The morphology, crystal structure, and elemental composition of biominerals are commonly different from chemically synthesized minerals, but the reasons for these are not fully understood. A facultative anaerobic bacterium, Enterobacter ludwigii SYB1, is used in experiments to document the hydrochemistry, mineral crystallization, and cell surface characteristics of biomineralization. It was found that carbonate anhydrase and ammonia production were major factors influencing the alkalinity and saturation of the closed biosystem. X-ray diffraction (XRD) spectra showed that calcite, monohydrocalcite (MHC), and dypingite formed in samples with bacterial cells. It was also found that the (222) plane of MHC was the preferred orientation compared to standard data. Scanning transmission electron microscopy (STEM) analysis of cell slices provides direct evidence of concentrated calcium and magnesium ions on the surface of extracellular polymeric substances (EPS). In addition, high-resolution transmission electron microscopy (HRTEM) showed that crystallized nanoparticles were formed within the EPS. Thus, the mechanism of the biomineralization induced by E. ludwigii SYB1 can be divided into three stages: (i) the production of carbonate anhydrase and ammonia increases the alkalinity and saturation state of the milieu, (ii) free calcium and magnesium ions are adsorbed and chelated onto EPS, and (iii) nanominerals crystallize and grow within the EPS. Seventeen kinds of amino acids were identified within both biotic MHC and the EPS of SYB1, while the percentages of glutamic and aspartic acid in MHC increased significantly (p < 0.05). Furthermore, the adsorption energy was calculated for various amino acids on seven diffracted crystal faces, with preferential adsorption demonstrated on (111) and (222) faces. At the same time, the lowest adsorption energy was always that of glutamic and aspartic acid for the same crystal plane. These results suggest that aspartic and glutamic acid always mix preferentially in the crystal lattice of MHC and that differential adsorption of amino acids on crystal planes can lead to their preferred orientation. Moreover, the mixing of amino acids in the mineral structure may also have a certain influence on the mineral lattice dislocations, thus enhancing the thermodynamic characteristics.

A mathematical first-order difference equation was designed to predict the dynamics of the phage-bacterium adsorption process in aquatic environments, under laboratory conditions. Our model requires knowledge of bacteria and bacteriophage concentrations and the measurements of bacterial size and velocity to predict both the number of bacteriophages adsorbed onto their bacterial host and the number of infected bacteria in a given specific time. It does not require data from previously performed adhesion experiments. The predictions generated by our model were validated in laboratory. Our model was initially conceived as an estimator for the effectiveness of the inoculation of phages as antibacterial therapy for aquaculture, is also suitable for a wide range of potential applications.

UL21 of herpes simplex virus type 1 (HSV-1) is an accessory gene that encodes a component of the tegument. Homologs of this protein have been identified in the alpha, beta, and gamma herpesvirus subfamilies, although their functions are unclear. To clarify the functions of UL21, we generated a UL21-null HSV-1 mutant. Growth analysis showed that the synthesis of infectious UL21-null HSV-1 in glial cells was delayed and that the overall yield was low. The plaque sizes of the UL21-null mutant were smaller than those of wild-type HSV-1. We identified several candidate UL21-interacting proteins, including intermediate filaments, by yeast two-hybrid screening. The distribution of glial fibrillary acidic protein (GFAP), which is the main component of intermediate filaments, was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells. These results will help clarify the function of UL21 and broaden our understanding of the life cycle of HSV.

