Biology subjects

Stein, L. Y.

Publications and source records attributed to Stein, L. Y..

7 recordsLinked to original sources

bioRxiv2026

Diverse phages of ammonia oxidizers with the potential to modulate nitrification

Agriculture is a major source of anthropogenic greenhouse-gas emissions and the largest source of nitrous oxide (N2O), an extremely potent greenhouse gas and ozone-depleting agent. Soil N2O emissions are largely driven by nitrification, in which ammonia-oxidizing microorganisms catalyze the oxidation of ammonia to nitrite. Nitrification not only mediates N2O fluxes but also reduces fertilization efficiency and contributes to eutrophication through nitrate leaching.\ Bacteriophage (phage)-based control of microbial communities is gathering interest; however, phages infecting ammonia-oxidizers are mostly uncharacterized, with only one lytic phage having been described, limiting the potential for developing phage-mediated nitrification control. Here, we characterized the largest set of cultivated phages infecting ammonia-oxidizing bacteria (AOB) to date: 45 dsDNA phages collected from urban wastewater, infecting four AOB species, with 16 demonstrating cross-genus host ranges and capable of eliminating nitrification activity in liquid cultures. Phylogenetic and taxonomic analyses revealed six proposed families of Caudoviricetes and several monophyletic clades, likely representing higher-level lineages. Structure-guided genome annotation revealed a diverse collection of auxiliary metabolic genes encoded by phage, ranging from a complete ABC transporter cassette to a large antimicrobial resistance gene cluster. These results unveil previously unrecognized diversity of cultivated AOB phages and their potential to alter host physiology. Our data describes a broad taxonomic and functional repertoire of cultured AOB phages, suggesting that viruses play a significant and complex role in nitrification. Moreover, we outline an effective methodological framework for isolating AOB phages from environmental samples. These results reframe our understanding of environmental nitrification and enable intensified cultivation, characterization and use of phages for its control.

microbiology↗

bioRxiv2025

Prolific release of S-layer units and associated proteins by the methanotrophic bacterium Methylomicrobium album BG8

Some methanotrophs synthesize S-layers that overlay their outer membrane. TEM imaging revealed that Methylomicrobium album BG8 constitutively sheds abundant S-layer units, a phenotype not found in 7 other methanotrophs, even though Methylotuvimicrobium buryatense 5GB1 produces a similar structure. Release of S-layer units occurred regardless of carbon (methane or methanol) or nitrogen (ammonium or nitrate) source, with 50X trace metals, under copper deprivation, and at all growth phases. The released S-layer units were isolated from the culture medium of M. album BG8 by density gradient centrifugation for proteome analysis. The proteome revealed the S-layer protein subunits, transporters for calcium uptake including TolC and Repeats-in-Toxin (RTX) proteins, transporters for uptake for cobalamin and siderophores, cell wall biogenesis proteins, and proteins with Type I secretion system (T1SS) target domains. M. album BG8 adapted to grow at pH 4 lost its S-layer and genome analysis revealed a frameshift mutation plus reduced expression of the S-layer unit gene plus the deletion and almost no expression of an S-layer-associated porin gene. Together, the results suggest that the biogenesis and secretion of M. album BG8 S-layer is mediated by its associated T1SS, the S-layer possesses metal acquisition functions, and low pH adaptation of M. album BG8 results in loss of S-layer, likely due to reduced, or incomplete, expression of S-layer units and loss of an associated porin. The involvement of the T1SS and shedding phenotype of the S-layer in M. album BG8 could be applied towards selective secretion of proteins and other factors of bioindustrial interest. ImportanceThe methanotrophic bacterium M. album BG8 produces and sheds large quantities of S-layer units into the culture medium regardless of carbon or nitrogen source, metal availability or growth phase. Of the 8 methanotrophic bacteria screened, only M. album BG8 possessed this phenotype. Proteomics analysis of density gradient purified culture supernatant identified the S-layer protein units and proteins involved in metal uptake and S-layer biogenesis, some with secretion signals for the T1SS. M. album BG8 adapted to grow at low pH lost production of its S-layer due to mutations in the genes encoding S-layer units and an associated porin. Better understanding of M. album BG8 S-layer production and its shedding phenotype could be harnessed for exporting expressed proteins and bioproducts of industrial interest for ease of collection and downstream processing.

