Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

The diversity and evolution of microbial dissimilatory phosphite oxidation.

Phosphite is the most energetically favorable chemotrophic electron donor known, with a half-cell potential (Eo') of -650 mV for the PO43-/PO33- couple. Since the discovery of microbial dissimilatory phosphite oxidation (DPO) in 2000, the environmental distribution, evolution, and diversity of DPO microorganisms (DPOMs) have remained enigmatic, as only two species have been identified. Here, metagenomic sequencing of phosphite-enriched microbial communities enabled the genome reconstruction and metabolic characterization of 21 additional DPOMs. These DPOMs spanned six classes of bacteria, including the Negativicutes, Desulfotomaculia, Synergistia, Syntrophia, Desulfobacteria, and Desulfomonilia_A Comparing the DPO genes from the genomes of enriched organisms with over 17,000 publicly available metagenomes revealed the global existence of this metabolism in diverse anoxic environments, including wastewaters, sediments, and subsurface aquifers. Despite their newfound environmental and taxonomic diversity, metagenomic analyses suggested that the typical DPOM is a chemolithoautotroph that occupies low-oxygen environments and specializes in phosphite oxidation coupled to CO2 reduction. Phylogenetic analyses indicated that the DPO genes form a highly conserved cluster that likely has ancient origins predating the split of monoderm and diderm bacteria. By coupling microbial cultivation strategies with metagenomics, these studies highlighted the unsampled metabolic versatility latent in microbial communities. We have uncovered the unexpected prevalence, diversity, biochemical specialization, and ancient origins of a unique metabolism central to the redox cycling of phosphorus, a primary nutrient on Earth.

How Do Standing Neutral, Supine Lateral, Standing Flexion, and Standing Extension Radiographs Compare in Detecting the Presence and Magnitude of Stable and Dynamic Spondylolisthesis?

BACKGROUND: Clinical guidelines recommend standing radiographs as the most appropriate imaging for detecting degenerative spondylolisthesis, although reliable evidence about the standing position is absent. To our knowledge, no studies have compared different radiographic views and pairings to detect the presence and magnitude of stable and dynamic spondylolisthesis. QUESTIONS/PURPOSES: (1) What is the percentage of new patients presenting with back or leg pain with stable (3 mm or greater listhesis on standing radiographs) and dynamic (3 mm or greater listhesis difference on standing-supine radiographs) spondylolisthesis? (2) What is the difference in the magnitude of spondylolisthesis between standing and supine radiographs? (3) What is the difference in the magnitude of dynamic translation among flexion-extension, standing-supine, and flexion-supine radiographic pairs? METHODS: This cross-sectional, diagnostic study was performed at an urban, academic institution between September 2010 and July 2016; 579 patients 40 years or older received a standard radiographic three-view series (standing AP, standing lateral, and supine lateral radiographs) at a new patient visit. Of those individuals, 89% (518 of 579) did not have any of the following: history of spinal surgery, evidence of vertebral fracture, scoliosis greater than 30°, or poor image quality. In the absence of a reliable diagnosis of dynamic spondylolisthesis using this three-view series, patients may have had flexion and extension radiographs, and approximately 6% (31 of 518) had flexion and extension radiographs. A total of 53% (272 of 518) of patients were female, and the patients had a mean age of 60 ± 11 years. Listhesis distance (in mm) was measured by two raters as displacement of the posterior surface of the superior vertebral body in relation to the posterior surface of the inferior vertebral body from L1 to S1; interrater and intrarater reliability, assessed with intraclass correlation coefficients, was 0.91 and 0.86 to 0.95, respectively. The percentage of patients with and the magnitude of stable spondylolisthesis was estimated on and compared between standing neutral and supine lateral radiographs. The ability of common pairs of radiographs (flexion-extension, standing-supine, and flexion-supine) to detect dynamic spondylolisthesis was assessed. No single radiographic view or pair was considered the gold standard because stable or dynamic listhesis on any radiographic view is often considered positive in clinical practice. RESULTS: Among 518 patients, the percentage of patients with spondylolisthesis was 40% (95% CI 36% to 44%) on standing radiographs alone, and the percentage of patients with dynamic spondylolisthesis was 11% (95% CI 8% to 13%) on the standing-supine pair. Standing radiographs detected greater listhesis than supine radiographs did (6.5 ± 3.9 mm versus 4.9 ± 3.8 mm, difference 1.7 mm [95% CI 1.2 to 2.1 mm]; p < 0.001). Among 31 patients, no single radiographic pairing identified all patients with dynamic spondylolisthesis. The listhesis difference detected between flexion-extension was no different from the listhesis difference detected between standing-supine (1.8 ± 1.7 mm versus 2.0 ± 2.2 mm, difference 0.2 mm [95% CI -0.5 to 1.0 mm]; p = 0.53) and flexion-supine (1.8 ± 1.7 mm versus 2.5 ± 2.2 mm, difference 0.7 mm [95% CI 0.0 to 1.5]; p = 0.06). CONCLUSION: This study supports current clinical guidelines that lateral radiographs should be obtained with patients in the standing position, because all cases of stable spondylolisthesis of 3 mm or greater were detected on standing radiographs alone. Each radiographic pair did not detect different magnitudes of listhesis, and no single pair detected all cases of dynamic spondylolisthesis. Clinical concern for dynamic spondylolisthesis may justify standing neutral, supine lateral, standing flexion, and standing extension views. Future studies could identify and evaluate a set of radiographic views that provides the greatest capacity to diagnose stable and dynamic spondylolisthesis. LEVEL OF EVIDENCE: Level III, diagnostic study.

