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bioRxiv2017

Neurotropism of enterovirus D68 isolates is independent of sialic acid and is not a recently acquired phenotype

Acute flaccid myelitis /acute flaccid paralysis (AFM/AFP) is a rare but serious illness of the nervous system, specifically affecting the grey matter of the spinal cord, motor controlling regions of the brain and the cranial nerve. Most cases of AFM/AFP are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years old. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM/AFP was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. Six of the seven EV-D68 isolates examined, including two from 1962 and four from the 2014 outbreak, replicated in neurons, and all replicated in astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as both sialic acid dependent and independent viruses replicated within neurons. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage, can infect both neurons and astrocytes, and that neurotropism is not a recently acquired characteristic as has been suggested.\n\nSignificanceRecently there has been an increase in the number of children infected with enterovirus D68 (EV-D68). Most infections are associated with mild flu-like symptoms, but neurological dysfunction may develop in a small number of children. How the biochemical and genetic differences among EV-D68 isolates relates to development of neurological disease remains an unanswered question. Assessing infection of multiple viral isolates in organotypic brain slice cultures from postnatal day 1 to 10 mice revealed that multiple isolates are neurotropic. Both neuraminidase sensitive and resistant viruses infected neurons, indicating that sialic acid binding does not play a role in EV-D68 neuropathogenesis. Establishment of a genetically and pharmacologically amenable system using organotypic brain slice cultures will provide insight into how EV-D68 neuropathologies develop.

microbiology↗

bioRxiv2017

Common Garlic (Allium sativum) has Potent Anti-Bacillus anthracis Activity among Various Commonly Used Spices and Herbs

Ethnopharmacological RelevanceGastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. Aim of the StudyEvaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. Materials and MethodsAqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. ResultsGarlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6-12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50{degrees}C for 12 h) and stability in gastric pH range (2-8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. ConclusionThe Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L. while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas. O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/162214v3_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@682faaorg.highwire.dtl.DTLVardef@114d64org.highwire.dtl.DTLVardef@1efe980org.highwire.dtl.DTLVardef@1caea0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2017

The Reconstruction of 2,631 Draft Metagenome-Assembled Genomes from the Global Oceans

Microorganisms play a crucial role in mediating global biogeochemical cycles in the marine environment. By reconstructing the genomes of environmental organisms through metagenomics, researchers are able to study the metabolic potential of Bacteria and Archaea that are resistant to isolation in the laboratory. Utilizing the large metagenomic dataset generated from 234 samples collected during the Tara Oceans circumnavigation expedition, we were able to assemble 102 billion paired-end reads into 562 million contigs, which in turn were co-assembled and consolidated in to 7.2 million contigs [≥]2kb in length. Approximately 1 million of these contigs were binned to reconstruct draft genomes. In total, 2,631 draft genomes with an estimated completion of [≥]50% were generated (1,491 draft genomes >70% complete; 603 high-quality genomes >90% complete). A majority of the draft genomes were manually assigned phylogeny based on sets of concatenated phylogenetic marker genes and/or 16S rRNA gene sequences. The draft genomes are now publically available for the research community at-large.

microbiology↗

bioRxiv2017

Utilising a Cohort Study of Hepatitis B Virus (HBV) Vaccine-Mediated Immunity in South African Children to Model Infection Dynamics: Can We Meet Global Targets for Elimination by 2030?

BackgroundSustainable Development Goals set a challenge for the elimination of hepatitis B virus (HBV) infection as a public health concern by the year 2030. Deployment of a robust prophylactic vaccine and enhanced interventions for prevention of mother to child transmission (PMTCT) are cornerstones of elimination strategy. However, in light of the estimated global burden of 290 million cases, enhanced efforts are required to underpin optimisation of public health strategy. Robust analysis of population epidemiology is particularly crucial for populations in Africa made vulnerable by HIV co-infection, poverty, stigma and poor access to prevention, diagnosis and treatment.\n\nMethodsWe here set out to evaluate the current and future role of HBV vaccination and PMTCT as tools for elimination. We first investigated the current impact of paediatric vaccination in a cohort of children with and without HIV infection in Kimberley, South Africa. Second, we used these data to inform a new model to simulate the ongoing impact of preventive interventions. By applying these two approaches in parallel, we are able to determine both the current impact of interventions, and the future projected outcome of ongoing preventive strategies over time.\n\nResultsExisting efforts have been successful in reducing paediatric prevalence of HBV infection in this setting to <1%, demonstrating the success of the existing vaccine campaign. Our model predicts that, if consistently deployed, combination efforts of vaccination and PMTCT can significantly reduce population prevalence (HBsAg) by 2030, such that a major public health impact is possible even without achieving elimination. However, the prevalence of HBV e-antigen (HBeAg)-positive carriers will decline more slowly, representing a persistent population reservoir. We show that HIV co-infection significantly reduces titres of vaccine-mediated antibody, but has a relatively minor role in influencing the projected time to elimination. Our model can also be applied to other settings in order to predict time to elimination based on specific interventions.\n\nConclusionsThrough extensive deployment of preventive strategies for HBV, significant positive public health impact is possible, although time to HBV elimination as a public health concern is likely to be substantially longer than that proposed by current goals.