microbiology↗

bioRxiv2024

Diverse bacteriohemerythrin genes of Methylomonas denitrificans FJG1 provide insight into the survival and activity of proteobacterial methanotrophs under hypoxia

Proteobacterial methanotrophs are dependent on the oxidation of methane for ATP production and assimilation of carbon into biomass. Interestingly, some types of gammaproteobacterial methanotrophs thrive in oxygen-depleted zones of lakes and other aquatic ecosystems despite their reliance on oxygen to support methane oxidation. The model gammaproteobacterial methanotroph, Methylomonas denitrificans FJG1, oxidizes methane coupled to nitrate reduction under hypoxia and highly upregulates its expression of bacteriohemerythrin (Bhr). Bhr is a homolog of eukaryotic hemerythrin, which is a protein associated with oxygen binding. Ten bhr homologs were identified in the genome of M. denitrificans FJG1, requiring phylogenetic and gene expression analyses to pinpoint which homolog is likely responsible for delivering oxygen to support methane oxidation to under low oxygen conditions. This study examined the prevalence and phylogeny of the 10 bhr homologs from M. denitrificans FJG1 in the genomes of other methanotrophs and across the Bacteria. The homolog denoted "bhr-00" was specific to methanotroph genomes, was highly expressed in M. denitrificans FJG1 under hypoxia, and its predicted structure was nearly identical to a purified oxygen-scavenging hemerythrin protein from Methylococcus capsulatus Bath. Other bhr homologs upregulated from denitrifying cultures of M. denitrificans FJG1 included those with gene neighborhoods related to oxygen sensing, denitrification and chemotaxis. Together, this study uncovered potential multifunctional roles of bacteriohemerythrin genes of M. denitrificans FJG1 under low oxygen conditions and identified the bhr homolog that most likely enables and supports oxygen delivery to methane monooxygenase enzymes in anoxic ecosystems. ImportanceAerobic gammaproteobacterial methanotrophs can survive and grow in anoxic lakes, but mechanisms that provide them with oxygen to support methane oxidation remain uncharacterized. Methylomonas denitrificans FJG1 encodes 10 copies of bacteriohemerthyrin (bhr), of which 7 are expressed at the mRNA level under low oxygen conditions. Comparing the 10 bhr homologs from M. denitrificans FJG1 with those from other methanotrophs and bacterial genomes shows that two are specific to methanotrophs. Gene neighbourhoods surrounding conserved bhr genes in methanotrophs suggest a range of potential functions including oxygen respiration, oxygen sensing, chemotaxis, and nitrate reduction. The results from this study illuminate a previously undescribed diversity of structures and potential functions of bhr homologs in M. denitrificans FJG1 and related methanotrophic bacteria. The results pinpoint a methanotroph-specific homolog, bhr-00, that is likely responsible for oxygen binding and delivery to methane monooxygenase enzymes to promote methane oxidation in low oxygen ecosystems.

microbiology↗

bioRxiv2023

Evidence for a core set of microbial lichen symbionts from a global survey of metagenomes

Lichens are the archetypal symbiosis and the one for which the term was coined. Although application of shotgun sequencing techniques has shown that many lichen symbioses can harbour more symbionts than the canonically recognized fungus and photobiont, no global census of lichen organismal composition has been undertaken. Here, we analyze the genome content of 437 lichen metagenomes from six continents, and show that four bacterial lineages occur in the majority of lichen symbioses, at a frequency on par with algal photobionts. A single bacterial genus, Lichenihabitans, occurs in nearly one-third of all lichens sampled. Genome annotations from the most common lichen bacterial symbionts suggest they are aerobic anoxygenic photoheterotrophs and produce essential vitamins, but do not fix nitrogen. We also detected secondary basidiomycete symbionts in about two-thirds of analyzed metagenomes. Our survey suggests a core set of four to seven microbial symbionts are involved in forming and maintaining lichen symbioses.