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage's ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage's "adsorptive host range." The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage's adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein's targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage's adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4's long tail fiber binding tip (gp37) to evaluate and quantify gp37's relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay's ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

The search for novel bioactive compounds from the natural environment has rapidly been gaining momentum with the increase in multi-drug resistant (MDR) pathogens. In the present study, the antimicrobial potential of novel actinomycetes has been evaluated by initial screening of six soil samples. Primary and secondary screening was performed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans, Candida tropicalis, Trichophyton rubrum, and other MDR bacterial and fungal test strains, thirteen active isolates were selected for further study. Microbial strains were identified on the basis of growth conditions and other biochemical characters. Five most active microbial strains were identified using 16S rRNA sequence homology and designated as Streptomyces xanthophaeus MTCC 11938, Streptomyces variabilis MTCC 12266, Streptomyces xanthochromogenes MTCC 11937, Streptomyces levis EU 124569, and Streptomyces sp. NCIM 5500. Four antibacterial and three antifungal compounds isolated from the above five isolates were purified and partially characterized using UV absorption and IR spectra. Two antibacterial metabolites, belong to chromone and peptide antibiotic, respectively. The antifungal compounds were found to be of non-polyene nature. In conclusion, we study the isolation of novel bacterial strains of actinomycetes for producing novel compounds having antibacterial and antifungal activities from the unexplored agro-ecological niches of India. Also, this study paves the way for further characterization of these isolates of Streptomyces sp. for their optimum utilization for antimicrobial purposes.

Bacterial spores spontaneously interact and tightly bind heterologous proteins. A variety of antigens and enzymes have been efficiently displayed on spores of Bacillus subtilis, the model system for spore formers. Adsorption on B. subtilis spores has then been proposed as a non-recombinant approach for the development of mucosal vaccine/drug delivery vehicles, biocatalysts, bioremediation, and diagnostic tools. We used spores of B. megaterium QM B1551 to evaluate their efficiency as an adsorption platform. Spores of B. megaterium are significantly larger than those of B. subtilis and of other Bacillus species and are surrounded by the exosporium, an outermost surface layer present only in some Bacillus species and lacking in B. subtilis. Strain QM B1551 of B. megaterium and a derivative strain totally lacking the exosporium were used to localize the adsorbed monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp., used as a model heterologous protein. Our results indicate that spores of B. megaterium adsorb mRFP more efficiently than B. subtilis spores, that the exosporium is essential for mRFP adsorption, and that most of the adsorbed mRFP molecules are not exposed on the spore surface but rather localized in the space between the outer coat and the exosporium.

The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying in silico molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.

Aflatoxin B1 (AfB1) is a carcinogenic mycotoxin that contaminates food and feed worldwide. We determined the AfB1-adsorption capability of non-viable Pleurotus eryngii mycelium, an edible fungus, as a potential means for removal of AfB1 from contaminated solutions. Lyophilized mycelium was produced and made enzymatically inert by sterilization at high temperatures. The material thus obtained was characterized by scanning electron microscopy with regard to the morpho-structural properties of the mycotoxin-adsorbing surfaces. The active surfaces appeared rough and sponge-like. The AfB1-mycelium system reached equilibrium at 37°C, 30 min, and pH 5-7, conditions that are compatible with the gastro-intestinal system of animals. The system remained stable for 48 h at room temperature, at pH 3, pH 7, and pH 7.4. A thermodynamic study of the process showed that this is a spontaneous and physical adsorption process, with a maximum of 85 ± 13% of removal efficiency of AfB1 by P. eryngii mycelium. These results suggest that biosorbent materials obtained from the mycelium of the mushroom P. eryngii could be used as a low-cost and effective feed additive for AfB1 detoxification.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O-antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior.

Flavin mononucleotide (FMN) and riboflavin are structurally similar flavins, except for the presence of a phosphate group on the FMN molecule. They are used by a variety of electroactive bacteria as extracellular electron shuttles in microbial Fe reduction and inevitably interact with Fe (hydr)oxides in the extracellular environment. It is currently unknown whether flavin/Fe (hydr)oxide interaction interferes with extracellular electron transfer (EET) to the mineral surface. In this study, we found that the goethite reduction rate was lower when mediated by FMN than by RF, suggesting that FMN was less effective in shuttling electrons between cells and minerals. Nevertheless, the phosphate group did not prevent the FMN molecule from accepting electrons from bacterial cells and transferring electrons to the mineral. Results of adsorption experiment, attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy, and bacterial attachment trend analyses showed that FMN exhibited strong adsorption on goethite surface by forming phosphate inner-sphere complex, which prevented bacterial cells from approaching goethite. Therefore, the interaction between FMN and goethite surface may increase the distance of electron transfer from bacterial cells to goethite and result in lower EET efficiency in comparison to those mediated by riboflavin. To our knowledge, these data reveal for the first time that the interaction between flavin and Fe (hydr)oxide affect flavin-mediated electron transfer to mineral surface and add a new dimension to our understanding of flavin-mediated microbial Fe reduction processes.

