected in the context of oceanographic environmental variables and structured habitat classifications. At all taxon rank levels, community structures of neritic, oceanic, estuarine biomes, as well as other exotic biomes (salt marsh, lake, mangrove), were readily distinguishable from each other. A strong structuring of the communities with chlorophyll a concentration and a weaker yet significant structuring with temperature and salinity were observed. Furthermore, there were significant correlations between community structures and habitat classification. These results were used for further investigation of one-to-one relationships between taxa and environment and provided indications for ecological preferences shaped by primary production for both cultured and uncultured bacterial and archaeal clades.

Microbial diversity in arctic freshwaters is structured by inoculation of microbes from soils

Microbes are transported in hydrological networks through many environments, but the nature and dynamics of underlying microbial metacommunities and the impact of downslope inoculation on patterns of microbial diversity across landscapes are unknown. Pyrosequencing of small subunit ribosomal RNA gene hypervariable regions to characterize microbial communities along a hydrological continuum in arctic tundra showed a pattern of decreasing diversity downslope, with highest species richness in soil waters and headwater streams, and lowest richness in lake water. In a downstream lake, 58% and 43% of the bacterial and archaeal taxa, respectively, were also detected in diverse upslope communities, including most of the numerically dominant lake taxa. In contrast, only 18% of microbial eukaryotic taxa in the lake were detected upslope. We suggest that patterns of diversity in surface waters are structured by initial inoculation from microbial reservoirs in soils followed by a species-sorting process during downslope dispersal of both common and rare microbial taxa. Our results suggest that, unlike for metazoans, a substantial portion of bacterial and archaeal diversity in surface freshwaters may originate in complex soil environments.

Molecular analysis of meso- and thermophilic microbiota associated with anaerobic biowaste degradation

BackgroundMicrobial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD.ResultsThe 454 sequencing showed that the diversity and number of bacterial taxa decreased with increasing organic load, while archaeal i.e. methanogenic taxa remained more constant. The number and diversity of fungal taxa increased during the process and varied less in composition with process temperature than bacterial and archaeal taxa, even though the fungal diversity increased with temperature as well. Evaluation of the microarray using AD sample DNA showed correlation of signal intensities with sequence read numbers of corresponding target groups. The sensitivity of the test was found to be about 1%.ConclusionsThe fungal community survives in anoxic conditions and grows with increasing organic loading, suggesting that Fungi may contribute to the digestion by metabolising organic nutrients for bacterial and methanogenic groups. The microarray proof of principle tests suggest that the method has the potential for semiquantitative detection of target microbial groups given that comprehensive sequence data is available for probe design.

Similarity of genes horizontally acquired by Escherichia coli and Salmonella enterica is evidence of a supraspecies pangenome

Most bacterial and archaeal genomes contain many genes with little or no similarity to other genes, a property that impedes identification of gene origins. By comparing the codon usage of genes shared among strains (primarily vertically inherited genes) and genes unique to one strain (primarily recently horizontally acquired genes), we found that the plurality of unique genes in Escherichia coli and Salmonella enterica are much more similar to each other than are their vertically inherited genes. We conclude that E. coli and S. enterica derive these unique genes from a common source, a supraspecies phylogenetic group that includes the organisms themselves. The phylogenetic range of the sharing appears to include other (but not all) members of the Enterobacteriaceae. We found evidence of similar gene sharing in other bacterial and archaeal taxa. Thus, we conclude that frequent gene exchange, particularly that of genetic novelties, extends well beyond accepted species boundaries.

RtcB, a Novel RNA Ligase, Can Catalyze tRNA Splicing and HAC1 mRNA Splicing in Vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

Analysis of 23S rRNA genes in metagenomes – A case study from the Global Ocean Sampling Expedition

As an evolutionary marker, 23S ribosomal RNA (rRNA) offers more diagnostic sequence stretches and greater sequence variation than 16S rRNA. However, 23S rRNA is still not as widely used. Based on 80 metagenome samples from the Global Ocean Sampling (GOS) Expedition, the usefulness and taxonomic resolution of 23S rRNA were compared to those of 16S rRNA. Since 23S rRNA is approximately twice as large as 16S rRNA, twice as many 23S rRNA gene fragments were retrieved from the GOS reads than 16S rRNA gene fragments, with 23S rRNA gene fragments being generally about 100 bp longer. Datasets for 16S and 23S rRNA sequences revealed similar relative abundances for major marine bacterial and archaeal taxa. However, 16S rRNA sequences had a better taxonomic resolution due to their significantly larger reference database. Reevaluation of the specificity of previously published PCR amplification primers and group specific fluorescence in situ hybridization probes on this metagenomic set of non-amplified 23S rRNA sequences revealed that out of 16 primers investigated, only two had more than 90% target group coverage. Evaluations of two probes, BET42a and GAM42a, were in accordance with previous evaluations, with a discrepancy in the target group coverage of the GAM42a probe when evaluated against the GOS metagenomic dataset.

Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.