Rachel Porter

All Publications

• CUPID-seq enables highly multiplexed amplicon sequencing via combinatorial in-line dual indexing. bioRxiv : the preprint server for biology Fu, B., Porter, R. L., Shi, H., Ea, A. C., Espeleta, A. M., Ambat, A., Relman, D. A., Huang, K. C., Xue, K. S. 2026

  Abstract

  Targeted amplicon sequencing is widely used to profile genetic variation in defined genomic regions. In microbial ecology, for example, amplicon sequencing of the 16S and 18S ribosomal RNA genes has been transformative for characterizing microbial communities. However, on high-capacity sequencing platforms with patterned flow cells, throughput is constrained by the requirement for unique dual indexes (UDIs), which increases primer costs and limits the number of samples that can be pooled per sequencing run. Here, we introduce CUPID-seq (Combinatorial, Unique, Phased, In-line Dual-indexed sequencing), a highly multiplexed amplicon-sequencing strategy that increases scalability through combinatorial indexing across two rounds of PCR. CUPID-seq introduces phased, in-line UDIs during Round 1 gene-specific amplification, enabling multiple samples to share the same Illumina UDI during Round 2 PCR while remaining uniquely identifiable. This design reduces upfront costs by up to 85% and reduces library preparation time and reagent use by up to 40%. We develop and validate CUPID-seq primers targeting the 16S V4 region and provide a computational workflow for demultiplexing in-line indexes. Although optimized here for 16S-based profiling, CUPID-seq can be readily adapted to other user-defined amplicons. By reducing cost and increasing multiplexing capacity, CUPID-seq enables users to leverage high-throughput sequencing platforms more effectively across diverse biological contexts.

  View details for DOI 10.64898/2026.05.20.726713

  View details for PubMedID 42239137

  View details for PubMedCentralID PMC13228431

• Circadian clock control of ribosome composition promotes rhythmic translation and termination fidelity CELL REPORTS Lamb, T. M., Castillo, K. D., Porter, R., Wu, C., Purvine, S. O., Best, G., Zink, E., Preh, E. O., Shen, L., Sachs, M. S., Bell-Pedersen, D. 2025; 44 (11): 116484

  Abstract

  Ribosome composition is dynamic, shifting with cell state and stress, but whether it varies with circadian time is unknown. Here, we uncover circadian clock-driven changes in ribosome composition in Neurospora crassa. Mass spectrometry of ribosomes across circadian time identified six ribosomal proteins and one associated factor under clock control. Rhythms in eL31 abundance were validated in purified ribosomes, and deletion of el31 disrupted translation rhythms in nearly half of rhythmically translated mRNAs. N. crassa eL31 promotes circadian control of translation termination and impacts elongation fidelity while maintaining Mg homeostasis, a key determinant of translational accuracy. These findings reveal that the circadian clock reprograms ribosome composition to orchestrate rhythmic translation and fidelity, temporally expanding the proteome beyond the static genome to align cellular function with time of day.

  View details for DOI 10.1016/j.celrep.2025.116484

  View details for Web of Science ID 001608069900001

  View details for PubMedID 41160737

• On the growth and form of bacterial colonies NATURE REVIEWS PHYSICS Porter, R., Trenado-Yuste, C., Martinez-Calvo, A., Su, M., Wingreen, N. S., Datta, S. S., Huang, K. 2025

  View details for DOI 10.1038/s42254-025-00849-x

  View details for Web of Science ID 001563653100001

• Competition for shared resources increases dependence on initial population size during coalescence of gut microbial communities. Proceedings of the National Academy of Sciences of the United States of America Goldman, D. A., Xue, K. S., Parrott, A. B., Lopez, J. A., Vila, J. C., Jeeda, R. R., Franzese, L. R., Porter, R. L., Gray, I. J., DeFelice, B. C., Petrov, D. A., Good, B. H., Relman, D. A., Huang, K. C. 2025; 122 (11): e2322440122

  Abstract

  The long-term success of introduced populations depends on both their initial size and ability to compete against existing residents, but it remains unclear how these factors collectively shape colonization dynamics. Here, we investigate how initial population (propagule) size shapes the outcome of community coalescence by systematically mixing eight pairs of in vitro microbial communities at ratios that vary over six orders of magnitude, and we compare our results to neutral ecological theory. Although the composition of the resulting cocultures deviated substantially from neutral expectations, each coculture contained species whose relative abundance depended on propagule size even after ~40 generations of growth. Using a consumer-resource model, we show that this dose-dependent colonization can arise when resident and introduced species have high niche overlap and consume shared resources at similar rates. Strain isolates displayed longer-lasting dose dependence when introduced into diverse communities than in pairwise cocultures, consistent with our model's prediction that propagule size should have larger, more persistent effects in diverse communities. Our model also successfully predicted that species with similar resource-utilization profiles, as inferred from growth in spent media and untargeted metabolomics, would show stronger dose dependence in pairwise coculture. This work demonstrates that transient, dose-dependent colonization dynamics can emerge from resource competition and exert long-term effects on the outcomes of community coalescence.

