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At least 991 records · Page 55

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DOE OSTI2021

Impact of river channel lateral migration on microbial communities across a discontinuous permafrost floodplain

Permafrost soils store approximately twice the amount of carbon currently present in Earth’s atmosphere and are acutely impacted by climate change due to the polar amplification of increasing global temperature. Many organic-rich permafrost sediments are located on large river floodplains, where river channel migration periodically erodes and re-deposits the upper tens of meters of sediment. Channel migration exerts a first-order control on the geographic distribution of permafrost and floodplain stratigraphy and thus may affect microbial habitats. Here, to examine how river channel migration in discontinuous permafrost environments affects microbial community composition, we used amplicon sequencing of the 16S rRNA gene on sediment samples from floodplain cores and exposed riverbanks along the Koyukuk River, a large tributary of the Yukon River in west-central Alaska. Microbial communities are sensitive to permafrost thaw: communities found in deep samples thawed by the river closely resembled near-surface active layer communities in non-metric multidimensional scaling analyses but did not resemble floodplain permafrost communities at the same depth. Microbial communities also displayed lower diversity and evenness in permafrost than in both the active layer and permafrost-free point bars recently deposited by river channel migration. Taxonomic assignments based on 16S and quantitative PCR for the methyl-coenzyme M reductase functional gene demonstrated that methanogens and methanotrophs are abundant in older permafrost-bearing deposits, but not in younger, non-permafrost point bar deposits. The results suggested that river migration, which regulates the distribution of permafrost, also modulates the distribution of microbes potentially capable of producing and consuming methane on the Koyukuk River floodplain.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Listeria monocytogenes in Stone Fruits Linked to a Multistate Outbreak: Enumeration of Cells and Whole-Genome Sequencing

In 2014, the identification of stone fruits contaminated with led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

In Heliobacterium modesticaldum , as in many Firmicutes , deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum , as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ~80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes , which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum . Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Transcriptional Control of hgcAB by an ArsR-Like Regulator in Pseudodesulfovibrio mercurii ND132

The hgcAB gene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg methylator Pseudodesulfovibrio mercurii ND132, we evaluated transcriptional control of hgcAB by a putative ArsR encoded upstream and cotranscribed with hgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosylhomocysteine (SAH)- responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR proteins in P. mercurii. Using quantitative PCR (qPCR) and RNA sequencing (RNA-seq) transcriptome analyses, we confirmed this ArsR regulates hgcAB transcription and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link between hgcAB activity and arsenic transformations, with significant upregulation of other ArsRregulated arsenic resistance operons alongside hgcAB. Interestingly, wild-type ND132 was less sensitive to As(V) (but not As(III)) than an hgcAB knockout strain, supporting the idea that hgcAB may be linked to arsenic resistance. Arsenic significantly impacted rates of Hg methylation by ND132; however, responses varied with culture conditions. Differences in growth and metabolic activity did not account for arsenic impacts on methylation. While arsenic significantly increased hgcAB expression, hgcAB gene and transcript abundance was not a good predictor of Hg methylation rates. Taken together, these results support the idea that Hg and As cycling are linked in P. mercurii ND132. Our results may hold clues to the evolution of hgcAB and the controls on Hg methylation in nature. This work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that control hgcAB expression. We show that hgcAB expression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosylhomocysteine in our model organism, Pseudodesulfovibrio mercurii ND132. Exposure to arsenic also significantly impacted Pseudodesulfovibrio mercurii ND132 mercury methylation rates. However, expression of hgcAB was not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls on hgcAB expression, which is needed to better predict environmental methylmercury production

RNA-seq↗

DOE OSTI2019

Comparative Genomics Guides Elucidation of Vitamin B 12 Biosynthesis in Novel Human-Associated Akkermansia Strains

Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a “next-generation probiotic.” However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B 12 ) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B 12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B 12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B 12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B 12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B 12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

An estimated 1.5 billion microbial infections occur globally each year and result in ~4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ~10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

