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In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.

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. To fill this gap, we argue that the research/scientific and biodefense/biosecurity communities should categorize agents based on how they impact their hosts to augment current list-based paradigms. Specifically, we propose integrating the results of multi-omics studies to identify bioagent-agnostic signatures (BASs) of disease—namely, patterns of biomarkers that accurately and reproducibly predict the impacts of infection or intoxication without prior knowledge of the causative agent. Here, we highlight three pathways that investigators might exploit as sources of signals to construct BASs and their applicability to this framework. The research community will need to forge robust interdisciplinary teams to surmount substantial experimental, technical, and data analytic challenges that stand in the way of our long-term vision. However, if successful, our functionality-based BAS model could present a means to more effectively surveil for and treat known and novel agents alike.

Bacillus anthracis is the causative agent of anthrax in animals and humans. The organism lies in a dormant state in the soil until introduced into an animal via, ingestion, cutaneous inoculation or inhalation. Once in the host, spores germinate into rapidly growing vegetative cells elaborating toxins. When animals die of anthrax, vegetative bacteria sporulate upon nutrient limitation in the carcass or soil while in the presence of air. After release into the soil environment, spores form a localized infectious zone (LIZ) at and around the carcass. Laboratory strains of B. anthracis produce fewer proteins associated with growth and sporulation compared to wild strains isolated from recent zoonotic disease events. We verified wild strains grow more rapidly than lab strains demonstrating a greater responsiveness to nutrient availability. Sporulation was significantly more rapid in these wild strains compared to lab strains, indicating wild strains are able to sporulate faster due to nutrient limitation while laboratory strains have a decrease in the speed at which they utilize nutrients and an increase in time to sporulation. These findings have implications for disease control at the LIZ as well as on the infectious cycle of this dangerous zoonotic pathogen.

In Escherichia coli, the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in the degS gene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS's protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS's basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS's protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σE-mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of ΔtolB ΔdegP mutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised.IMPORTANCE The Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP's periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( ha positive [ ha + ] or orfX + ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene ( bont ) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type ( ha + or orfX + ) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

The toxic protein ricin (also known as RCA60), found in the seed of the castor plant (Ricinus communis) is frequently encountered in law enforcement investigations. The ability to detect ricin by analyzing its proteolytic (tryptic) peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well established. However, ricin is just one member of a family of proteins in R. communis with closely related amino acid sequences, including R. communis agglutinin I (RCA120) and other ricin-like proteins (RLPs). Inferring the presence of ricin from its constituent peptides requires an understanding of the specificity, or uniqueness to ricin, of each peptide. Here we describe the set of ricin-derived tryptic peptides that can serve to uniquely identify ricin in distinction to closely-related RLPs and to proteins from other species. Other ricin-derived peptide sequences occur only in the castor plant, and still others are shared with unrelated species. We also characterized the occurrence and relative abundance of ricin and related proteins in an assortment of forensically relevant crude castor seed preparations. We find that whereas ricin and RCA120 are abundant in castor seed extracts, other RLPs are not represented by abundant unique peptides. Therefore, the detection of peptides shared between ricin and RLPs (other than RCA120) in crude castor seed extracts most likely reflects the presence of ricin in the sample.

The rapid pace of bacterial evolution enables organisms to adapt to the laboratory environment with repeated passage and thus diverge from naturally-occurring environmental ("wild") strains. Distinguishing wild and laboratory strains is clearly important for biodefense and bioforensics; however, DNA sequence data alone has thus far not provided a clear signature, perhaps due to lack of understanding of how diverse genome changes lead to convergent phenotypes, difficulty in detecting certain types of mutations, or perhaps because some adaptive modifications are epigenetic. Monitoring protein abundance, a molecular measure of phenotype, can overcome some of these difficulties. We have assembled a collection of Yersinia pestis proteomics datasets from our own published and unpublished work, and from a proteomics data archive, and demonstrated that protein abundance data can clearly distinguish laboratory-adapted from wild. We developed a lasso logistic regression classifier that uses binary (presence/absence) or quantitative protein abundance measures to predict whether a sample is laboratory-adapted or wild that proved to be ~98% accurate, as judged by replicated 10-fold cross-validation. Protein features selected by the classifier accord well with our previous study of laboratory adaptation in Y. pestis. The input data was derived from a variety of unrelated experiments and contained significant confounding variables. We show that the classifier is robust with respect to these variables. The methodology is able to discover signatures for laboratory facility and culture medium that are largely independent of the signature of laboratory adaptation. Going beyond our previous laboratory evolution study, this work suggests that proteomic differences between laboratory-adapted and wild Y. pestis are general, potentially pointing to a process that could apply to other species as well. Additionally, we show that proteomics datasets (even archived data collected for different purposes) contain the information necessary to distinguish wild and laboratory samples. This work has clear applications in biomarker detection as well as biodefense.

