Biogenic FeS accelerates reductive dechlorination of carbon tetrachloride by Shewanella putrefaciens CN32

Dissimilatory metal reducing bacteria (DMRB) widely exist in the subsurface environment and are involved in various contaminant degradation and element geochemical cycling processes. Recent studies suggest that DMRB can biosynthesize metal nanoparticles during metal reduction, but it is unclear yet how such biogenic nanomaterials would affect their decontamination behaviors. In this study, we found that the dechlorination rates of carbon tetrachloride (CT) by Shewanella putrefaciens CN32 was significantly increased by 8 times with the formation of biogenic ferrous sulfide (FeS) nanoparticles. The pasteurized biogenic FeS enabled 5 times faster dechlorination than abiotic FeS that had larger sizes and irregular structure, confirming a significant contribution of the biogenic FeS to CT bioreduction resulting from its good dispersion and relatively high dechlorination activity. This study highlights a potentially important role of biosynthesized nanoparticles in environmental bioremediation.     

c-Type Cytochrome-Dependent Formation of U(IV) Nanoparticles by Shewanella oneidensis

Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracelluar UO ₂ nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO ₂ nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO ₂-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO ₂ nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO ₂ nanoparticles. In the environment, such association of UO ₂ nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O ₂ or transport in soils and sediments.     

FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation by Pseudomonas putida GB-1

Biomineralization in heterogeneous aqueous systems results from a complex association between pre-existing surfaces, bacterial cells, extracellular biomacromolecules, and neoformed precipitates. Fourier transform infrared (FTIR) spectroscopy was used in several complementary sample introduction modes (attenuated total reflectance [ATR], diffuse reflectance [DRIFT], and transmission) to investigate the processes of cell adhesion, biofilm growth, and biological Mn-oxidation by Pseudomonas putida strain GB-1. Distinct differences in the adhesive properties of GB-1 were observed upon Mn oxidation. No adhesion to the ZnSe crystal surface was observed for planktonic GB-1 cells coated with biogenic MnO _x , whereas cell adhesion was extensive and a GB-1 biofilm was readily grown on ZnSe, CdTe, and Ge crystals prior to Mn-oxidation. IR peak intensity ratios reveal changes in biomolecular (carbohydrate, phosphate, and protein) composition during biologically catalyzed Mn-oxidation. In situ monitoring via ATR-FTIR of an active GB-1 biofilm and DRIFT data revealed an increase in extracellular protein (amide I and II) during Mn(II) oxidation, whereas transmission mode measurements suggest an overall increase in carbohydrate and phosphate moieties. The FTIR spectrum of biogenic Mn oxide comprises Mn-O stretching vibrations characteristic of various known Mn oxides (e.g., “acid” birnessite, romanechite, todorokite), but it is not identical to known synthetic solids, possibly because of solid-phase incorporation of biomolecular constituents. The results suggest that, when biogenic MnO _x accumulates on the surfaces of planktonic cells, adhesion of the bacteria to other negatively charged surfaces is hindered via blocking of surficial proteins.     

Highly Diverse Cyanobactins in Strains of the Genus Anabaena

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26).The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain d-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20).Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids.     

Vibriophages Differentially Influence Biofilm Formation by Vibrio anguillarum Strains

Vibrio anguillarum is an important pathogen in marine aquaculture, responsible for vibriosis. Bacteriophages can potentially be used to control bacterial pathogens; however, successful application of phages requires a detailed understanding of phage-host interactions under both free-living and surface-associated growth conditions. In this study, we explored in vitro phage-host interactions in two different strains of V. anguillarum (BA35 and PF430-3) during growth in microcolonies, biofilms, and free-living cells. Two vibriophages, ΦH20 (Siphoviridae) and KVP40 (Myoviridae), had completely different effects on the biofilm development. Addition of phage ΦH20 to strain BA35 showed efficient control of biofilm formation and density of free-living cells. The interactions between BA35 and ΦH20 were thus characterized by a strong phage control of the phage-sensitive population and subsequent selection for phage-resistant mutants. Addition of phage KVP40 to strain PF430-3 resulted in increased biofilm development, especially during the early stage. Subsequent experiments in liquid cultures showed that addition of phage KVP40 stimulated the aggregation of host cells, which protected the cells against phage infection. By the formation of biofilms, strain PF430-3 created spatial refuges that protected the host from phage infection and allowed coexistence between phage-sensitive cells and lytic phage KVP40. Together, the results demonstrate highly variable phage protection mechanisms in two closely related V. anguillarum strains, thus emphasizing the challenges of using phages to control vibriosis in aquaculture and adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.     

Complete Genome Sequencing of Four Geographically Diverse Strains of Batai Virus

Batai virus (BATV) is a widely distributed but poorly studied member of the Orthobunyavirus genus in the family Bunyaviridae and is of particular interest as a known participant in natural reassortment events. Both research and surveillance efforts on this and other related viruses have been hampered by the lack of available full-length sequence data covering all three genomic segments. Here, we report the complete genome sequence of four BATV strains (MM2222, Chittoor/IG-20217, UgMP-6830, and MS50) isolated from various geographical locations. Based on these data, we have determined that strain MS50 is in fact unrelated to BATV and likely represents as a novel genotype in the genus Orthobunyavirus.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.     