Tungstate Control of Microbial Sulfidogenesis and Souring of the Engineered Environment.

Sulfide accumulation in oil reservoir fluids (souring) from the activity of sulfate-reducing microorganisms (SRM) is of grave concern because of the associated health and facility failure risks. Here, we present an assessment of tungstate as a selective and potent inhibitor of SRM. Dose-response inhibitor experiments were conducted with a number of SRM isolates and enrichments at 30-80 °C and an increase in the effectiveness of tungstate treatment at higher temperatures was observed. To explore mixed inhibitor treatment modes, we tested synergy or antagonism between several inhibitors with tungstate, and found synergism between WO42- and NO2-, while additive effects were observed with ClO4- and NO3-. We also evaluated SRM inhibition by tungstate in advective upflow oil-sand-packed columns. Although 2 mM tungstate was initially sufficient to inhibit sulfidogenesis, subsequent temporal CaWO4 precipitation resulted in loss of the bioavailable inhibitor from solution and a concurrent increase in effluent sulfide. Mixing 4 mM sodium carbonate with the 2 mM tungstate was enough to promote tungstate solubility to reach inhibitory concentrations, without precipitation, and completely inhibit SRM activity. Overall, we demonstrate the effectiveness of tungstate as a potent SRM inhibitor, particularly at higher temperatures, and propose a novel carbonate-tungstate formulation for application to soured oil reservoirs.

Adaptation of Desulfovibrio alaskensis G20 to perchlorate, a specific inhibitor of sulfate reduction.

Hydrogen sulfide produced by sulfate-reducing microorganisms (SRM) poses significant health and economic risks, particularly during oil recovery. Previous studies identified perchlorate as a specific inhibitor of SRM. However, constant inhibitor addition to natural systems results in new selective pressures. Consequently, we investigated the ability of Desulfovibrio alaskensis G20 to evolve perchlorate resistance. Serial transfers in increasing concentrations of perchlorate led to robust growth in the presence of 100 mM inhibitor. Isolated adapted strains demonstrated a threefold increase in perchlorate resistance compared to the wild-type ancestor. Whole genome sequencing revealed a single base substitution in Dde_2265, the sulfate adenylyltransferase (sat). We purified and biochemically characterized the Sat from both wild-type and adapted strains, and showed that the adapted Sat was approximately threefold more resistant to perchlorate inhibition, mirroring whole cell results. The ability of this mutation to confer resistance across other inhibitors of sulfidogenesis was also assayed. The generalizability of this mutation was confirmed in multiple evolving G20 cultures and in another SRM, D. vulgaris Hildenborough. This work demonstrates that a single nucleotide polymorphism in Sat can have a significant impact on developing perchlorate resistance and emphasizes the value of adaptive laboratory evolution for understanding microbial responses to environmental perturbations.

GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP.

Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 µm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

High-Throughput Screening To Identify Potent and Specific Inhibitors of Microbial Sulfate Reduction.

The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive, and corrosive. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to identify potent and selective inhibitors of SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Zinc pyrithione is the most potent inhibitor of sulfidogenesis that we identified, and is several orders of magnitude more potent than commonly used industrial biocides. Both zinc and copper pyrithione are also moderately selective against SRM. The high-throughput (HT) approach we present can be readily adapted to target SRM in diverse environments and similar strategies could be used to quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant to efforts to engineer environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms.