Phosphorus is an essential nutrient for all living organisms. In bacteria, the preferential phosphorus source is phosphate, which is often a limiting macronutrient in many areas of the ocean. The geochemical cycle of phosphorus is strongly interconnected with the cycles of other elements and especially iron, because phosphate tends to adsorb onto iron minerals, such as iron oxide formed in oxic marine environments. Although the response to either iron or phosphate limitation has been investigated in several bacterial species, the metabolic interplay between these two nutrients has rarely been considered. In this study we evaluated the impact of phosphate limitation on the iron metabolism of the marine bacterium Pseudovibrio sp. FO-BEG1. We observed that phosphate limitation led to an initial decrease of soluble iron in the culture up to three times higher than under phosphate surplus conditions. Similarly, a decrease in soluble cobalt was more pronounced under phosphate limitation. These data point toward physiological changes induced by phosphate limitation that affect either the cellular surface and therefore the metal adsorption onto it or the cellular metal uptake. We discovered that under phosphate limitation strain FO-BEG1, as well as selected strains of the Roseobacter clade, secreted iron-chelating molecules. This leads to the hypothesis that these bacteria might release such molecules to dissolve iron minerals, such as iron-oxyhydroxide, in order to access the adsorbed phosphate. As the adsorption of phosphate onto iron minerals can significantly decrease phosphate concentrations in the environment, the observed release of iron-chelators might represent an as yet unrecognized link between the biogeochemical cycle of phosphorus and iron, and it suggests another biological function of iron-chelating molecules in addition to metal-scavenging.

Infections caused by antibiotic-resistant Escherichia coli are a threat to human and animal health globally. Phage therapy has made great progress for the treatment of drug-resistant infections, but it is still unclear whether E. coli resistance to antibiotics could change the lysis ability of phages. In this study, we demonstrate that over expression of AmpC, an important β-lactamase for ampicillin resistance, promotes lysis of E. coli by phage utilizing OmpA as a receptor. E. coli strains expressing more AmpC showed higher levels of OmpA, an E. coli outer membrane protein known to serve as a receptor for T-even phages, which resulted in increased adsorption and lysis by the phage tested in this study. These data demonstrate that increased ampicillin resistance can increase the sensitivity of E. coli to some lytic phage, which provides evidence for the feasibility of synergistic application of phage and antibiotics.

Mayaro virus (MAYV) is an emergent arbovirus first described in forest regions of the American continent, with recent and increasing notification of urban area circulation. Similar to Chikungunya (CHIKV) and other arthritogenic Alphavirus, MAYV-induced disease shows a high prevalence of persistent arthralgia, and myalgia. Despite this, knowledge regarding pathogenesis and characteristics of host immune response of MAYV infections are still limited. Here, using different ages of wild-type (WT), adult Type I Interferon receptor deficient (IFNAR-/-), and adult recombination activation gene-1 deficient (RAG-/-) mice, we have investigated the dependence of age, innate and adaptive immunity for the control of MAYV replication, tissue damage, and inflammation in mice. We have found that MAYV induces clinical signal and replicates in young WT mice, which gain the ability to restrict MAYV replication with aging. In addition, we observed that mice age and type I interferon response are related to restriction of MAYV infection and muscular inflammation in mice. Moreover, MAYV continues to replicate persistently in RAG-/- mice, being detected at blood and tissues 40 days post infection, indicating that adaptive immunity is essential to MAYV clearance. Despite chronic replication, infected adult RAG-/- mice did not develop an apparent signal of muscle damage in early and late infection. On the other hand, MAYV infection in young WT and adult IFNAR-/- mice triggers an increase in the expression of pro-inflammatory mediators, such as TNF, IL-6, KC, IL-1β, MCP-1, and RANTES, in muscle tissue, and decreases TGF-β expression, that were not significantly modulated in adult WT and RAG-/- mice. Taken together, our data demonstrated that age, innate and adaptive immunity are important to restrict MAYV replication and that adaptive immunity is also involved in MAYV-induced tissue damage. These results contribute to the comprehension of MAYV pathogenesis, and describe translational mice models for further studies of MAYV infection, vaccine tests, and therapeutic strategies against this virus.