microbiology↗

bioRxiv2017

Polioviruses that bind a chimeric Pvr-nectin-2 protein identify capsid residues involved in receptor interaction

Amino acid changes in the CC\"D region in poliovirus receptor domain 1 disrupt poliovirus binding. To examine further the role of the CC\"D region in poliovirus infection, we substituted this region of Pvr into the corresponding region of a murine homolog, nectin-2. The chimeric receptor, nectin-2Pvr(c'c\"d), rendered transformed L cells susceptible to infection with poliovirus P1/Mahoney, but not with polioviruses P2/Lansing and P3/Leon, due to lack of binding. Twenty-four variants of P2/Lansing were selected that replicate in nectin-2Pvr(c'c\"d) producing cell lines. Sequence analysis revealed 30 amino acid changes at 28 capsid residues. One change, K1103R, is found in nearly all isolates and is located at one end of the VP1 BC loop. Other alterations are located on the canyon surface, at the protomer interface, and along the perimeter of the canyon south wall. Unlike poliovirus-Pvr binding, the VP1 BC loop is required for infection of cells producing nectin-2Pvr(c'c\"d).

microbiology↗

bioRxiv2017

Qinichelins, novel catecholate-hydroxamate siderophores synthesized via a multiplexed convergent biosynthesis pathway

The explosive increase in genome sequencing and the advances in bioinformatic tools have revolutionized the rationale for natural product discovery from actinomycetes. In particular, this has revealed that actinomycete genomes contain numerous orphan gene clusters that have the potential to specify many yet unknown bioactive specialized metabolites, representing a huge unexploited pool of chemical diversity. Here, we describe the discovery of a novel group of catecholate-hydroxamate siderophores termed qinichelins (2-5) from Streptomyces sp. MBT76. Correlation between the metabolite levels and the protein expression profiles identified the biosynthetic gene cluster (BGC; named qch) most likely responsible for qinichelin biosynthesis. The structure of the molecules was elucidated by bioinformatics, mass spectrometry and NMR. Synthesis of the qinichelins requires the interplay between four gene clusters, for its synthesis and for precursor supply. This biosynthetic complexity provides new insights into the challenges scientists face when applying synthetic biology approaches for natural product discovery.\n\nPride repository reviewer account details:\n\nURL: https://www.ebi.ac.uk/pride/archive/login\n\nProject accession: PXD006577\n\nUsername: reviewer35793@ebi.ac.uk\n\nPassword: 3H0iM1FK

microbiology↗

bioRxiv2017

Hemagglutinin Glycan Clock Guides Human Influenza A Virus Evolution

Adaptive immunity to influenza A virus is limited by frequent mutations in the immunodominant head of hemagglutinin (HA). Over the last century, the upward trend in HA-head glycosylation indicates glycan addition can increase fitness, but its role in viral evolution remains unclear. Here, we report glycan evolution follows a clock-like rhythm, pacing the timeline, trajectory, and replacement of HA. Following pandemic introduction, glycans are added to HA at 4- to 6-year intervals, until a functional glycan limit is reached, after which, at 9- to 12-year intervals, glycans are either swapped between different sites, or the HA is replaced by a novel pandemic virus. Using this, we predicted the appearance of the newest glycan on pH1N1 HA. Phylogeographic reconstruction suggests these highly fit strains originated in the Middle East, before rapidly replacing all strains globally. Going forward, we can use this simple algorithm to forecast future glycan evolution and identify seasons with higher pandemic potential.

microbiology↗

bioRxiv2017

DUSP1 promotes apoptosis and suppresses cell migration, while leaving the JIP1-protected cytokine production intact, during paramyxovirus infection.

