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Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes
The first step in transcription initiation in eukaryotes is mediated by the TATA-binding protein, a subunit of the transcription factor IID complex. We have cloned and sequenced the gene for a presumptive homolog of this eukaryotic protein from Thermococcus celer, a member of the Archaea (formerly archaebacteria). The protein encoded by the archaeal gene is a tandem repeat of a conserved domain, corresponding to the repeated domain in its eukaryotic counterparts. Molecular phylogenetic analyses of the two halves of the repeat are consistent with the duplication occurring before the divergence of the archael and eukaryotic domains. In conjunction with previous observations of similarity in RNA polymerase subunit composition and sequences and the finding of a transcription factor IIB-like sequence in Pyrococcus woesei (a relative of T. celer) it appears that major features of the eukaryotic transcription apparatus were well-established before the origin of eukaryotic cellular organization. The divergence between the two halves of the archael protein is less than that between the halves of the individual eukaryotic sequences, indicating that the average rate of sequence change in the archael protein has been less than in its eukaryotic counterparts. To the extent that this lower rate applies to the genome as a whole, a clearer picture of the early genes (and gene families) that gave rise to present-day genomes is more apt to emerge from the study of sequences from the Archaea than from the corresponding sequences from eukaryotes.
Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes
The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.
Filling gaps in bacterial catabolic pathways with computation and high-throughput genetics
To discover novel catabolic enzymes and transporters, we combined high-throughput genetic data from 29 bacteria with an automated tool to find gaps in their catabolic pathways. GapMind for carbon sources automatically annotates the uptake and catabolism of 62 compounds in bacterial and archaeal genomes. For the compounds that are utilized by the 29 bacteria, we systematically examined the gaps in GapMind’s predicted pathways, and we used the mutant fitness data to find additional genes that were involved in their utilization. We identified novel pathways or enzymes for the utilization of glucosamine, citrulline, myo-inositol, lactose, and phenylacetate, and we annotated 299 diverged enzymes and transporters. We also curated 125 proteins from published reports. For the 29 bacteria with genetic data, GapMind finds high-confidence paths for 85% of utilized carbon sources. In diverse bacteria and archaea, 38% of utilized carbon sources have high-confidence paths, which was improved from 27% by incorporating the fitness-based annotations and our curation. GapMind for carbon sources is available as a web server ( http://papers.genomics.lbl.gov/carbon ) and takes just 30 seconds for the typical genome.
Functional Horizontal Gene Transfer From Bacteria to Eukaryotes
The antiquity of bacteria and archaea in part explains why they, along with viruses, encode most of the genetic and biochemical diversity on Earth. Eukaryotic life evolved into a world teeming with prokaryotes, and so bacteria (especially) have inevitably affected eukaryotic biology as parasitic, commensal, or beneficial symbionts. But along with these important organismal interactions, the ubiquity and diversity of bacteria have also made them frequent sources of horizontally transferred DNA into eukaryotic genomes. Here we survey the role of bacterial genes throughout the eukaryotic lineage. We review what steps horizontal gene transfers (HGTs) take in becoming functional, what bacterial groups these HGTs come from, and what functions these HGTs typically bestow on their eukaryotic recipient. We classify HGTs into two broad types: those that maintain preexisting functions and those that add new functionality to the recipient. We find that genes involved in host nutrition, protection, and adaptation to extreme environments are the most common HGTs from bacterial to eukaryotic genomes.
Microbial Adaptation to Spaceflight Is Correlated With Bacteriophage-Encoded Functions
Evidence from the International Space Station suggests microbial populations are rapidly adapting to the spacecraft environment; however, the mechanism of this adaptation is not understood. Bacteriophages are prolific mediators of bacterial adaptation on Earth. Here we survey 245 genomes sequenced from bacterial strains isolated on the International Space Station for dormant (lysogenic) bacteriophages. Our analysis indicates phage-associated genes are significantly different between spaceflight strains and their terrestrial counterparts. Additionally, we identify 283 complete prophages, those that could initiate bacterial lysis and infect additional hosts, of which 21% are novel. These prophage regions encode functions that correlate with increased persistence in extreme environments, such as spaceflight, to include antimicrobial resistance and virulence, DNA damage repair, and dormancy. Our results correlate microbial adaptation in spaceflight to bacteriophage-encoded functions that may impact human health in spaceflight.