The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently taken up by another host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature, providing a basis for the type of response by public health officials and investigators.

Brucellosis is a common infection of feral swine throughout the United States. With the recent expansion of feral swine populations across the country, this disease poses an increasing threat to agriculture and hunters. The standard approach to Brucella surveillance in feral swine has been serological testing, which gives an indication of past exposure and is a rapid method of determining populations where Brucella is present. More in-depth analyses require bacterial isolation to determine the Brucella species and biovar involved. Ultimately, for a comprehensive understanding of Brucella epizootiology in feral swine, incorporation of genotyping assays has become essential. Fortunately, the past decade has given rise to an array of genetic tools for assessing Brucella transmission and dispersal. This review aims to synthesize what is known about brucellosis in feral swine and will cover prospective genomic techniques that may be utilized to develop more complete understanding of the disease and its transmission history.

The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement.

The Cpx and sigma(E) regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the sigma(E) pathway monitors the biogenesis of beta-barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of beta-barrel OMP mis-assembly, by utilizing mutants expressing either a defective beta-barrel OMP assembly machinery (Bam) or assembly defective beta-barrel OMPs. Analysis of specific mRNAs showed that Delta cpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the sigma(E) pathway. The synthetic conditional lethal phenotype of Delta cpxR in mutant Bam or beta-barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant beta-barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly-defective beta-barrel OMP species. Together, these results showed that both the Cpx and sigma(E) regulons are required to reduce envelope stress caused by aberrant beta-barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.

Recent studies have revealed that prokaryotes and viruses are abundant in extreme environments. However, almost nothing is known about viral contributions to community dynamics and ecosystem function in such environments. In the present study, sediment/mat and water samples from 2 separate thermal areas (Mickey and Borax) within the Alvord Desert Basin, Oregon, USA, were analyzed for prokaryotic and viral abundance, viral decay and production. Springs sampled at Mickey ranged in temperature from 61 to 94°C, and those at Borax from 61 to 96°C; all springs were near-neutral in pH. The total number of virus-like particles (10 6 particles ml -1 , Yo-Pro-1) exceeded total prokaryotic cell counts (DAPI staining, 10 5 cells ml -1 ). Virus to bacterium ratios for these systems ranged from 4.81 to 18.87. Viral production and decay rates were determined for sediment/mat samples and water from each hot spring; viral turnover rates ranged from 0.27 to 1.00 h -1 . Lysis rates of approximately 9.50% of cells h -1 in Borax springs vs. 21.70% in Mickey springs were extrapolated from viral production rates. Decay rates determined from hot springs at Mickey ranged from 0.09 to 0.20 h -1 , corresponding to turnover times of 5 to 11 h, while decay rates at Borax corresponded to viral turnover times of 12 to 14 h. Exposure of water samples to mitomycin C yielded lysogeny rates of 18 to 21% for the total prokaryotic communities in any given sample. Together, these data show that viruses exert an important influence on microbial communities and play a significant role in extreme thermal environments.

A variety of naturally occurring bacteria produce enzymes that cometabolically degrade trichloroethene (TCE), including organisms with aerobic oxygenases. Groundwater contaminated with TCE was collected from the aerobic region of the Test Area North site of the Idaho National Laboratory. Samples were evaluated with enzyme activity probes, and resulted in measurable detection of toluene oxygenase activity (6-79% of the total microbial cells). Wells from both inside and outside contaminated plume showed activity. Toluene oxygenase-specific PCR primers determined that toluene-degrading genes were present in all groundwater samples evaluated. In addition, bacterial isolates were obtained and possessed toluene oxygenase enzymes, demonstrated activity, and were dominated by the phylotype Pseudomonas. This study demonstrated, through the use of enzymatic probes and oxygenase gene identification, that indigenous microorganisms at a contaminated site were cometabolically active. Documentation such as this can be used to substantiate observations of natural attenuation of TCE-contaminated groundwater plumes.

RseA sequesters RpoE (σ(E)) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σ(E) to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σ(E) regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σ(E) levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σ(E)-dependent RybB::LacZ construct showed only a weak activation of the σ(E) pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σ(E) and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrA(L222Q), it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σ(E)-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σ(E)-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σ(E) may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σ(E) levels.