The host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. This host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. Post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. Here, we investigated the role of the dual-specificity phosphatase 1 (DUSP1) in the host defense against human respiratory syncytial virus (RSV), a pathogenic virus of the Pneumoviridae family, and Sendai virus (SeV), a model virus being developed as a vector for anti-RSV vaccine. We found that DUSP1 is upregulated before being subjected to proteasomal degradation. DUSP1 does not inhibit the antiviral response, but negatively regulates virus-induced JNK/p38 MAPK phosphorylation. Interaction with the JNK-interacting protein 1 scaffold protein prevents dephosphorylation of JNK by DUSP1, likely explaining that AP-1 activation and downstream cytokine production are protected from DUSP1 inhibition. Importantly, DUSP1 promotes SeV-induced apoptosis and suppresses cell migration in RSV-infected cells. Collectively, our data unveil a previously unrecognized selective role of DUSP1 in the regulation of tissue damage and repair during infections by RSV and SeV.

microbiology↗

bioRxiv2017

Interplay between antibiotic efficacy and drug-induced lysis underlie enhanced biofilm formation at subinhibitory drug concentrations

Subinhibitory concentrations of antibiotics have been shown to enhance biofilm formation in multiple bacterial species. While antibiotic exposure has been associated with modulated expression in many biofilm-related genes, the mechanisms of drug-induced biofilm formation remain a focus of ongoing research efforts and may vary significantly across species. In this work, we investigate antibiotic-induced biofilm formation in E. faecalis, a leading cause of nosocomial infections. We show that biofilm formation is enhanced by subinhibitory concentrations of cell wall synthesis inhibitors, but not by inhibitors of protein, DNA, folic acid, or RNA synthesis. Furthermore, enhanced biofilm is associated with increased cell lysis, an increase in extracellular DNA (eDNA), and an increase in the density of living cells in the biofilm. In addition, we observe similar enhancement of biofilm formation when cells are treated with non-antibiotic surfactants that induce cell lysis. These findings suggest that antibiotic-induced biofilm formation is governed by a trade-off between drug toxicity and the beneficial effects of cell lysis. To understand this trade-off, we developed a simple mathematical model that predicts changes to antibiotic-induced biofilm formation due to external perturbations, and we verify these predictions experimentally. Specifically, we demonstrate that perturbations that reduce eDNA (DNase treatment) or decrease the number of living cells in the planktonic phase (a second antibiotic) decrease biofilm induction, while chemical inhibitors of cell lysis increase relative biofilm induction and shift the peak to higher antibiotic concentrations. Overall, our results offer experimental evidence linking cell wall synthesis inhibitors, cell lysis, increased eDNA, and biofilm formation in E. faecalis while also providing a predictive, quantitative model that sheds light on the interplay between cell lysis and antibiotic efficacy in developing biofilms.

microbiology↗

bioRxiv2017

Exploration of Western Himalayan region for identification of gold nanoparticles synthesizing bacteria

The development of eco-friendly methods for the synthesis of nanomaterial shape and size is an important area of research in the field of nanotechnology. In present study an indigenous bacterial strain GPI-1 has been isolated from a local gold mine Khaltunala. It was characterized morphological, biochemically and also by using 16S rrna gene technology and was identified as Bacillus flexus GPI-1, the phylogeny of this bacterial strain was determined using various bioinformatics tools viz BLASTn and MEGA 5.0. To achieve maximum invitro gold nanoparticles synthesis various parameters such as pH, incubation temperature, incubation time period and wavelength were optimized as 6.8, 37{degrees}C, 36 hrs, 560 nm respectively. Stable and cubical gold nanoparticles (GNPs) formation with 40-45 nm dimensions were synthesized successfully under invitro conditions upon exposure of gold chloride trihydrates (HAuCl4) solution to the supernatant of Bacillus flexus strain GPI-1. These gold nanoparticles have been characterized by Transmission electron microscope, Fourier transform infrared spectroscopy (FTIR). Thus in the present study successful biosynthesis method of stable and cubical gold nanoparticles in the size range of 12-30 nm using this efficient Bacillus flexus GPI-1 strain. Thus in the present study leading to development of an easy bioprocess for synthesis of GNPs of desired size and shape, has been reported and this green route of biosynthesis of GNPs is a simple, economically viable and an eco-friendly process. The use of gold nanoparticles in biomedical research like X-ray computed tomography and magnetic resonance imaging, cancer research, drug delivery applications.

microbiology↗

bioRxiv2017

A phylogenetic and evolutionary analysis of antimycin biosynthesis

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of pharmaceuticals. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XLsrelated anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 65 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ or IPform, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ and IPform biosynthetic pathways.