Shewanella putrefaciens Adhesion and Biofilm Formation on Food Processing Surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10⁸ CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10² CFU/cm²) than in a batch system (reaching 10⁷ CFU/cm²), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

Formation of a New Biogenic Aldehyde Adduct by Incubation of Tryptamine with Rat Brain Tissue

Tryptamine was degraded by incubation with rat brain homogenate to an unknown product. The reaction was stimulated by the nonionic detergents Triton X-100 and Lubrol PX and less by the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS). The same results were obtained with pig brain and bovine brain. The monoamine oxidase inhibitor pargyline inhibited the reaction strongly, indicating the participation of the enzyme on the reaction. Addition of 17,000 g supernatant from rat brain homogenate increased the formation effectively whereas phospholipids or chloroform/methanol (7:3) extract from the 17,000 g supernatant showed only little or no effect. Chromatographic and electrophoretic properties as well as the chemical reaction of the product with specific reagents suggest that the compound consists of an indole part and an amino acid part. The product could be identified by fast atom bombardment mass spectrometry and by comparison with the synthetic substance (4R)-2-(3-indolylmethyl)-1,3-thiazolidine-4-carboxylic acid. It is formed by the enzymatic oxidation of tryptamine producing indole-3-acetaldehyde which spontaneously cyclizes with free L-cysteine from the tissue. The results suggest that the reaction of biogenic aldehydes with brain macromolecules may proceed via an analogous reaction.     

Genotypic Prediction of Tropism of Highly Diverse HIV-1 Strains from Cameroon

Background

The use of CCR5 antagonists involves determination of HIV-1 tropism prior to initiation of treatment. HIV-1 tropism can be assessed either by phenotypic or genotypic methods. Genotypic methods are extensively used for tropism prediction. However, their validation in predicting tropism of viral isolates belonging to group M non-B subtypes remains challenging. In Cameroon, the genetic diversity of HIV-1 strains is the broadest reported worldwide. To facilitate the integration of CCR5 antagonists into clinical practice in this region, there is a need to evaluate the performance of genotypic methods for predicting tropism of highly diverse group M HIV-1 strains.

Methods

Tropism of diverse HIV-1 strains isolated from PBMCs from Cameroon was determined using the GHOST cell assay. Prediction, based on V3 sequences from matched plasma samples, was determined using bioinformatics algorithms and rules based on position 11/25 and net charge applied independently or combined according to Delobel''s and Garrido''s rules. Performance of genotypic methods was evaluated by comparing prediction generated with tropism assigned by the phenotypic assay.

Results

Specificity for predicting R5-tropic virus was high, ranging from 83.7% to 97.7% depending on the genotypic methods used. Sensitivity for X4-tropic viruses was fairly low, ranging from 33.3% to 50%. In our study, overall, genotypic methods were less able to accurately predict X4-tropic virus belonging to subtype CRF02_AG. In addition, it was found that of the methods we used the Garrido rule has the highest sensitivity rate of over 50% with a specificity of 93%.

Conclusion

Our study demonstrated that overall, genotypic methods were less sensitive for accurate prediction of HIV-1 tropism in settings where diverse HIV-1 strains co-circulate. Our data suggest that further optimization of genotypic methods is needed and that larger studies to determine their utility for tropism prediction of diverse HIV-1 strains may be warranted.     

Lysophosphatidylcholine Reversibly Arrests Pore Expansion during Syncytium Formation Mediated by Diverse Viral Fusogens

Using lysophosphatidylcholine, a curvature-inducing lysolipid, we have isolated a reversible, “stalled pore” phenotype during syncytium formation induced by the p14 fusion-associated small transmembrane (FAST) protein and influenza virus hemagglutinin (HA) fusogens. This is the first evidence that lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores.     

Biogenic Magnetite Formation through Anaerobic Biooxidation of Fe(II)

The presence of isotopically light carbonates in association with fine-grained magnetite is considered to be primarily due to the reduction of Fe(III) by Fe(III)-reducing bacteria in the environment. Here, we report on magnetite formation by biooxidation of Fe(II) coupled to denitrification. This metabolism offers an alternative environmental source of biogenic magnetite.     

Genetically Diverse Clostridium difficile Strains Harboring Abundant Prophages in an Estuarine Environment

Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in health care settings across the world. Despite its pathogenic capacity, it can be carried asymptomatically and has been found in terrestrial and marine ecosystems outside hospital environments. Little is known about these environmental strains, and few studies have been conducted on estuarine systems. Although prophage abundance and diversity are known to occur within clinical strains, prophage carriage within environmental strains of C. difficile has not previously been explored. In this study, we isolated C. difficile from sites sampled in two consecutive years in an English estuarine system. Isolates were characterized by PCR ribotype, antibiotic resistance, and motility. The prevalence and diversity of prophages were detected by transmission electron microscopy (TEM) and a phage-specific PCR assay. We show that a dynamic and diverse population of C. difficile exists within these sediments and that it includes isolates of ribotypes which are associated with severe clinical infections and those which are more frequently isolated from outside the hospital environment. Prophage carriage was found to be high (75%), demonstrating that phages play a role in the biology of these strains.     

Diverse Tetracycline Resistance Genotypes of Megasphaera elsdenii Strains Selectively Cultured from Swine Feces

A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 μg/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.