47 OTHER INSTRUMENTATION↗

DOE OSTI2017

Predominance and metabolic potential of Halanaerobium spp. in produced water from hydraulically fractured Marcellus Shale wells

Here, microbial activity in the produced water from hydraulically fractured oil and gas wells may potentially interfere with hydrocarbon production and cause damage to the well and surface infrastructure via corrosion, sulfide release, and fouling. In this study, we surveyed the microbial abundance and community structure of produced water sampled from 42 Marcellus Shale wells in southwestern Pennsylvania (well age ranged from 150 to 1,846 days) to better understand the microbial diversity of produced water. We sequenced the V4 region of the 16S rRNA gene to assess taxonomy and utilized quantitative PCR (qPCR) to evaluate the microbial abundance across all 42 produced water samples. Bacteria of the order Halanaerobiales were found to be the most abundant organisms in the majority of the produced water samples, emphasizing their previously suggested role in hydraulic fracturing-related microbial activity. Statistical analyses identified correlations between well age and biocide formulation and the microbial community, in particular, the relative abundance of Halanaerobiales . We further investigated the role of members of the order Halanaerobiales in produced water by reconstructing and annotating a Halanaerobium draft genome (named MDAL1), using shotgun metagenomic sequencing and metagenomic binning. The recovered draft genome was found to be closely related to the species H. congolense, an oil field isolate, and Halanaerobium sp. strain T82-1, also recovered from hydraulic fracturing produced water. Reconstruction of metabolic pathways revealed Halanaerobium sp. strain MDAL1 to have the potential for acid production, thiosulfate reduction, and biofilm formation, suggesting it to have the ability to contribute to corrosion, souring, and biofouling events in the hydraulic fracturing infrastructure.

04 OIL SHALES AND TAR SANDS↗

DOE OSTI2015

Microbial Community Analysis with Ribosomal Gene Fragments from Shotgun Metagenomes

Shotgun metagenomic sequencing does not depend on gene-targeted primers or PCR amplification; thus, it is not affected by primer bias or chimeras. However, searching rRNA genes from large shotgun Illumina data sets is computationally expensive, and no approach exists for unsupervised community analysis of small-subunit (SSU) rRNA gene fragments retrieved from shotgun data. We present a pipeline, SSUsearch, to achieve the faster identification of short-subunit rRNA gene fragments and enabled unsupervised community analysis with shotgun data. It also includes classification and copy number correction, and the output can be used by traditional amplicon analysis platforms. Shotgun metagenome data using this pipeline yielded higher diversity estimates than amplicon data but retained the grouping of samples in ordination analyses. We applied this pipeline to soil samples with paired shotgun and amplicon data and confirmed bias against Verrucomicrobia in a commonly used V6-V8 primer set, as well as discovering likely bias against Actinobacteria and for Verrucomicrobia in a commonly used V4 primer set. In conclusion, this pipeline can utilize all variable regions in SSU rRNA and also can be applied to large-subunit (LSU) rRNA genes for confirmation of community structure.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. In order to facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense , a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense , gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2018

Increased mortality in mice following immunoprophylaxis therapy with high dosage of nicotinamide in Burkholderia persistent infections

Bacterial persistence, known as non-inherited antibacterial resistance, is a contributing factor to the establishment of long-lasting chronic bacterial infections. In this study, we examined the ability of nicotinamide (NA) to potentiate different classes of antibiotics against Burkholderia thailandensis persister cells. Here we demonstrate that addition of NA in in vitro models of B. thailandensis infection resulted in a significant depletion of the persister population in response to various classes of antibiotics. We applied microfluidic bioreactors with a continuous media flow to study the effect of supplementation with a NA gradient on the recovery of B. thailandensis persister populations. A co-culture of human neutrophils pre-activated with 50 µM NA and B. thailandensis resulted in the most efficient reduction in the persister population. Applying single cell RNA FISH analysis and quantitative PCR, we found that NA inhibited gene expression of the stringent response regulator relA , implicated in the regulation of the persister metabolic state. In conclusion, we also demonstrate that a therapeutic dose of NA (250mg/kg), previously applied as immunoprophylaxis against antibiotic-resistant bacterial species, produced adverse effects in an in vivo murine infection model of the highly pathogenic Burkholderia pseudomallei , indicating that therapeutic dose and metabolite effects have to be carefully evaluated and tailored for every case of potential clinical application.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Proteomic and Mutant Analysis of the CO 2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO 2 + HCO 3 - + CO 3 2- ) with CO 2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854 ; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853 -encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea . In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea , the Euryarchaeota . Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment

We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Impact of Baloxavir Resistance-Associated Substitutions on Influenza Virus Growth and Drug Susceptibility

Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Here, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

The dynamics of SIV 2-LTR Circles in the Presence and Absence of CD8 + Cells

CD8 + cells play a key role in HIV/SIV infection, but their specific mechanism(s) of action in controlling the virus are unclear. 2-LTR circles are extrachromosomal products generated upon failed integration of HIV/SIV. To understand the specific effects of CD8 + cells on infected cells, we analyzed the dynamics of 2-LTR circles in SIVmac251-infected rhesus macaques (RM) treated with an integrase inhibitor (INT). Twenty RMs underwent CD8 + cell depletion, received RAL monotherapy or a combination of both. Blood, lymph nodes (LNs) and gut biopsies were routinely sampled. Plasma viral loads (pVLs) and 2-LTR circles from PBMCs and LN lymphocytes were measured with qRT-PCR. In the CD8 depletion group, an ~1 log increase in pVLs and a slow increase in PBMC 2-LTRs occurred following depletion. In the INT group, a strong decline in pVLs upon treatment initiation and no change in 2-LTR levels were observed. In the INT and CD8 + cell depletion group, a similar increase in pVLs following CD8 depletion was observed, with a modest decline following INT initiation, and 2-LTR circles significantly increased in PBMCs and LNs. Analyzing the 2-LTR data across all treatment groups with a mathematical model indicates that the data best supports an effect of CD8 + cells in killing cells prior to viral integration. Sensitivity analyses of these results confirm that effect, but also allow for additional effects, which the data does not discriminate well. Overall, we show that INT does not significantly increase the levels of 2-LTR circles. However, CD8 + cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT. CD8 + T cells play an essential role in controlling HIV and simian immunodeficiency virus (SIV) infection, but the specific mechanisms involved remain poorly understood. Due to failed viral infection, HIV and SIV can form 2-LTR extrachromosomal circles that can be quantified. We present novel data on the dynamics of these 2-LTR forms in a SIV-infected macaque model under three different treatment conditions: depletion of CD8 + cells; administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles; and combination of the two treatments. We used a new mathematical model to help interpret the data, and the results suggest that CD8 + cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8 + cells in SIV infection. Here, confirmation of our results would be an important step in understanding immune control of HIV.

2-LTR circles↗

DOE OSTI2015

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli , Phaeobacter inhibens , Pseudomonas stutzeri , Shewanella amazonensis , and Shewanella oneidensis . To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens , we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Metabolic regulation as a consequence of anaerobic 5-methylthioadenosine recycling in Rhodospirillum rubrum

