Computational and Systems Biology Advances to Enable Bioagent Agnostic Signatures.

Enumerated threat agent lists have long driven biodefense priorities. The global SARS-CoV-2 pandemic demonstrated the limitations of searching for known threat agents as compared to a more agnostic approach. Recent technological advances are enabling agent-agnostic biodefense, especially through the integration of multi-modal observations of host-pathogen interactions directed by a human immunological model.

Beyond the List: Bioagent-Agnostic Signatures Could Enable a More Flexible and Resilient Biodefense Posture Than an Approach Based on Priority Agent Lists Alone.

As of 2021, the biothreat policy and research communities organize their efforts around lists of priority agents, which elides consideration of novel pathogens and biotoxins. For example, the Select Agents and Toxins list is composed of agents that historic biological warfare programs had weaponized or that have previously caused great harm during natural outbreaks. Similarly, lists of priority agents promulgated by the World Health Organization and the National Institute of Allergy and Infectious Diseases are composed of previously known pathogens and biotoxins.

The small protein MgtS and small RNA MgrR modulate the PitA phosphate symporter to boost intracellular magnesium levels.

In response to low levels of magnesium (Mg ), the PhoQP two component system induces the transcription of two convergent genes, one encoding a 31-amino acid protein denoted MgtS and the second encoding a small, regulatory RNA (sRNA) denoted MgrR. Previous studies showed that the MgtS protein interacts with and stabilizes the MgtA Mg importer to increase intracellular Mg levels, while the MgrR sRNA base pairs with the eptB mRNA thus affecting lipopolysaccharide modification. Surprisingly, we found overexpression of the MgtS protein also leads to induction of the PhoRB regulon.

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance.

The AcrAB-TolC multidrug efflux pump confers resistance to a wide variety of antibiotics and other compounds in Escherichia coli. Here we show that AcrZ (formerly named YbhT), a 49-amino-acid inner membrane protein, associates with the AcrAB-TolC complex. Co-purification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB.

Competition assays using barcoded deletion strains to gain insight into small RNA function.

Ordered collections of barcoded deletion mutants can be screened rapidly in mixed cultures to uncover resistance and sensitivity phenotypes associated with the loss of a gene. As such, they are invaluable tools for assigning gene function in the post-genomic era. In this protocol, we describe methodologies for creating and employing barcoded deletion mutants in competitive screens as well as how to analyze the ensuing results.

Phosphorylation of Spo0A by the histidine kinase KinD requires the lipoprotein med in Bacillus subtilis.

The response regulatory protein Spo0A of Bacillus subtilis is activated by phosphorylation by multiple histidine kinases via a multicomponent phosphorelay. Here we present evidence that the activity of one of the kinases, KinD, depends on the lipoprotein Med, a mutant of which has been known to cause a cannibalism phenotype. We show that the absence of Med impaired and the overproduction of Med stimulated the transcription of two operons (sdp and skf) involved in cannibalism whose transcription is known to depend on Spo0A in its phosphorylated state (Spo0A∼P).

An expanding universe of small proteins.

Historically, small proteins (sproteins) of less than 50 amino acids, in their final processed forms or genetically encoded as such, have been understudied. However, both serendipity and more recent focused efforts have led to the identification of a number of new sproteins in both Gram-negative and Gram-positive bacteria. Increasing evidence demonstrates that sproteins participate in a wide array of cellular processes and exhibit great diversity in their mechanisms of action, yet general principles of sprotein function are emerging.

Small RNAs and small proteins involved in resistance to cell envelope stress and acid shock in Escherichia coli: analysis of a bar-coded mutant collection.

More than 80 small regulatory RNAs (sRNAs) and 60 proteins of 16 to 50 amino acids (small proteins) are encoded in the Escherichia coli genome. The vast majority of the corresponding genes have no known function. We screened 125 DNA bar-coded mutants to identify novel cell envelope stress and acute acid shock phenotypes associated with deletions of genes coding for sRNAs and small proteins.

Parallel pathways of repression and antirepression governing the transition to stationary phase in Bacillus subtilis.

The AbrB protein of the spore-forming bacterium Bacillus subtilis is a repressor of numerous genes that are switched on during the transition from the exponential to the stationary phase of growth. The gene for AbrB is under the negative control of the master regulator for entry into sporulation, Spo0A-P. It has generally been assumed that derepression of genes under the negative control of AbrB is achieved by Spo0A-P-mediated repression of abrB followed by rapid degradation of the AbrB protein.

A three-protein signaling pathway governing immunity to a bacterial cannibalism toxin.

We describe a three-protein signal-transduction pathway that governs immunity to a protein toxin involved in cannibalism by the spore-forming bacterium Bacillus subtilis. Cells of B. subtilis enter the pathway to sporulate under conditions of nutrient limitation but delay becoming committed to spore formation by killing nonsporulating siblings and feeding on the dead cells.

Direct measurement of acid efflux from isolated guinea pig pancreatic ducts.

Objectives: The current studies used the technique of microphysiometry to directly determine the effects of stimulators and inhibitors of pancreatic duct secretion on acid efflux from isolated pancreatic ducts.

Methods: Main and interlobular ducts were isolated from guinea pig pancreata by collagenase digestion and manual selection. Segments were placed in the chambers of a microphysiometer, which uses a silicon chip-based, light-addressable potentiometric sensor to determine the proton concentration in the superfusing solution.

Cannibalism by sporulating bacteria.

Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation. Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse. The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis.

Extracellular acidification parallels insulin secretion in INS-1 and HIT-T15 beta-cell lines.

As an alternative to manual assays that track insulin secretion, we tested a silicon-based biosensor that allows automated monitoring of extracellular acidification. Glucose stimulation of INS-1 and HIT-T15 cells resulted in a rapid increase in extracellular acidification in a biphasic and concentration-dependent fashion much like insulin secretion (EC(50) INS-1=5 mM and HIT-T15=1 mM). This response was attenuated by verapamil (10 microM) and stimulated by administration of glybenclamide (100 nM) or KCl-induced (40 mM) depolarization.