We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.

Nitrogen fixation, a prokaryotic, O 2 -inhibited process that reduces N 2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O 2 consumption by respiration still exceeded the rate of O 2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis-and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N 2 fixation in hot spring mats and factors that can markedly influence the extent of N 2 fixation over the diel cycle.

Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-m vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O 2 , pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O 2 concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.

Based on the Stable Ecotype Model, evolution leads to the divergence of ecologically distinct populations (e.g., with different niches and/or behaviors) of ecologically interchangeable membership. In this study, pyrosequencing was used to provide deep sequence coverage of Synechococcus psaA genes and transcripts over a large number of habitat types in the Mushroom Spring microbial mat. Putative ecological species [putative ecotypes (PEs)], which were predicted by an evolutionary simulation based on the Stable Ecotype Model (Ecotype Simulation), exhibited distinct distributions relative to temperature-defined positions in the effluent channel and vertical position in the upper 1 mm-thick mat layer. Importantly, in most cases variants predicted to belong to the same PE formed unique clusters relative to temperature and depth in the mat in canonical correspondence analysis, supporting the hypothesis that while the PEs are ecologically distinct, the members of each ecotype are ecologically homogeneous. PEs responded differently to experimental perturbations of temperature and light, but the genetic variation within each PE was maintained as the relative abundances of PEs changed, further indicating that each population responded as a set of ecologically interchangeable individuals. Compared to PEs that predominate deeper within the mat photic zone, the timing of transcript abundances for selected genes differed for PEs that predominate in microenvironments closer to upper surface of the mat with spatiotemporal differences in light and O2 concentration. All of these findings are consistent with the hypotheses that Synechococcus species in hot spring mats are sets of ecologically interchangeable individuals that are differently adapted, that these adaptations control their distributions, and that the resulting distributions constrain the activities of the species in space and time.