Speeding genomic island discovery through systematic design of reference database composition
Background Genomic islands (GIs) are mobile genetic elements that integrate site-specifically into bacterial chromosomes, bearing genes that affect phenotypes such as pathogenicity and metabolism. GIs typically occur sporadically among related bacterial strains, enabling comparative genomic approaches to GI identification. For a candidate GI in a query genome, the number of reference genomes with a precise deletion of the GI serves as a support value for the GI. Our comparative software for GI identification was slowed by our original use of large reference genome databases (DBs). Here we explore smaller species-focused DBs. Results With increasing DB size, recovery of our reliable prophage GI calls reached a plateau, while recovery of less reliable GI calls (FPs) increased rapidly as DB sizes exceeded ~500 genomes; i.e., overlarge DBs can increase FP rates. Paradoxically, relative to prophages, FPs were both more frequently supported only by genomes outside the species and more frequently supported only by genomes inside the species; this may be due to their generally lower support values. Setting a DB size limit for our SMA ll R anked T ailored (SMART) DB design speeded runtime ~65-fold. Strictly intra-species DBs would tend to lower yields of prophages for small species (with few genomes available); simulations with large species showed that this could be partially overcome by reaching outside the species to closely related taxa, without an FP burden. Employing such taxonomic outreach in DB design generated redundancy in the DB set; as few as 2984 DBs were needed to cover all 47894 prokaryotic species. Conclusions Runtime decreased dramatically with SMART DB design, with only minor losses of prophages. We also describe potential utility in other comparative genomics projects.
Dataset for the Danczak et al., 2025 manuscript about bacterial-fungal interactions
We generated genome-resolved multiomics data from a series of metagenomic and metatranscriptomic sequencing. Specifically, we acquired, functionally annotated, and taxonomically classified both bacterial and eukaryotic metagenome assembled genomes (MAGs). For bacterial MAGs, we assembled eukaryotic float metagenomic sequencing data from JGI using MEGAHIT, binned and refined MAGs using MetaWRAP and dRep, functionally annotated MAGs using eggNOG mapper, and assigned taxonomy using GTDB-tk. For eukaryotic MAGs, we first identified potentially eukaryotic contigs from a coassembly of eukaryotic float metagenomic sequencing data from JGI using EukRep and Whokaryote, binned MAGs using MetaBAT2, functionally annotated MAGs using eggNOG mapper, and assigned taxonomy using Eukulele. Bulk metatranscriptomic reads were mapped to bacterial MAGs and polyA-metatranscriptomic read were mapped to eukaryotic MAGs using bbmap.
Discovery and Functional Characterization of Genomic Islands for Non-Model Bacterial Systems
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CRISPR–Cas9 bends and twists DNA to read its sequence
In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.
Ecological divergence of syntopic marine bacterial species is shaped by gene content and expression
Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species’ divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species’ pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare.
Activity, structure, and diversity of Type II proline-rich antimicrobial peptides from insects
Abstract Apidaecin 1b (Api), the first characterized Type II Proline-rich antimicrobial peptide (PrAMP), is encoded in the honey bee genome. It inhibits bacterial growth by binding in the nascent peptide exit tunnel of the ribosome after the release of the completed protein and trapping the release factors. By genome mining, we have identified 71 PrAMPs encoded in insect genomes as pre-pro-polyproteins. Having chemically synthesized and tested the activity of 26 peptides, we demonstrate that despite significant sequence variation in the N-terminal sequence, the majority of the PrAMPs that retain the conserved C-terminal sequence of Api are able to trap the ribosome at the stop codons and induce stop codon readthrough—all hallmarks of Type II PrAMP mode of action. Some of the characterized PrAMPs exhibit superior antibacterial activity in comparison with Api. The newly solved crystallographic structures of the ribosome complexed with Api and with the more active peptide Fva1 from the stingless bee demonstrate the universal placement of the PrAMPs’ C-terminal pharmacophore in the post-release ribosome despite variations in their N-terminal sequence.
Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway
Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.
Evolutionary Flexibility and Rigidity in the Bacterial Methylerythritol Phosphate (Mep) Pathway
Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.
High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome
We present Microbe-seq, a high-throughput single-microbe method that yields strain-resolved genomes from complex microbial communities. We encapsulate individual microbes into droplets with microfluidics and liberate their DNA, which we amplify, tag with droplet-specific barcodes, and sequence. We use Microbe-seq to explore the human gut microbiome; we collect stool samples from a single individual, sequence over 20,000 microbes, and reconstruct nearly-complete genomes of almost 100 bacterial species, including several with multiple subspecies strains. We use these genomes to probe genomic signatures of microbial interactions: we reconstruct the horizontal gene transfer (HGT) network within the individual and observe far greater exchange within the same bacterial phylum than between different phyla. We probe bacteria-virus interactions; unexpectedly, we identify a significant in vivo association between crAssphage, an abundant bacteriophage, and a single strain of Bacteroides vulgatus. Microbe-seq contributes high-throughput culture-free capabilities to investigate genomic blueprints of complex microbial communities with single-microbe resolution.
Linking soil microbial genomic features to forest-to-pasture conversion in the Amazon
Genome-resolved metagenomics was applied to recover soil archaeal and bacterial metagenome-assembled genomes (MAGs) from three forests and three pastures of the Brazilian Eastern Amazon.
Hyporheic zone, river, and groundwater metagenome resolved genomes and rpS3 genes in East River Watershed, Colorado USA Summer 2020, 2021
Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from water filter collected across 8 locations along the East River Watershed, CO, and 1 nearby groundwater well. The purpose was to look for connectivity and similarities across the network and to see the impact of the groundwater. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed community composition and strain similarities between the sites and we also compared it to previous metagenomic studies within the watershed looking at floodplain (Matheus Carnevali et al. 2021) and hillslope (Lavy et al. 2019) microbiomes. Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from filters across 8 locations during August 2020 and July 2021. This resulted in 32 samples. The groundwater sample was sequenced at UC Berkley's QB3. The other 31 samples were sequenced at University of Maryland. Metagenomes were assembled using four autobinners and the best bins were selected using dasTool. The genomes were dereplicated at 95% with dRep and the subset of winning genomes were manually curated based on visual inspection of taxonomic profile, GC content, coverage, and a set of 51 bacterial single copy genes (BSCG), and 38 archaeal signal copy genes (ASCG). The dataset includes a zip file of 311 genomes (HZ_River_SW_MAGS_Dereplicated_95.zip). The dataset additionally includes a zipped file of ribosomal protein small subunit 3 (rpS3) proteins from the hyporheic zone and river data (rpS3_Proteins_HZ_River.zip), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a location metadata file (locations.csv). This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.
Organic carbon oxidation state shapes fermentative methanogenic microbiomes and controls greenhouse gas fluxes
Organic compounds with a negative nominal oxidation state of carbon (NOSC) are thermodynamically recalcitrant in anaerobic ecosystems, but few studies have measured the influence of NOSC on carbon degradation rates, gaseous product yields, or microbiome composition. We amended anaerobic rice paddy sediment microcosms with water-soluble monomeric organic carbon compounds varying in NOSC. Consistent with thermodynamic and stoichiometric predictions, negative NOSC compounds are catabolized more slowly but produce more methane per mole of carbon. Negative NOSC microbiomes have higher alpha diversity, more syntrophs and methanogens, and fewer fermentative bacteria. Strikingly, fermentative bacterial taxa display genomically encoded NOSC catabolic preferences both in the lab and field. Negative NOSC-preferring fermenters have longer predicted doubling times, consistent with the thermodynamic recalcitrance of their preferred substrates. We propose that microbial NOSC catabolic preferences may reflect the thermodynamic niche of microorganisms and we anticipate that extending research on microbial catabolic preferences to a greater variety of organic carbon substrates and diverse microbiomes will improve our understanding of microbial carbon cycling and trait evolution.