Hypoxic Adaptation by Efg1 Regulates Biofilm Formation by Candida albicans▿

Hypoxia is encountered frequently by Candida albicans during systemic infection of the human host. We tested if hypoxia allows biofilm formation by C. albicans, which is a major cause of perseverance and antifungal resistance in C. albicans infections. Using an in vitro biofilm system, we unexpectedly discovered that several positive regulators of biofilm formation during normoxia, including Tec1, Ace2, Czf1, Och1, and Als3, had little or no influence on biofilm development during hypoxia, irrespective of the carbon dioxide level, indicating that C. albicans biofilm pathways differ depending on the oxygen level. In contrast, the Efg1 and Flo8 regulators were required for both normoxic and hypoxic biofilm formation. To explore the role of Efg1 during hypoxic and/or biofilm growth, we determined transcriptome kinetics following release of EFG1 expression by a system under transcriptional control of a doxycycline-inducible promoter. During hypoxia, Efg1 rapidly induced expression of all major classes of genes known to be associated with normoxic biofilm formation, including genes involved in glycolysis, sulfur metabolism, and antioxidative and peroxisome activities, as well as genes for iron uptake. The results suggest that hypoxic adaptation mediated by the Efg1 and Flo8 regulators is required even during normoxic biofilm development, while hypoxic biofilm formation in deep tissues or in organs may generate foci of C. albicans infections

Application of Recognition of Individual Genes-Fluorescence In Situ Hybridization (RING-FISH) To Detect Nitrite Reductase Genes (nirK) of Denitrifiers in Pure Cultures and Environmental Samples ▿ †

Denitrification is an alternative type of anaerobic respiration in which nitrate is reduced to gaseous products via nitrite. The key step in this process is the reduction of nitrite to nitric oxide, which is catalyzed by two structurally different but functionally equivalent forms of nitrite reductase encoded by the nirK and nirS genes. Cultivation-independent studies based on these functional marker genes showed that in the environment there was a dominance of organisms with nirK and nirS genes presumably derived from organisms that have not been cultured yet. However, the phylogenetic affiliation of these organisms has not been resolved since the ability to denitrify is widespread in phylogenetically unrelated organisms. To unravel the phylogeny of the organisms from which the nitrite reductase (nirK) genes originated, one option is to use a special variant of whole-cell hybridization termed recognition of individual genes-fluorescence in situ hybridization (RING-FISH). In RING-FISH a multiply labeled transcript polynucleotide probe is used to detect a single gene on the bacterial chromosome during FISH. Here, RING-FISH was used with laboratory cultures and environmental samples, such as activated sludge. Furthermore, probe-based cell sorting using magnetic beads could also be carried out with mixtures of pure cultures, which led to effective depletion of the nirK-negative organism but capture of the nirK-positive organism, which was demonstrated by terminal restriction fragment length polymorphism analysis based on 16S rRNA genes. The results indicate that RING-FISH coupled with probe-based cell sorting could be used with environmental samples, which could provide a means for phylogenetic classification of nirK-type denitrifiers. Thus, the results of RING-FISH could increase our understanding of the phylogeny and function of denitrifying microorganisms in the environment

Functional analysis of the conserved transcriptional regulator CfWor1 in C

Fungal Wor1-like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. ¿cfwor1 mutants produce sclerotium-like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A CE.CfWor1 transformant that constitutively expresses CfWor1 produces fewer spores with altered morphology and is also reduced in virulence. RNA-seq and RT-qrtPCR analyses suggest that reduced virulence of ¿cfwor1 mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the CE.CfWor1 transformant is likely due to downregulation of effector genes. Complementation of a non-virulent ¿fosge1 (Wor1-homologue) mutant of Fusarium oxysporum f. sp. lycopersici with CfWor1 restored expression of the SIX effector genes in this fungus, but not its virulence. Chimeric proteins of CfWor1/FoSge1 also only partially restored defects of the ¿fosge1 mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that CfWor1 primarily regulates development of C.¿fulvum, which indirectly affects the expression of a subset of virulence genes