Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m2 for wet sampling and 100.5 ± 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

The bacterial protein WhiD belongs to the Wbl family of iron–sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer–dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD–σHrdB4 complex as ~0.7 μM, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex

The bldC developmental locus of Streptomyces coelicolor encodes a member of a family of small DNA-binding proteins related to the DNA-binding domains of the MerR family.

The bldC locus, required for formation of aerial hyphae in Streptomyces coelicolor, was localized by map-based cloning to the overlap between cosmids D17 and D25 of a minimal ordered library. Subcloning and sequencing showed that bldC encodes a member of a previously unrecognized family of small (58- to 78-residue) DNA-binding proteins, related to the DNA-binding domains of the MerR family of transcriptional activators. BldC family members are found in a wide range of gram-positive and gram-negative bacteria. Constructed {Delta}bldC mutants were defective in differentiation and antibiotic production. They failed to form an aerial mycelium on minimal medium and showed severe delays in aerial mycelium formation on rich medium. In addition, they failed to produce the polyketide antibiotic actinorhodin, and bldC was shown to be required for normal and sustained transcription of the pathway-specific activator gene actII-orf4. Although {Delta}bldC mutants produced the tripyrrole antibiotic undecylprodigiosin, transcripts of the pathway-specific activator gene (redD) were reduced to almost undetectable levels after 48 h in the bldC mutant, in contrast to the bldC+ parent strain in which redD transcription continued during aerial mycelium formation and sporulation. This suggests that bldC may be required for maintenance of redD transcription during differentiation. bldC is expressed from a single promoter. S1 nuclease protection assays and immunoblotting showed that bldC is constitutively expressed and that transcription of bldC does not depend on any of the other known bld genes. The bldC18 mutation that originally defined the locus causes a Y49C substitution that results in instability of the protein

Survival of ice nucleation-active and genetically engineered inactive strains of Pseudomonas syringae

The survival of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared under starvation and freezing conditions. In starvation experiments, both strains displayed similar survival curves and recovery from starvation was nearly identical for the two strains. In freezing experiments, oat seedlings colonized by each strain were subjected to freezing temperatures. Populations of the INA strain increased 15-fold in the 18 hours after oat plants incurred frost damage at {dollar}-{dollar}5 and {dollar}-{dollar}12{dollar}\sp\circ{dollar}C. Plants colonized by the non-INA strain were undamaged at {dollar}-{dollar}5{dollar}\sp\circ{dollar}C and exhibited no changes in population size. As temperatures were lowered, plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases. At {dollar}-{dollar}12{dollar}\sp\circ{dollar}C, both strains behaved identically. The data show a relationship between frost damage to plants and subsequent increased bacterial population size, indicating a potential competitive advantage for INA strains of P. syringae in mild freezing environments

DevA, a GntR-like transcriptional regulator required for development in streptomyces coelicolor

The gram-positive filamentous bacterium Streptomyces coelicolor has a complex developmental cycle with three distinct phases: growth of the substrate mycelium, development of reproductive structures called aerial hyphae, and differentiation of these aerial filaments into long chains of exospores. During a transposon mutagenesis screen, we identified a novel gene (devA) required for proper development. The devA mutant produced only rare aerial hyphae, and those that were produced developed aberrant spore chains that were much shorter than wild-type chains and had misplaced septa. devA encodes a member of the GntR superfamily, a class of transcriptional regulators that typically respond to metabolite effector molecules. devA forms an operon with the downstream gene devB, which encodes a putative hydrolase that is also required for aerial mycelium formation on R5 medium. S1 nuclease protection analysis showed that transcription from the single devA promoter was temporally associated with vegetative growth, and enhanced green fluorescent protein transcriptional fusions showed that transcription was spatially confined to the substrate hyphae in the wild type. In contrast, devAB transcript levels were dramatically upregulated in a devA mutant and the devA promoter was also active in aerial hyphae and spores in this background, suggesting that DevA might negatively regulate its own production. This suggestion was confirmed by gel mobility shift assays that showed that DevA binds its own promoter region in vitro

Agreement on "being sufficiently active for health" measured objectively by pedometer and subjectively by IPAQ questionnaires

Background: Measuring physical activity is complex and to achieve reliable and accurate measurements is challenging. Physical activity can be assessed subjectively using a variety of self report questionnaires, diaries, or logs or objectively using devices such as pedometers and accelerometers. This study aimed to test agreement on "being sufficiently active for health" measured objectively by pedometer and subjectively by International Physical Activity Questionnaires. Methods: Local government workers wore a Yamax Digi-walker pedometer for a one week period after which time they completed the Long Version of the International Physical Activity Questionnaire to allow a comparison between objective and subjective measures of physical activity. Results: Analysis of pedometer data showed that 49% of participants were sufficiently active for health (10,000 steps in a 24 hours period was used to define "sufficiently active for health). IPAQ classification of physical activity revealed much higher perceived levels of physical activity with 18.9% classified as achieving medium levels of activity and 73% achieving high levels of activity. Conclusions: In this study pedometers were shown to be more accurate measurements of physical activity with the self-report survey vastly over-estimating the actual physical activity achieved. Use of subjective measures of physical activity may under-estimate the true picture of physical activity behaviour in populations and wherever possible more objective measures should be used

Characterization of Microbial Activity

The overall goal of this study is to investigate the phenomena that affect the fate and transport of radionuclides in the environment. The objective of this task, “Characterization of Microbial Activity”, is to develop a molecular biological method for the characterization of the microbial population indigenous to the Yucca Mountain Project site, with emphasis in detection and measurement of species or groups of microorganisms that could be involved in actinide and/or metal reduction, and subsurface transport. Subtasks consist of QA planning and preparation, and literature review. This task is part of a cooperative agreement between the UNLV Research Foundation and the U.S. Department of Energy (#DE-FC28-04RW12237) titled “Yucca Mountain Groundwater Characterization”