Rhodospirillum rubrum possesses a novel oxygen-independent, aerobic methionine salvage pathway (MSP) for recycling methionine from 5-methylthioadenosine (MTA), the MTA-isoprenoid shunt. This organism can also metabolize MTA as a sulfur source under anaerobic conditions, suggesting that the MTA-isoprenoid shunt may also function anaerobically as well. In this study, deep proteomics profiling, directed metabolite analysis, and reverse transcriptase quantitative PCR (RT-qPCR) revealed metabolic changes in response to anaerobic growth on MTA versus sulfate as sole sulfur source. The abundance of protein levels associated with methionine transport, cell motility, and chemotaxis increased in the presence of MTA over that in the presence of sulfate. Purine salvage from MTA resulted primarily in hypoxanthine accumulation and a decrease in protein levels involved in GMP-to-AMP conversion to balance purine pools. Acyl coenzyme A (acyl-CoA) metabolic protein levels for lipid metabolism were lower in abundance, whereas poly-β-hydroxybutyrate synthesis and storage were increased nearly 10-fold. The known R. rubrum aerobic MSP was also shown to be upregulated, to function anaerobically, and to recycle MTA. This suggested that other organisms with gene homologues for the MTA-isoprenoid shunt may also possess a functioning anaerobic MSP. In support of our previous findings that ribulose-1,5-carboxylase/oxygenase (RubisCO) is required for an apparently purely anaerobic MSP, RubisCO transcript and protein levels both increased in abundance by over 10-fold in cells grown anaerobically on MTA over those in cells grown on sulfate, resulting in increased intracellular RubisCO activity. Lastly, these results reveal for the first time global metabolic responses as a consequence of anaerobic MTA metabolism compared to using sulfate as the sulfur source.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Linking the Dynamic Response of the Carbon Dioxide-Concentrating Mechanism to Carbon Assimilation Behavior in Fremyella diplosiphon

ABSTRACT Cyanobacteria use a carbon dioxide (CO 2 )-concentrating mechanism (CCM) that enhances their carbon fixation efficiency and is regulated by many environmental factors that impact photosynthesis, including carbon availability, light levels, and nutrient access. Efforts to connect the regulation of the CCM by these factors to functional effects on carbon assimilation rates have been complicated by the aqueous nature of cyanobacteria. Here, we describe the use of cyanobacteria in a semiwet state on glass fiber filtration discs—cyanobacterial discs—to establish dynamic carbon assimilation behavior using gas exchange analysis. In combination with quantitative PCR (qPCR) and transmission electron microscopy (TEM) analyses, we linked the regulation of CCM components to corresponding carbon assimilation behavior in the freshwater, filamentous cyanobacterium Fremyella diplosiphon . Inorganic carbon (C i ) levels, light quantity, and light quality have all been shown to influence carbon assimilation behavior in F. diplosiphon . Our results suggest a biphasic model of cyanobacterial carbon fixation. While behavior at low levels of CO 2 is driven mainly by the C i uptake ability of the cyanobacterium, at higher CO 2 levels, carbon assimilation behavior is multifaceted and depends on C i availability, carboxysome morphology, linear electron flow, and cell shape. Carbon response curves (CRCs) generated via gas exchange analysis enable rapid examination of CO 2 assimilation behavior in cyanobacteria and can be used for cells grown under distinct conditions to provide insight into how CO 2 assimilation correlates with the regulation of critical cellular functions, such as the environmental control of the CCM and downstream photosynthetic capacity. IMPORTANCE Environmental regulation of photosynthesis in cyanobacteria enhances organismal fitness, light capture, and associated carbon fixation under dynamic conditions. Concentration of carbon dioxide (CO 2 ) near the carbon-fixing enzyme RubisCO occurs via the CO 2 -concentrating mechanism (CCM). The CCM is also tuned in response to carbon availability, light quality or levels, or nutrient access—cues that also impact photosynthesis. We adapted dynamic gas exchange methods generally used with plants to investigate environmental regulation of the CCM and carbon fixation capacity using glass fiber-filtered cells of the cyanobacterium Fremyella diplosiphon . We describe a breakthrough in measuring real-time carbon uptake and associated assimilation capacity for cells grown in distinct conditions (i.e., light quality, light quantity, or carbon status). These measurements demonstrate that the CCM modulates carbon uptake and assimilation under low-C i conditions and that light-dependent regulation of pigmentation, cell shape, and downstream stages of carbon fixation are critical for tuning carbon uptake and assimilation.

59 BASIC BIOLOGICAL SCIENCES↗