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ABSTRACT Most of Earth’s iron is mineral-bound, but it is unclear how and to what extent iron-oxidizing microbes can use solid minerals as electron donors. A prime candidate for studying mineral-oxidizing growth and pathways is Sideroxydans lithotrophicus ES-1, a robust, facultative iron oxidizer with multiple possible iron oxidation mechanisms. These include Cyc2 and Mto pathways plus other multiheme cytochromes and cupredoxins, and so we posit that the mechanisms may correspond to different Fe(II) sources. Here, S. lithotrophicus ES-1 was grown on dissolved Fe(II)-citrate and magnetite. S. lithotrophicus ES-1 oxidized all dissolved Fe²⁺ released from magnetite and continued to build biomass when only solid Fe(II) remained, suggesting it can utilize magnetite as a solid electron donor. Quantitative proteomic analyses of S. lithotrophicus ES-1 grown on these substrates revealed global proteome remodeling in response to electron donor and growth state and uncovered potential proteins and metabolic pathways involved in the oxidation of solid magnetite. While the Cyc2 iron oxidases were highly expressed on both dissolved and solid substrates, MtoA was only detected during growth on solid magnetite, suggesting this protein helps catalyze oxidation of solid minerals inS. lithotrophicusES-1. A set of cupredoxin domain-containing proteins were also specifically expressed during solid iron oxidation. This work demonstrated that the iron oxidizer S. lithotrophicus ES-1 utilized additional extracellular electron transfer pathways when growing on solid mineral electron donors compared to dissolved Fe(II). IMPORTANCEMineral-bound iron could be a vast source of energy to iron-oxidizing bacteria, but there is limited physiological evidence of this metabolism, and it has been unknown whether the mechanisms of solid and dissolved Fe(II) oxidation are distinct. In iron-reducing bacteria, multiheme cytochromes can facilitate iron mineral reduction, and here, we link a multiheme cytochrome-based pathway to mineral oxidation, expanding the known functionality of multiheme cytochromes. Given the growing recognition of microbial oxidation of minerals and cathodes, increasing our understanding of these mechanisms will allow us to recognize and trace the activities of mineral-oxidizing microbes. This work shows how solid iron minerals can promote microbial growth, which, if widespread, could be a major agent of geologic weathering and mineral-fueled nutrient cycling in sediments, aquifers, and rock-hosted environments. 

Stochastic nature of gene expression leads to the complex formation of the bacterial transcriptome and proteome. In contrast to typical transcriptome studies, we employ a near wild-type, Syn1.0, of the naturally genome-reducedMycoplasmas, and the dramatically further genome-reduced JCVI-syn3A thus avoiding additional contributions from many non-essential cellular functions. To aid in profiling the transcriptional landscape within these bacteria, we present a bioinformatic analysis of the genetic sequence motifs implicated in modulating the stochastic gene expression events, coupled with genome-wide short-read (Illumina) and long-read (Oxford Nanopore Technologies and Pacfic Biosciences) RNA sequencing. The bioinformatic analysis coupled with information from structural studies assigns strengths of the Shine-Dalgarno signatures and identifies both transcription initiation and termination sites, leading to predictions of RNA isoforms in Syn1.0 (and related organisms). The long-read and short-read RNA sequencing characterized the predicted transcriptional activity, and the long-read methods provide direct insight into the RNA isoform complexity within Syn1.0. Comparison of the RNA sequencing results with that of the bioinformatic analysis highlights the inability of bioinformatics alone to capture the results of bacterial transcription without including effects of RNA degradation. This study emphasizes the need for comparative analysis and potential dangers of genome reduction, exemplified through the discovery of altered gene expression patterns of JCVI-syn1.0 and JCVI-syn3A, achieved via the union of our transcriptome study with their proteomics data. Analysis of the transcriptomics data sets through a Jupyter notebook allows any genomic region to be easily examined. Table of Content Image 

We present an efficient, effective, and economical approach, named E3technology, for proteomics sample preparation. By immobilizing silica microparticles into the polytetrafluoroethylene matrix, we develop a robust membrane medium, which could serve as a reliable platform to generate proteomics-friendly samples in a rapid and low-cost fashion. We benchmark its performance using different formats and demonstrate them with a variety of sample types of varied complexity, quantity, and volume. Our data suggest that E3technology provides proteome-wide identification and quantitation performance equivalent or superior to many existing methods. We further propose an enhanced single-vessel approach, named E4technology, which performs on-filter in-cell digestion with minimal sample loss and high sensitivity, enabling low-input and low-cell proteomics. Lastly, we utilized the above technologies to investigate RNA-binding proteins and profile the intact bacterial cell proteome. 

ABSTRACT Extracellular vesicles (EVs) are small particles that are released by cells and mediate cell–cell communication by transferring bioactive molecules such as RNA. RNA cargo of EVs, including coding and non‐coding RNAs, can change the behaviour of recipient cells, affecting processes including gene expression, proliferation, and Fapoptosis. CircRNAs are stable and resistant to degradation and have been shown to be enriched in EVs. They play key roles in gene regulation and are also emerging as promising biomarkers for disease diagnosis due to their stability and disease‐specific expression. Although microRNAs (miRNAs) are the most well studied RNA cargo of EVs, very little is known about the mechanisms of enrichment of circular RNAs (circRNAs) as well as long linear RNAs. Here, we take a comprehensive genome‐wide approach to investigate the role of structuredness and shape along with GC%, size, exon count and coding potential, in the sorting and enrichment of circular and long linear RNAs into EVs. We developed a model using these parameters to predict the likelihood of EV packaging of RNA and it was validated by using single molecule RNA imaging of EV bound RNAs. Furthermore, we found that structuredness could explain the relative enrichment of circRNAs over their linear counterparts. These results were validated on existing public databases of circular and linear RNAs in EVs. By identifying and analysing these factors, we aim to better understand the complex mechanisms behind EV‐mediated RNA transfer and its impact on cell communication in both health and disease. This mechanistic understanding of RNA enrichment in EVs is crucial for engineering EVs with selective RNA cargo.