microbiology↗

bioRxiv2022

Lysogenized phages of methanotrophic bacteria show a broad and untapped genetic diversity

Methanotrophs are a unique class of bacteria with the ability to metabolize single-carbon compounds such as methane. They play an important role in the global methane cycle and have great potential as industrial platforms for the bioconversion of methane from industrial waste streams into valuable products, such as biofuels and bioplastics. However, many aspects of methanotroph biology have yet to be elucidated, including the prevalence and impact of lysogenized bacteriophages (phages), which can greatly affect both the ecology and the industrial performance of these bacteria. The present study investigates the presence of putative prophages in three gammaproteobacterial (Methylobacter marinus A45, Methylomicrobium album BG8, Methylomonas denitrificans FJG1) and two alphaproteobacterial (Methylosinus trichosporium OB3b, Methylocystis sp. Rockwell) methanotrophs using four programs predicting putative phage sequences (PhageBoost, PHASTER, Phigaro, and Island Viewer). Mitomycin C was used to trigger induction of prophages, which was monitored through infection dynamics. Successfully induced phages from M. marinus A45 (MirA1, MirA2), M. album BG8 (MirB1), and M. trichosporium OB3b (MirO1) were isolated and characterized using transmission electron microscopy. Subsequently, bioinformatic analyses (BLAST and phylogenetics) were performed on three induced phages to obtain a profile of their respective genetic makeup. Their broad diversity and differences from previously known phages, based on whole genome and structural gene sequences, suggest they each represent a new phage family, genus and species: "Britesideviridae Inducovirus miraone", "Patronusviridae Enigmavirus miratwo", and "Kainiviridae Tripudiumvirus miroone" represented by isolates MirA1, MirA2, and MirO1, respectively.

microbiology↗

bioRxiv2021

Integrative genome-scale metabolic modeling reveals versatile metabolic strategies for methane utilization in Methylomicrobium album BG8

Methylomicrobium album BG8 is an aerobic methanotrophic bacterium that can mitigate environmental methane emission, and is a promising microbial cell factory for the conversion of methane to value-added chemicals. However, the lack of a genome-scale metabolic model (GEM) of M. album BG8 has hindered the development of systems biology and metabolic engineering of this methanotroph. To fill this gap, a high-quality GEM was constructed to facilitate a system-level understanding on the biochemistry of M. album BG8. Next, experimental time-series growth and exometabolomics data were integrated into the model to generate context-specific GEMs. Flux balance analysis (FBA) constrained with experimental data derived from varying levels of methane, oxygen, and biomass were used to model the metabolism of M. album BG8 and investigate the metabolic states that promote the production of biomass and the excretion of carbon dioxide, formate, and acetate. The experimental and modeling results indicated that the system-level metabolic functions of M. album BG8 require a ratio > 1:1 between the oxygen and methane specific uptake rates for optimal growth. Integrative modeling revealed that at a high ratio of oxygen-to-methane uptake flux, carbon dioxide and formate were the preferred excreted compounds; at lower ratios, however, acetate accounted for a larger fraction of the total excreted flux. The results of this study reveal a trade-off between biomass production and organic compound excretion and provide evidence that this trade-off is linked to the ratio between the oxygen and methane specific uptake rates.

microbiology↗

bioRxiv2021

Transcriptomic and metabolomic responses to carbon and nitrogen sources in Methylomicrobium album BG8

Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled its phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated haoAB hydroxylamine dehydrogenase and the overall central metabolic activity; whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species. IMPORTANCEMethanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism varies across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.

microbiology↗