Project description:The thermophilic fungus Chaetomium thermophilum has been successfully used in the past for biochemical and high resolution structural studies of protein complexes, but subsequent functional analysis of these assemblies were hindered due to the lack of genetic tools in this thermophile, which are typically amenable in several other mesophilic eukaryotic model organisms, in particular the yeast Saccharomycers cerevisiae. Hence, we aimed to develop a regulatable gene-expression system in C. thermophilum, which might facilitate such in vivo studies, based on what we know about the galactose-inducible GAL promoter in yeast. To identify sugar-regulatable promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes induced by xylose but repressed by glucose. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of the thermostable YFP (yellow fluorescent protein) reporter. In this way, we could demonstrate xylose-dependent YFP expression by either western blotting or life cell imaging fluorescence microscopy. Prompted by these results, we finally expressed a well-characterized dominant-negative ribosome assembly factor mutant, rsa4 E117>D, under the control of the XDH promoter, which allowed us to induce a nuclear export defect of the pre-60S subunit when C. thermophilum cells were grown in xylose but not glucose containing medium. Altogether, our study recognized xylose-regulatable promoters in Chaetomium thermophilum, which may foster functional studies of genes of interest in this thermophilic eukaryotic model organism.
Project description:Although the bacterium Symbiobacterium thermophilum has a genome with a high guanine-cytosine (GC) content (69%), it belongs to a low GC content bacterial group. We detected only 18 low GC content regions with 5 or more consecutive genes whose GC contents were below 65% in the genome of this organism. S. thermophilum has 66 transposase genes, which are markers of transposable genetic elements, and 38 (58%) of them were located in the low GC content regions, suggesting that Symbiobacterium has a similar gene silencing system as Salmonella. The top hit (best match) analyses for each Symbiobacterium protein showed that putative horizontally transferred genes and vertically inherited genes are scattered across the genome. Approximately 25% of the 3338 Symbiobacterium proteins have the highest similarity with the protein of a phylogenetically distant organism. The putative horizontally transferred genes also have a high GC content, suggesting that Symbiobacterium has gained many DNA fragments from phylogenetically distant organisms during the early stage of Firmicutes evolution. After acquiring genes, Symbiobacterium increased the GC content of the horizontally transferred genes and thereby maintained a genome with a high GC content.
Project description:Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.