Despite the environmental and economic cost of microbial sulfidogenesis in industrial operations, few compounds are known as selective inhibitors of respiratory sulfate reducing microorganisms (SRM), and no study has systematically and quantitatively evaluated the selectivity and potency of SRM inhibitors. Using general, high-throughput assays to quantitatively evaluate inhibitor potency and selectivity in a model sulfate-reducing microbial ecosystem as well as inhibitor specificity for the sulfate reduction pathway in a model SRM, we screened a panel of inorganic oxyanions. We identified several SRM selective inhibitors including selenate, selenite, tellurate, tellurite, nitrate, nitrite, perchlorate, chlorate, monofluorophosphate, vanadate, molydate, and tungstate. Monofluorophosphate (MFP) was not known previously as a selective SRM inhibitor, but has promising characteristics including low toxicity to eukaryotic organisms, high stability at circumneutral pH, utility as an abiotic corrosion inhibitor, and low cost. MFP remains a potent inhibitor of SRM growing by fermentation, and MFP is tolerated by nitrate and perchlorate reducing microorganisms. For SRM inhibition, MFP is synergistic with nitrite and chlorite, and could enhance the efficacy of nitrate or perchlorate treatments. Finally, MFP inhibition is multifaceted. Both inhibition of the central sulfate reduction pathway and release of cytoplasmic fluoride ion are implicated in the mechanism of MFP toxicity.

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria.

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

Phylogenetic distribution and experimental characterization of corrinoid production and dependence in soil bacterial isolates.

Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids-the vitamin B12 family of cofactors-are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microorganisms involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12.

Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests that corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community- and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

Virtually delivered Mindfulness-Oriented Recovery Enhancement (MORE) reduces daily pain intensity in patients with lumbosacral radiculopathy: a randomized controlled trial.

Introduction: Lumbosacral radiculopathy (LR), also known as sciatica, is a common type of radiating neurologic pain involving burning, tingling, and numbness in the lower extremities. It has an estimated lifetime prevalence as high as 43%. Objectives: The objective of this randomized controlled trial was to evaluate the impact of virtually delivered Mindfulness-Oriented Recovery Enhancement (MORE) on patients with LR during the COVID-19 pandemic. Methods: Potentially eligible patients were identified using electronic health record queries and phone screenings. Participants were then randomized to MORE or treatment-as-usual (TAU) for 8 weeks, with pain intensity assessed daily. At baseline and follow-up visits, participants completed questionnaires assessing the primary outcome, disability, as well as quality of life, depression, mindful reinterpretation of pain, and trait mindfulness. Results: In our study, patients undergoing virtual delivery of MORE had greater improvements in daily pain intensity (P = 0.002) but not in disability (P = 0.09), depression (P = 0.26), or quality of life (P = 0.99 and P = 0.89, SF-12 physical and mental component scores, respectively), relative to TAU patients. In addition, patients in MORE experienced significantly greater increases in mindful reinterpretation of pain (P = 0.029) and trait mindfulness (P = 0.035). Conclusion: Among patients with lumbar radiculopathy, MORE significantly reduced daily pain intensity but did not decrease disability or depression symptoms. Given the long duration of symptoms in our sample, we hypothesize the discrepancy between changes in daily pain intensity and disability is due to fear avoidance behaviors common in patients with chronic pain. As the first trial of a mindfulness intervention in patients with LR, these findings should inform future integrative approaches to LR treatment, particularly when considering the increasing use of virtual interventions throughout the COVID-19 pandemic.

Fe(II) oxidation is an innate capability of nitrate-reducing bacteria that involves abiotic and biotic reactions.

Phylogenetically diverse species of bacteria can catalyze the oxidation of ferrous iron [Fe(II)] coupled to nitrate (NO(3)(-)) reduction, often referred to as nitrate-dependent iron oxidation (NDFO). Very little is known about the biochemistry of NDFO, and though growth benefits have been observed, mineral encrustations and nitrite accumulation likely limit growth. Acidovorax ebreus, like other species in the Acidovorax genus, is proficient at catalyzing NDFO. Our results suggest that the induction of specific Fe(II) oxidoreductase proteins is not required for NDFO. No upregulated periplasmic or outer membrane redox-active proteins, like those involved in Fe(II) oxidation by acidophilic iron oxidizers or anaerobic photoferrotrophs, were observed in proteomic experiments. We demonstrate that while "abiotic" extracellular reactions between Fe(II) and biogenic NO(2)(-)/NO can be involved in NDFO, intracellular reactions between Fe(II) and periplasmic components are essential to initiate extensive NDFO. We present evidence that an organic cosubstrate inhibits NDFO, likely by keeping periplasmic enzymes in their reduced state, stimulating metal efflux pumping, or both, and that growth during NDFO relies on the capacity of a nitrate-reducing bacterium to overcome the toxicity of Fe(II) and reactive nitrogen species. On the basis of our data and evidence in the literature, we postulate that all respiratory nitrate-reducing bacteria are innately capable of catalyzing NDFO. Our findings have implications for a mechanistic understanding of NDFO, the biogeochemical controls on anaerobic Fe(II) oxidation, and the production of NO(2)(-), NO, and N(2)O in the environment.

