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Small proteins are traditionally overlooked due to computational and experimental difficulties in detecting them. To systematically identify small proteins, we carried out a comparative genomics study on 1,773 human-associated metagenomes from four different body sites. We describe >4,000 conserved protein families, the majority of which are novel; ∼30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain and almost half are not represented in reference genomes. We identify putative housekeeping, mammalian-specific, defense-related, and protein families that are likely to be horizontally transferred. We provide evidence of transcription and translation for a subset of these families. Our study suggests that small proteins are highly abundant and those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.

Bugs as Drugs: Therapeutic Microbes for the Prevention and Treatment of Disease Robert A. Britton, Patrice D. Cani, editors; ASM Press, Washington, DC, USA, 2018; ISBN-10: 1555819699; ISBN-13: 978-1555819699; e-ISBN: [...]

Drug-resistant bacterial infections continue to occupy the headlines, amid increasingly desperate calls for new antibiotics to treat infectious diseases. Some of the most alarming reports concern 'Gram-positive' pathogens (1), which [...]

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers' sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. [ABSTRACT FROM AUTHOR]

Enterococcus faecalis is a resident of the human gut, though upon translocation to the blood or body tissues, it can be pathogenic. Here we discover and characterize two peptide-based quorum-sensing systems that transcriptionally modulate de novo purine biosynthesis in E. faecalis . Using a comparative genomic analysis, we find that most enterococcal species do not encode this system; E. moraviensis , E. haemoperoxidus and E. caccae , three species that are closely related to E. faecalis , encode one of the two systems, and only E. faecalis encodes both systems. We show that these systems are important for the intracellular survival of E. faecalis within macrophages and for the fitness of E. faecalis in a murine wound infection model. Taken together, we combine comparative genomics, microbiological, bacterial genetics, transcriptomics, targeted proteomics and animal model experiments to describe a paired quorum sensing mechanism that directly influences central metabolism and impacts the pathogenicity of E. faecalis ., Competing Interests: Declaration of Interests Cesar de la Fuente-Nunez provides consulting services to Invaio Sciences and is a member of the Scientific Advisory Boards of Nowture S.L., Peptidus, and Phare Bio. De la Fuente is also on the Advisory Board of the Peptide Drug Hunting Consortium (PDHC). The de la Fuente Lab has received research funding or in-kind donations from United Therapeutics, Strata Manufacturing PJSC, and Procter & Gamble, none of which were used in support of this work.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker., Importance: Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

Small-molecule screening campaigns of model bacteria have been conducted extensively in biotechnology and pharmaceutical companies to search for novel compounds with antibacterial activity. Recently, there has been increasing interest in running such high-throughput screens within academic settings to answer questions in biology. In this respect, whole-cell screening has the particular advantage of identifying compounds with physical and chemical properties compatible with microbial cell permeation, thereby providing probes with which to study diverse aspects of microbial cell physiology and biochemistry. The focus of this chapter is to describe a general method of running a high-throughput screen against a model bacterium to identify small molecules with growth inhibitory activity. Once the primary bioactives have been identified, the determination of their dose-response relationships with the target microbe further characterizes their growth inhibitory effect.