ALBERT

Description: Soil ice content is an important component for winter soil hydrology. The sensible heat balance (SHB) method using measurements from heat pulse probes (HPPs) is a possible way to determine transient soil ice content. In a previous study, in situ soil ice content estimates with the SHB method were inaccurate, due to thermal conductivity errors and the use of relatively long time steps for calculations. The objective of this study is to reexamine the SHB method for soil ice content determination. A soil freezing and thawing laboratory experiment was performed with soil columns and heat exchangers. Transient soil ice contents in the soil columns during soil freezing and thawing were determined with the SHB method. The SHB method was able to determine dynamic changes in soil ice contents during initial freezing and final thawing for soil temperatures between –5 and 0°C when latent heat values associated with ice formation or with thawing were relatively large. During an extended freezing period, when soil temperatures were below –5°C, the small associated latent heat fluxes were below the sensitivity of the SHB method, and the SHB method did not provide accurate estimates of ice contents with time. However, the soil ice contents during the extended freezing period could be estimated well from changes in volumetric heat capacity ( C ) determined with HPP. Thus, combining the SHB method for initial freezing and final thawing, with a change in C method for extended freezing periods, allowed determination of dynamic soil ice contents for the entire range of freezing and thawing soil temperatures investigated. HPPs were able to measure soil ice contents.

Description: Phylogenomic approaches have the potential to improve confidence about the inter-relationships of species in the order Mucorales within the fungal tree of life. Rhizopus species are especially important as plant and animal pathogens and bioindustrial fermenters for food and metabolite production. A dataset of 192 orthologous genes was used to construct a phylogenetic tree of 21 Rhizopus strains, classified into four species isolated from habitats of industrial, medical and environmental importance. The phylogeny indicates that the genus Rhizopus consists of three major clades, with R. microsporus as the basal species and the sister lineage to R. stolonifer and two closely related species R. arrhizus and R. delemar . A comparative analysis of the mating type locus across Rhizopus reveals that its structure is flexible even between different species in the same genus, but shows similarities between Rhizopus and other mucoralean fungi. The topology of single-gene phylogenies built for two genes involved in mating is similar to the phylogenomic tree. Comparison of the total length of the genome assemblies showed that genome size varies by as much as threefold within a species and is driven by changes in transposable element copy numbers and genome duplications.

Description: Calcineurin modulates environmental stress survival and virulence of the human fungal pathogen Cryptococcus neoformans . Previously, we identified 44 putative calcineurin substrates, and proposed that the calcineurin pathway is branched to regulate targets including Crz1, Pbp1, and Puf4 in C. neoformans . In this study, we characterized Had1, which is one of the putative calcineurin substrates belonging to the ubiquitously conserved haloacid dehalogenase β-phosphoglucomutase protein superfamily. Growth of the had1 mutant was found to be compromised at 38° or higher. In addition, the had1 mutant exhibited increased sensitivity to cell wall perturbing agents, including Congo Red and Calcofluor White, and to an endoplasmic reticulum stress inducer dithiothreitol. Virulence studies revealed that the had1 mutation results in attenuated virulence compared to the wild-type strain in a murine inhalation infection model. Genetic epistasis analysis revealed that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways that orchestrate stress survival and fungal virulence. Overall, our results demonstrate that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C. neoformans .

Description: Determining soil ice content during freezing and thawing is important and challenging for both engineering and environmental issues. The thermo-time domain reflectometry (T-TDR) probe, which can monitor unfrozen soil water content and soil thermal properties simultaneously, has the potential to measure ice content in partially frozen soils. The objective of this study was to identify an optimum heat application strategy for measuring soil thermal properties with T-TDR probes in partially frozen soil while minimizing ice melting during the process. The optimized heating schemes were then applied for monitoring soil ice content dynamics during freezing and thawing. The results indicated that the heat pulse method failed at temperatures between –5 and 0°C because of temperature field disturbances from latent heat of fusion. When soil temperatures were ≤ –5°C, ice melting during heat pulse applications could be limited effectively with a combination of 60-s heat-pulse duration and 450 J m –1 heating strength, or a 90-s heat-pulse duration and heating strength of 450 to 900 J m –1 . With the optimized heating scheme, T-TDR probes were able to measure soil ice content changes at ≤ –5°C during freezing and thawing, and the errors were within ±0.05 m 3 m –3 in sandy loams and within ±0.1 m 3 m –3 in soils with high clay content. At temperatures between –5 and 0°C, soil ice contents could not be measured accurately with the heat-pulse method directly, but they could be estimated coarsely from water content before freezing, TDR measured unfrozen water content, and T-TDR measured total water content at temperatures below –5°C.

Description: Mucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. Nonetheless, the importance of these fungi as pathogens and model systems has increased. M. circinelloides is one of a few genetically tractable organisms in the basal fungi, but it is far from a robust genetic system when compared to model fungi in the subkingdom Dikarya. One problem is the organism is resistant to drugs utilized to select for dominant markers in other fungal transformation systems. Thus, we developed a blaster recyclable marker system by using the pyrG gene (encoding an orotidine-5'-phosphate decarboxylase, ortholog of URA3 in Saccharomyces cerevisiae ). A 237-bp fragment downstream of the pyrG gene was tandemly incorporated into the upstream region of the gene, resulting in construction of a pyrG-dpl237 marker. To test the functionality of the pyrG-dpl237 marker, we disrupted the carRP gene that is involved in carotenoid synthesis in pyrG – mutant background. The resulting carRP :: pyrG-dpl237 mutants exhibit a white colony phenotype due to lack of carotene, whereas wild type displays yellowish colonies. The pyrG marker was then successfully excised, generating carRP-dpl237 on 5-FOA medium. The mutants became auxotrophic and required uridine for growth. We then disrupted the calcineurin B regulatory subunit cnbR gene in the carRP :: dpl237 strain, generating mutants with the alleles carRP :: dpl237 and cnbR :: pyrG . These results demonstrate that the recyclable marker system is fully functional, and therefore the pyrG-dpl237 marker can be used for sequential gene deletions in M. circinelloides .