microbiology↗

bioRxiv2017

Hepatitis B Virus Infection as a Neglected Tropical Disease

BACKGROUND BACKGROUND CURRENT STRATEGIES FOR HBV... APPLICATION OF NTD CRITERIA... RECOMMENDATIONS BASED ON NTD... CONCLUSIONS SUPPORTING INFORMATION LEGEND REFERENCES The Global Hepatitis Health Sector Strategy is aiming for elimination of viral hepatitis as a public health threat by 2030 [1], while enhanced elimination efforts for hepatitis are also promoted under the broader remit of global Sustainable Development Goals (SDGs) [2]. This is an enormous challenge for hepatitis B virus (HBV) given the estimated global burden of 260 million chronic carriers, of whom the majority are unaware of their infection [3] (Figure 1).\n\nWe here present HBV within the framework for ...

microbiology↗

bioRxiv2017

Degradation of benzene by the heavy-metal resistant bacterium Cupriavidus metallidurans CH34 reveals its catabolic potential for aromatic compounds

Benzene, toluene, ethylbenzene and the three xylene isomers are monoaromatic contaminants widely distributed on polluted sites. Some microorganisms have developed mechanisms to degrade these compounds, but their aerobic and anaerobic degradation is inhibited in presence of heavy metals, such as mercury or lead. In this report, the degradation of benzene and other aromatic compounds catalyzed by the metal resistant bacterium Cupriavidus metallidurans CH34 was characterized. A metabolic reconstruction of aromatic catabolic pathways was performed based on bioinformatics analyses. Functionality of the predicted pathways was confirmed by growing strain CH34 on benzene, toluene, o-xylene, p-cymene, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, homogentisate, catechol, naphthalene, and 2-aminophenol as sole carbon and energy sources. Benzene catabolic pathway was further characterized. Results showed that firstly benzene is transformed into phenol and, thereafter, into catechol. Benzene is degraded under aerobic conditions via a combined pathway catalyzed by three Bacterial Multicomponent Monooxygenases: a toluene-2-monoxygenase (TomA012345), a toluene-4-monooxygenase (TmoABCDEF) and a phenol-2-hydroxylase (PhyZABCDE). A catechol-2,3-dioxygenase (TomB) expressed at early exponential phase cleaves the catechol ring in meta-position; an ortho-cleavage of catechol is accomplished by a catechol-1,2-dioxygenase (CatA) at late exponential phase instead. This study additionally shows that C. metallidurans CH34 is capable of degrading benzene in presence of heavy metals, such as Hg(II) or Pb(II). This capability of degrading aromatic compounds in presence of heavy metals is rather unusual among environmental bacteria; therefore, C. metallidurans CH34 seems to be a promising candidate for developing novel bioremediation process for multi-contaminated environments.\n\nHIGHLIGHTSO_LIThe strain Cupriavidus metallidurans CH34 is capable to degrade benzene aerobically\nC_LIO_LIBenzene oxydation is mediated by bacterial multicomponent monoxygenases\nC_LIO_LIStrain CH34 is able to grow using a broad range of aromatic compounds as sole carbon and energy source\nC_LIO_LIBenzene degradation occurs even in presence of heavy metals such as mercury and lead\nC_LI

microbiology↗

bioRxiv2017

Evolution within the fungal genus Verticillium is characterized by chromosomal rearrangement and gene loss

The fungal genus Verticillium contains ten species, some of which are notorious plant pathogens causing vascular wilt diseases in host plants, while others are known as saprophytes and opportunistic plant pathogens. Whereas the genome of V. dahliae, the most notorious plan pathogen of the genus, has been well characterized, evolution and speciation of other members of the genus received little attention thus far. Here, we sequenced the genomes of the nine haploid Verticillium spp. to study evolutionary trajectories of their divergence from a last common ancestor. Frequent occurrence of chromosomal rearrangement and gene family loss was identified. In addition to ~11,000 core genes that are shared among all species, only 200-600 species-specific genes occur. Intriguingly, these species-specific genes show different features than core genes.

microbiology↗

bioRxiv2017

Resilience mechanisms of small intestinal lactobacilli to the toxicity of soybean oil fatty acids