  View details for DOI 10.1073/pnas.2322440122

  View details for PubMedID 40063808

• Degradation of polypropylene by fungi Coniochaeta hoffmannii and Pleurostoma richardsiae. Microbiological research Porter, R., Černoša, A., Fernández-Sanmartín, P., Cortizas, A. M., Aranda, E., Luo, Y., Zalar, P., Podlogar, M., Gunde-Cimerman, N., Gostinčar, C. 2023; 277: 127507

  Abstract

  The urgent need for better disposal and recycling of plastics has motivated a search for microbes with the ability to degrade synthetic polymers. While microbes capable of metabolizing polyurethane and polyethylene terephthalate have been discovered and even leveraged in enzymatic recycling approaches, microbial degradation of additive-free polypropylene (PP) remains elusive. Here we report the isolation and characterization of two fungal strains with the potential to degrade pure PP. Twenty-seven fungal strains, many isolated from hydrocarbon contaminated sites, were screened for degradation of commercially used textile plastic. Of the candidate strains, two identified as Coniochaeta hoffmannii and Pleurostoma richardsiae were found to colonize the plastic fibers using scanning electron microscopy (SEM). Further experiments probing degradation of pure PP films were performed using C. hoffmannii and P. richardsiae and analyzed using SEM, Raman spectroscopy and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). The results showed that the selected fungi were active against pure PP, with distinct differences in the bonds targeted and the degree to which each was altered. Whole genome and transcriptome sequencing was conducted for both strains and the abundance of carbohydrate active enzymes, GC content, and codon usage bias were analyzed in predicted proteomes for each. Enzymatic assays were conducted to assess each strain's ability to degrade naturally occurring compounds as well as synthetic polymers. These investigations revealed potential adaptations to hydrocarbon-rich environments and provide a foundation for further investigation of PP degrading activity in C. hoffmannii and P. richardsiae.

  View details for DOI 10.1016/j.micres.2023.127507

  View details for PubMedID 37793281

• Fatty Acid Synthesis Knockdown Promotes Biofilm Wrinkling and Inhibits Sporulation in Bacillus subtilis. mBio Arjes, H. A., Gui, H., Porter, R., Atolia, E., Peters, J. M., Gross, C., Kearns, D. B., Huang, K. C. 2022: e0138822

  Abstract

  Many bacterial species typically live in complex three-dimensional biofilms, yet much remains unknown about differences in essential processes between nonbiofilm and biofilm lifestyles. Here, we created a CRISPR interference (CRISPRi) library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain NCIB 3610 and investigated growth, biofilm colony wrinkling, and sporulation phenotypes of the knockdown library. First, we showed that gene essentiality is largely conserved between liquid and surface growth and between two media. Second, we quantified biofilm colony wrinkling using a custom image analysis algorithm and found that fatty acid synthesis and DNA gyrase knockdown strains exhibited increased wrinkling independent of biofilm matrix gene expression. Third, we designed a high-throughput screen to quantify sporulation efficiency after essential gene knockdown; we found that partial knockdowns of essential genes remained competent for sporulation in a sporulation-inducing medium, but knockdown of essential genes involved in fatty acid synthesis exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. We conclude that a subset of essential genes are particularly important for biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. IMPORTANCE For many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles held together by extracellular matrix. To examine how essential gene function varies between vegetative growth and the developmental states of biofilm formation and sporulation, we created and screened a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the biofilm-capable Bacillus subtilis strain 3610. High-throughput assays and computational algorithms identified a subset of essential genes involved in biofilm wrinkling and sporulation and indicated that fatty acid synthesis plays important and multifaceted roles in bacterial development.

  View details for DOI 10.1128/mbio.01388-22

  View details for PubMedID 36069446

• Circadian Clock Control of Translation Initiation Factor eIF2α Activity Requires eIF2γ-Dependent Recruitment of Rhythmic PPP-1 Phosphatase in Neurospora crassa. mBio Ding, Z., Lamb, T. M., Boukhris, A., Porter, R., Bell-Pedersen, D. 2021; 12 (3)

  Abstract

  The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of N. crassa PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of N. crassa eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that N. crassa eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events.IMPORTANCE Circadian clock control of mRNA translation contributes to the daily cycling of a significant proportion of the cellular protein synthesis, but how this is accomplished is not understood. We discovered that the clock in the model fungus Neurospora crassa regulates rhythms in protein synthesis by controlling the phosphorylation and dephosphorylation of a conserved translation initiation factor eIF2α. During the day, N. crassa eIF2α is phosphorylated and inactivated by CPC-3 kinase. At night, a clock-controlled phosphatase, PPP-1, dephosphorylates and activates eIF2α, leading to increased nighttime protein synthesis. Translation requires significant cellular energy; thus, partitioning translation to the night by the clock provides a mechanism to coordinate energy metabolism with protein synthesis and cellular growth.

  View details for DOI 10.1128/mBio.00871-21

  View details for PubMedID 34006661

  View details for PubMedCentralID PMC8262944