Surfaceomics and surface-enhanced Raman spectroscopy of environmental microbes: matching cofactors with redox-active surface proteins.

Trypsin shaving is a targeted proteomic method for identifying cell-surface exposed proteins on bacterial cells. For the identification of redox-active cell-surface proteins, trypsin-shaving datasets can be matched with surface-enhanced Raman spectra of intact cells to identify the cofactors associated with the cell-surface proteins. Together, these approaches could help resolve questions about the presence of cell-surface electron transport components in environmental microorganisms, especially microbes that oxidize and reduce metals and metalloids as electron donors and acceptors.

Activation of the insulin-like growth factor-1 receptor alters p27 regulation by the epidermal growth factor receptor in oral squamous carcinoma cells.

BACKGROUND: Although oral squamous cell carcinomas (OSCCs) commonly overexpress the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors (TKIs) exhibit poor efficacy clinically. Activation of the insulin-like growth factor-1 receptor (IGF1R) induces resistance of OSCC cells to EGFR-TKIs in vitro. This study seeks to evaluate the changes in cell cycle status in OSCC cells in response to gefitinib and IGF1R activation. METHODS: SCC-25 OSCC cells were used for in vitro analyses. RESULTS: Gefitinib caused a 50% reduction in S-phase population, and IGF1R activation caused a 2.8-fold increase; combined treatment yielded a baseline S-phase population. Gefitinib treatment increased the cyclin-dependent kinase inhibitor p27, and this was not abrogated by IGF1R activation. pT157-p27 was noted by immunoblot to be decreased on gefitinib treatment, but this was reversed with IGF1R activation. T157 phosphorylation contributes to cytoplasmic localization of p27 where it can promote cell proliferation and cell motility. Using both subcellular fractionation and immunofluorescence microscopy techniques, IGF1R stimulation was noted to increase the relative cytoplasmic localization of p27; this persisted when combined with gefitinib. CONCLUSIONS: IGF1R activation partially reverses the cell cycle arrest caused by gefitinib in OSCC cells. While IGF1R stimulation does not eliminate the gefitinib-induced increase in total p27, its phosphorylation state and subcellular localization are altered. This may contribute to the ability of the IGF1R to rescue OSCC cells from EGFR-TKI treatment and may have important implications for the use of p27 as a biomarker of cell cycle arrest and response to therapy.

H-NOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri.

The bioluminescent bacterium Vibrio fischeri initiates a specific, persistent symbiosis in the light organ of the squid Euprymna scolopes. During the early stages of colonization, V. fischeri is exposed to host-derived nitric oxide (NO). Although NO can be both an antimicrobial component of innate immunity and a key signaling molecule in eukaryotes, potential roles in beneficial host-microbe associations have not been described. V. fischeri hnoX encodes a heme NO/oxygen-binding (H-NOX) protein, a member of a family of bacterial NO- and/or O(2)-binding proteins of unknown function. We hypothesized that H-NOX acts as a NO sensor that is involved in regulating symbiosis-related genes early in colonization. Whole-genome expression studies identified 20 genes that were repressed in an NO- and H-NOX-dependent fashion. Ten of these, including hemin-utilization genes, have a promoter with a putative ferric-uptake regulator (Fur) binding site. As predicted, in the presence of NO, wild-type V. fischeri grew more slowly on hemin than a hnoX deletion mutant. Host-colonization studies showed that the hnoX mutant was also 10-fold more efficient in initially colonizing the squid host than the wild type; similarly, in mixed inoculations, it outcompeted the wild-type strain by an average of 16-fold after 24 h. However, the presence of excess hemin or iron reversed this dominance. The advantage of the mutant in colonizing the iron-limited light-organ tissues is caused, at least in part, by its greater ability to acquire host-derived hemin. Our data suggest that V. fischeri normally senses a host-generated NO signal through H-NOX(Vf) and modulates the expression of its iron uptake capacity during the early stages of the light-organ symbiosis.

Geochemical constraints on bacteriophage infectivity in terrestrial environments.

Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in the environment, we do not know how this variability influences the impact of phage predation on microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. Many alkaline earth metals and alkali metals enabled lytic infection but the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate-reducing microbiome, we found that the ability of lytic phages to influence nitrate reduction end-products depended upon the carbon source as well as ionic strength. For all phage:host pairs, the ion EC50s for phage infection exceeded the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where phages most influence terrestrial microbial functional ecology in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and nutrients are available to support the growth of specific phage hosts.