Flagellotropic bacteriophages are interesting candidates as therapeutics against pathogenic bacteria dependent on flagellar motility for colonization and causing disease. Yet, phage resistance other than loss of motility has been scarcely studied. Here we developed a soft agar assay to study flagellotropic phage F341 resistance in motile Campylobacter jejuni. We found that phage adsorption was prevented by diverse genetic mutations in the lipooligosaccharides forming the secondary receptor of phage F341. Genome sequencing showed phage F341 belongs to the Fletchervirus genus otherwise comprising capsular-dependent C. jejuni phages. Interestingly, phage F341 encodes a hybrid receptor binding protein (RBP) predicted as a short tail fiber showing partial similarity to RBP1 encoded by capsular-dependent Fletchervirus, but with a receptor binding domain similar to tail fiber protein H of C. jejuni CJIE1 prophages. Thus, C. jejuni prophages may represent a genetic pool from where lytic Fletchervirus phages can acquire new traits like recognition of new receptors.

Influenza A virus causes periodic outbreaks and seriously threatens human health. The drug-resistant mutants have shown an epidemic trend because of the abuse of chemical drugs. Aloe polysaccharides (APS) extracted from Aloe vera leaves have evident effects on the therapy of virus infection. However, the activity of APS in anti-influenza virus has yet to be investigated. Here, we refined polysaccharides from A. vera leaf. In vitro test revealed that APS could inhibit the replication of a H1N1 subtype influenza virus, and the most obvious inhibitory effect was observed in the viral adsorption period. Transmission electron microscopy indicated that APS directly interacted with influenza virus particles. Experiments on PR8 (H1N1) virus infection in mice demonstrated that APS considerably ameliorated the clinical symptoms and the lung damage of the infected mice, and significantly reduced the virus loads and mortality. Our findings provided a theoretical basis for the development of novel natural anti-influenza agents.

Natural attenuation is an effective and feasible technology for controlling groundwater contamination. This study investigated the potential effectiveness and mechanisms of natural attenuation of 1,1,1-trichloroethane (TCA) contaminants in shallow groundwater in Shanghai by using a column simulation experiment, reactive transport model, and 16S rRNA gene clone library. The results indicated that the majority of the contaminant mass was present at 2-6 m in depth, the contaminated area was approximately 1000 m × 1000 m, and natural attenuation processes were occurring at the site. The effluent breakthrough curves from the column experiments demonstrated that the effectiveness of TCA natural attenuation in the groundwater accorded with the advection-dispersion-reaction equation. The kinetic parameter of adsorption and biotic dehydrochlorination of TCA was 0.068 m(3)/kg and 0.0045 d(-1). The contamination plume was predicted to diminish and the maximum concentration of TCA decreased to 280 μg/L. The bacterial community during TCA degradation in groundwater belonged to Trichococcus, Geobacteraceae, Geobacter, Mucilaginibacter, and Arthrobacter.