Over the past century, soybean oil (SBO) consumption in the United States increased dramatically. The main SBO fatty acid, linoleic acid (18:2), inhibits in vitro the growth of lactobacilli, beneficial members of the small intestinal microbiota. Human-associated lactobacilli have declined in prevalence in Western microbiomes, but how dietary changes may have impacted their ecology is unclear. Here, we compared the in vitro and in vivo effects of 18:2 on Lactobacillus reuteri and L. johnsonii. Directed evolution in vitro in both species led to strong 18:2 resistance with mutations in genes for lipid biosynthesis, acid stress, and the cell membrane or wall. Small-intestinal Lactobacillus populations in mice were unaffected by chronic and acute 18:2 exposure, yet harbored both 18:2- sensitive and resistant strains. This work shows that extant small intestinal lactobacilli are protected from toxic dietary components via the gut environment as well as their own capacity to evolve resistance.

microbiology↗

bioRxiv2017

Conditionally rare taxa contribute but do not account for prokaryotic community changes in soils

Conditionally rare taxa (CRT) are thought to greatly impact microbial community turnover across many environments, but little is known about their role in soils. Here, we investigate the contribution of CRT to whole community variation over space and time in a series of geographically distinct soils dedicated to three agricultural practices of differing intensities and sampled over a full seasonal cycle. We demonstrate that soil CRT do not account for observed total community changes, but that these rare taxa can be modified by spatiotemporal filters.

microbiology↗

bioRxiv2017

Isolation and characterization of BpL1, a broad acting lytic bacteriophage against Brucella

We have isolated a new broad acting lytic brucellaphage (BpL1) from the sewage of a dairy farm. The phage lysed all the 12 Brucella abortus field isolates, B. abortus strain 99 and Brucella melitensis but did not lyse any of the heterologous species tested viz. Staphylococcus aureus, Pasteurella multocida, Escherichia coli, and Salmonella species. Streaking the plaques on Brucella lawn gave clear zones along the streak lines. The plaques were circular with a diameter of 0.5- 3.0 mm. At a concentration of 10-4 the phage count was 4.5 x 106 plaques per ml. It was a tailed phage with icosahedral head (62.2 nm in diameter and 73.71 nm in length), and the head to tail length was 229.21 nm. The phage belonged to the order Caudovirales and family Siphoviridae. It was inactivated within one hour at 55{degrees}C and within 4 hours at -20{degrees}C. Treatment at pH 2 for 4 hours and at pH 4 for 12 hours inactivated it. It was inactivated after 4 hours exposure to sunlight, and within 4 minutes by UV light. Chloroform and Sodium Dodecyl Sulfate inactivated it within 15 minutes. Lysozyme inactivated it within 1 hour whereas RNase treatment did not affect its activity.

microbiology↗

bioRxiv2017

Cellular reductase activity in uncultivated Thiomargarita spp. assayed using a redox-sensitive dye

The largest known bacteria, Thiomargarita spp., have yet to be isolated in pure culture, but their large size allows for individual cells to be followed in time course experiments, or to be individually sorted for omics-based investigations. Here we report a novel application of a tetrazolium-based dye that measures the flux of reductase production from catabolic pathways to investigate the metabolic activity of individual cells of Thiomargarita spp. When coupled to microscopy, staining of the cells with a tetrazolium-formazan dye allows for metabolic responses in Thiomargarita spp. to be to be tracked in the absence of observable cell division. Additionally, the metabolic activity of Thiomargarita spp. cells can be differentiated from the metabolism of other microbes in specimens that contain adherent bacteria. The results of our redox-dye-based assay suggests that Thiomargarita is the most metabolically versatile under anoxic conditions where it appears to express cellular reductase activity in response to the electron donors succinate, acetate, citrate, formate, thiosulfate, H2, and H2S. Under hypoxic conditions, formazan staining results suggest the metabolism of succinate, and likely acetate, citrate, and H2S. Cells incubated under oxic conditions showed the weakest formazan staining response, and then only to H2S, citrate, and perhaps succinate. These results provide experimental validation of recent genomic studies of Ca. Thiomargarita nelsonii that suggest metabolic plasticity and mixotrophic metabolism. The cellular reductase response of bacteria attached to the exteriors of Thiomargarita also supports the possibility of trophic interactions between these largest of known bacteria and attached epibionts.\n\nIMPORTANCEThe metabolic potentials of many microorganisms that cannot be grown in the laboratory are known only from genomic data. Genomes of Thiomargarita spp. suggest that these largest of known bacteria are mixotrophs, combining lithotrophic metabolisms with organic carbon degradation. Our use of a redox-sensitive tetrazolium dye to query the metabolism of these bacteria provides an independent line of evidence that corroborates the apparent metabolic plasticity of Thiomargarita observed in recently produced genomes. Finding new cultivation- independent means of testing genomic results is critical to testing genome-derived hypotheses on the metabolic potentials of uncultivated microorganisms.

microbiology↗