Biology subjects

Hanson, T. E.

Publications and source records attributed to Hanson, T. E..

4 recordsLinked to original sources

bioRxiv2026

A Sample to Results Workflow for Compositional Analysis of Multiplexed Amplicon Sequencing Experiments

Microbial communities play key roles in the transformation and cycling of elements ranging from required macronutrients to toxic metalloids. Next-generation sequencing has been applied across multiple ecosystems to probe the interplay of microbial community structure and functional potential with respect to elemental cycling. Shotgun metagenomics collects marker gene sequences without amplification and is costly for large numbers of samples and deep coverage. Conversely, amplicon sequencing of taxonomic marker genes, e.g. 16S and 18S rRNA, is cost-effective for large numbers of samples, but provides limited functional insight. A middle ground between the two approaches is needed to analyze community structure and functional potential within a sample while remaining cost-effective with high throughput. To address this need, we developed a standardized workflow for multiplexed amplicon sequencing from sample collection through data analysis for diverse sample types, including freshwater, sediments, and soils, that produces data and publication-ready figures for multiple taxonomic and functional genes for carbon, nitrogen, phosphorus, sulfur, and arsenic cycling for each sample analyzed. The workflows utility was shown by analyzing 11 taxonomic and functional gene amplicons sequenced from 25 samples with high technical replicate similarity. The workflow is named CAMASE for Compositional Analysis of Multiplex Amplicon Sequencing Experiments. This proof-of-concept shows that CAMASE economically produces standard amplicon sequencing outputs (ASV/OTU counts and taxonomy, PCA, and relative abundance plots) for hundreds of amplicon by sample combinations and provides specific recommendations for implementation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/741237v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@14c746corg.highwire.dtl.DTLVardef@6f9890org.highwire.dtl.DTLVardef@894038org.highwire.dtl.DTLVardef@d72b2a_HPS_FORMAT_FIGEXP M_FIG C_FIG Samples are collected in a preservative and material collected on filters prior to DNA extraction. Target gene amplicons are produced in parallel with internal barcodes enabling sequencing in a single run followed by compositional data analysis. All wet lab protocols, code markdowns, and templates for required metadata files are available at https://hansonlabgit.dbi.udel.edu/aprange/CAMASE. Created in BioRender. Bennett, A. (2026) https://BioRender.com/ymnojt0

microbiology↗

bioRxiv2024

Chlorobaculum tepidum Outer Membrane Vesicles Are Likely a Significant Route for Biogenic Sulfur Transport.

Outer membrane-derived vesicles (OMVs) have been studied in different phyla of Gramnegative bacteria, most extensively in the Pseudomonadota, where they have been shown to participate in diverse biological and environmental processes. To date, the production of OMVs has not been reported in the Chlorobiaceae within the phylum Chlorobiota. Chlorobaculum. tepidum is the model organism for the Chlorobiaceae that synthesizes and consumes insoluble extracellular sulfur (S(0)) globules by an unknown mechanism. Here, we report evidence implicating outer membrane vesicles in biogenic S(0) globule synthesis. We demonstrate that Cba. tepidum secretes OMVs in the extracellular milieu, and that OMV concentration and size vary with growth conditions, particularly sulfide concentration. A core of 31 proteins involved in diverse biological processes such as cell wall biogenesis, inorganic ion transport and metabolism were found to be shared between OMVs, extracellular S(0) globules and Cba. tepidum intact cells. Multiple analytical methods indicated that OMVs contain S(0) and that OMVs and biogenic S(0) globules share protein and polysaccharide signatures, including lipooligosaccharides. Together these lines of evidence indicate that Cba. tepidums OMVs are one component of sulfur transport between cells and extracellular sulfur globules. IMPORTANCEAll living cells must exchange material with their environment while maintaining cellular integrity. This is a particular challenge for materials that are not water soluble, yet many bacteria utilize insoluble materials for energy conservation and as nutrients for growth. Here we show that Cba. tepidum makes outer membrane vesicles and that these vesicles are likely involved in the exchange of material with extracellular elemental sulfur globules formed and consumed by Cba. tepidum as part of its energy metabolism based on oxidizing reduced sulfur compounds like hydrogen sulfide. These data expand our basic understanding of Cba. tepidums metabolism. As elemental sulfur is an industrial by-product with a limited number of uses, the information here may help enable the use of additional sulfur compounds by Cba. tepidum to drive the synthesis of biomass and/or specialty biochemicals from waste elemental sulfur by this autotrophic bacterium.

microbiology↗

bioRxiv2017

N-methyl-bacillithiol, a new metabolite discovered in the Chlorobiaceae, indicates that bacillithiol and derivatives are widely phylogenetically distributed.

Low-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae. Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed that Chlorobaculum tepidum contained a novel LMW thiol with a mass of 412 {+/-} 1 Da corresponding to a molecular formula of C14H24N2O10S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, the Cba. tepidum thiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in all Chlorobi examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes. A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobi, Bacteroidetes, Deinococcus-Thermus, and Firmicutes, but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria.\n\nSignificance StatementHere, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor in Cba. tepidum and the gene, nmbA, encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailed in vivo and in vitro dissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.

microbiology↗

bioRxiv2017

dRNA-seq implicates sulfide as master regulator of S(0) metabolism in Chlorobaculum tepidum and other green sulfur bacteria

The green sulfur bacteria (Chlorobiaceae) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S(0), and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum, the model system for the Chlorobiaceae, both produces and consumes extracellular S(0) globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e. specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA-seq (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S(0), and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide mediated repression is the dominant regulatory mode in Cba. tepidum. First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the gene CT1277, encoding a putative regulatory protein, leads to constitutive over-expression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae. Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other Chlorobiaceae.\n\nImportanceElemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum both produces and consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur, and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This study identifies new genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this study paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements to control gene expression, a key element of strain engineering.

microbiology↗