Gene-associated with retinoid-interferon-induced mortality 19 (GRIM-19) targets multiple signaling pathways involved in cell death and growth. However, the role of GRIM-19 in the pathogenesis of hepatitis virus infections remains unexplored. Here, we investigated the restrictive effects of GRIM-19 on the replication of hepatitis C virus (HCV). We found that GRIM-19 protein levels were reduced in HCV-infected Huh7 cells and Huh7 cells harboring HCV replicons. Moreover, ectopically expressed GRIM-19 caused a reduction in both intracellular viral RNA levels and secreted viruses in HCVcc-infected cell cultures. The restrictive effect on HCV replication was restored by treatment with siRNA against GRIM-19. Interestingly, GRIM-19 overexpression did not alter the level of phosphorylated STAT3 or its subcellular distribution. Strikingly, forced expression of GRIM-19 attenuated an increase in intracellular lipid droplets after oleic acid (OA) treatment or HCVcc infection. GRIM-19 overexpression abrogated fatty acid-induced upregulation of sterol regulatory element-binding transcription factor-1 (SREBP-1c), resulting in attenuated expression of its target genes such as fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC). Treatment with OA or overexpression of SREBP-1c in GRIM-19-expressing, HCVcc-infected cells restored HCV replication. Our results suggest that GRIM-19 interferes with HCV replication by attenuating intracellular lipid accumulation and therefore is an anti-viral host factor that could be a promising target for HCV treatment.

Stenotrophomonas maltophilia (S. maltophilia) is a common opportunistic pathogen that is resistant to many antibiotics. Bacteriophages are considered to be an effective alternative to antibiotics for the treatment of drug-resistant bacterial infections. In this study, we isolated and characterized a phage, BUCT603, infecting drug-resistant S. maltophilia. Genome sequencing showed BUCT603 genome was composed of 44,912 bp (32.5% G + C content) with 64 predicted open reading frames (ORFs), whereas no virulence-related genes, antibiotic-resistant genes or tRNA were identified. Whole-genome alignments showed BUCT603 shared 1% homology with other phages in the National Center for Biotechnology Information (NCBI) database, and a phylogenetic analysis indicated BUCT603 can be classified as a new member of the Siphoviridae family. Bacteriophage BUCT603 infected 10 of 15 S. maltophilia and used the TonB protein as an adsorption receptor. BUCT603 also inhibited the growth of the host bacterium within 1 h in vitro and effectively increased the survival rate of infected mice in a mouse model. These findings suggest that bacteriophage BUCT603 has potential for development as a candidate treatment of S. maltophilia infection.

Bacteriophage 919TP is a temperate phage of Vibrio cholerae serogroup O1 El Tor and is used as a subtyping phage in the phage-biotyping scheme in cholera surveillance in China. In this study, sequencing of the 919TP genome showed that it belonged to the Vibrio phage K139 family. The mechanisms conferring resistance to 919TP infection of El Tor strains were explored to help understand the subtyping basis of phage 919TP and mutations related to 919TP resistance. Among the test strains resistant to phage 919TP, most contained the temperate 919TP phage genome, which facilitated superinfection exclusion to 919TP. Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms, since they did not contain LPS receptor mutations or temperate K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection.

A screen of bacteriophages infecting a panel of Campylobacter jejuni PT14 gene knock-out mutants identified a role for the minor flagellin encoded by the flaB gene, in the defense of the host against CP8unalikevirus bacteriophage CP_F1 infection. Inactivation of the flaB gene resulted in an increase in the susceptibility of PT14 cultures to infection by CP_F1 and an increase in bacteriophage yields. Infection of wild type PT14 with CP_F1 produces turbid plaques in bacterial lawns, from which 78% of the resistant isolates recovered exhibit either attenuation or complete loss of motility. CP_F1 produces clear plaques on the flaB mutant with no regrowth in the lysis zones. Complementation of the mutant restored overgrowth and the development of resistance at the expense of motility. Further analyses revealed an increase in bacteriophage adsorption constant of nearly 2-fold and burst-size 3-fold, relative to the wild type. Motility analysis showed no major reduction in swarming motility in the flaB mutant. Thus, we propose a new role for FlaB in the defense of campylobacters against bacteriophage infection.