Genomic Analysis of \u3ci\u3eExophiala SP\u3c/i\u3e. ETNP2018, a Marine Fungal Strain Isolated From an Oxygen Minimum Zone

Microbial metabolic activity is central to biogeochemical cycling in virtually all ecosystems. Fungi are important parts of microbial communities, serving as decomposers, parasites, and symbionts. Most of our understanding of fungal ecology is derived from the study of isolated, cultured terrestrial fungi. However, the marine environment includes thousands of divergent, uncultured fungi. A number of these species, which include saprobes, parasites, and endophytes, can survive and sporulate in a wide range of salinities. Other such species face the additional challenge of limited nutrient accessibility. The genus Exophiala includes agents of mycoses in both humans and in marine animals. This research seeks to characterize the genome of Exophiala sp. ETNP2018, a novel strain of marine fungi isolated from the Eastern Tropical North Pacific (ETNP) oxygen minimum zone, revealing the genetic adaptations of this strain to its unique environment, including low oxygen conditions and nutrient limitation. Structural and functional annotation, as well as a comparison to the representative species of its genus (Exophiala aquamarina), serves to elucidate the lifestyle of this novel strain. Furthermore, this research will, through the identification of protein-encoding sequences, determine the mechanisms used by this species to break down and metabolize biomass in the ocean

A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection

Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection

Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface

INFRARED SPECTROSCOPY OF THE AR−C2HDAR-C_{2}HD COMPLEX: POTENTIAL ENERGY SURFACES

Author Institution: Department of Chemistry, University of North CarolinaThe argon-acetylene complex has been studied by a number of experimental and theoretical groups, with the aim in mind of determining an accurate potential energy surface for this system. Both microwave and infrared supectroscopy have provided detailed rotational and vibrational constants for this system. In addition, scattering experiments have been reported and ab initio calculation performed. Even with all of this, there are still some fundamental questions that remain unanswered with regards to the shape of the potential surface. We will review some of this work to outline the current situation. In the present study we have obtained high resolution infrared spectra of the Ar-$C_{2}HD$ complex with the aim of providing additional molecular constants that could be used to help constrain the potential. Collocation calculations are reported, using a number of potential surfaces, for this and the normal isotopomer. This additional data helps to answer some of these open questions

Photothermal Therapy of Glioblastoma Multiforme Using Multiwalled Carbon Nanotubes Optimized for Diffusion in Extracellular Space

Glioblastoma multiforme (GBM) is the most common and most lethal primary brain tumor with a 5 year overall survival rate of approximately 5%. Currently, no therapy is curative and all have significant side effects. Focal thermal ablative therapies are being investigated as a new therapeutic approach. Such therapies can be enhanced using nanotechnology. Carbon nanotube mediated thermal therapy (CNMTT) uses lasers that emit near-infrared radiation to excite carbon nanotubes (CNTs) localized to the tumor to generate heat needed for thermal ablation. Clinical translation of CNMTT for GBM will require development of effective strategies to deliver CNTs to tumors, clear structure–activity and structure–toxicity evaluation, and an understanding of the effects of inherent and acquired thermotolerance on the efficacy of treatment. In our studies, we show that a dense coating of phospholipid-poly­(ethylene glycol) on multiwalled CNTs (MWCNTS) allows for better diffusion through brain phantoms, while maintaining the ability to achieve ablative temperatures after laser exposure. Phospholipid-poly­(ethylene glycol)-coated MWCNTs do not induce a heat shock response (HSR) in GBM cell lines. Activation of the HSR in GBM cells via exposure to subablative temperatures or short-term treatment with an inhibitor of heat shock protein 90 (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)), induces a protective heat shock response that results in thermotolerance and protects against CNMTT. Finally, we evaluate the potential for CNMTT to treat GBM multicellular spheroids. These data provide preclinical insight into key parameters needed for translation of CNMTT including nanoparticle delivery, cytotoxicity, and efficacy for treatment of thermotolerant GBM

Design of artificial cell–cell communication using gene and metabolic networks

Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell–cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell–cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling ΔpH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp(2) promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model

Stable production of hyaluronic acid in Streptococcus zooppidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h(-1)) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci. (c) 2005 Wiley Periodicals, Inc