Biomedical subjects
Publications and source records attributed to Rolf Daniel.
We report on the complete genome sequence of the carboxydotrophic hydrogenogenic, thermophilic, and alkalitolerant acetogen Thermincola carboxydiphila (DSM 17129T). The genome harbors a circular chromosome with a size of 3.519 Mbp and a GC content of 45.6%. The genome contains 3,490 genes in total.
Colletotrichum graminicola causes significant losses of the staple crop maize worldwide. The fungus produces two distinct asexual spore types, oval and falcate conidia, which show unique processes in development and plant interaction. Based on genome resequencing of our laboratory strain (CgM2/M1.001), we investigated the gene expression profiles of oval and falcate conidia during development and early leaf infection using RNA-seq. Our results reveal specific gene expression profiles between the two spore types, indicating fundamental differences in their developmental programs that reflect different modes of infection. We identified expression patterns discriminating both conidia types from mycelium and spore type-specific ones for genes encoding transcription factors, conserved fungal developmental genes, transporters, genes of secondary metabolite clusters, and pathogenicity-related functions, including effectors and carbohydrate-active enzymes (CAZymes). Our study shows that despite the identical genomic basis, oval and falcate conidia show unique transcriptomes across vegetative development and early plant interaction. Taking together, these results provide new insights into the molecular mechanisms determining the biology of C. graminicola and its interaction with the plant host.
Phylogenetic surveys of soil ecosystems have shown that the number of prokaryotic species found in a single sample exceeds that of known cultured prokaryotes. Soil metagenomics, which comprises isolation of soil DNA and the production and screening of clone libraries, can provide a cultivation-independent assessment of the largely untapped genetic reservoir of soil microbial communities. This approach has already led to the identification of novel biomolecules. However, owing to the complexity and heterogeneity of the biotic and abiotic components of soil ecosystems, the construction and screening of soil-based libraries is difficult and challenging. This review describes how to construct complex libraries from soil samples, and how to use these libraries to unravel functions of soil microbial communities.
BACKGROUND: Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Recently, it has been proposed that autoinducer-2 (AI-2), a furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine (SAH) to homocysteine, serves as a universal signal for interspecies communication. RESULTS: In this study, 138 completed genomes were examined for the genes involved in the synthesis and detection of AI-2. Except for some symbionts and parasites, all organisms have a pathway to recycle SAH, either using a two-step enzymatic conversion by the Pfs and LuxS enzymes or a one-step conversion using SAH-hydrolase (SahH). 51 organisms including most Gamma-, Beta-, and Epsilonproteobacteria, and Firmicutes possess the Pfs-LuxS pathway, while Archaea, Eukarya, Alphaproteobacteria, Actinobacteria and Cyanobacteria prefer the SahH pathway. In all 138 organisms, only the three Vibrio strains had strong, bidirectional matches to the periplasmic AI-2 binding protein LuxP and the central signal relay protein LuxU. The initial two-component sensor kinase protein LuxQ, and the terminal response regulator luxO are found in most Proteobacteria, as well as in some Firmicutes, often in several copies. CONCLUSIONS: The genomic analysis indicates that the LuxS enzyme required for AI-2 synthesis is widespread in bacteria, while the periplasmic binding protein LuxP is only present in Vibrio strains. Thus, other organisms may either use components different from the AI-2 signal transduction system of Vibrio strains to sense the signal of AI-2, or they do not have such a quorum sensing system at all.
Thirteen different chitin-degrading bacteria were isolated from soil and sediment samples. Five of these strains (SGE2, SGE4, SSL3, MG1, and MG3) exhibited antifungal activity against phytopathogenic fungi. Analyses of the 16S rRNA genes and the substrate spectra revealed that the isolates belong to the genera Bacillus or Streptomyces. The closest relatives were Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B. ehimensis (MG1), and Streptomyces griseus (MG3). The chitinases present in the culture supernatants of the five isolates revealed optimal activity between 45 degrees C and 50 degrees C and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6 (SGE4), and 5-7 (MG3). The crude chitinase preparations of all five strains possessed antifungal activity. The chitinase of MG3 (ChiIS) was studied further, since the crude enzyme conferred strong growth suppression of all fungi tested and was very active over the entire pH range tested. The chiIS gene was cloned and the gene product was purified. The deduced protein consisted of 303 amino acids with a predicted molecular mass of 31,836 Da. Sequence analysis revealed that ChiIS of MG3 is similar to chitinases of Streptomyces species, which belong to family 19 of glycosyl hydrolases. Purified ChiIS showed remarkable antifungal activity and stability.
Soil microorganisms have been the most valuable source of natural products, providing industrially important antibiotics and biocatalysts. But, of late, the discovery rate of novel biomolecules using traditional cultivation techniques has been extremely low, as most soil microorganisms cannot be cultured in this way. The development of novel cultivation-dependent and molecular cultivation-independent approaches has paved the way for a new era of product recovery from soil microorganisms. In particular, gene-mining based on the construction and screening of complex libraries derived from the soil metagenome provides opportunities to fully explore and exploit the enormous genetic and metabolic diversity of soil microorganisms. This strategy has already resulted in the isolation of novel biocatalysts and bioactive molecules.
Modern biotechnology has a steadily increasing demand for vitamins, antibiotics and, in particular, novel biocatalysts for use in the production of flavors, agrochemicals, pharmaceuticals and high-value fine chemicals. Novel experimental approaches are being developed in attempts to identify such molecules. However, it is known that up to 99.8% of the microbes present in many environments are not readily culturable; hence, they cannot be exploited for biotechnology. The 'metagenome technology' offers a solution to this problem by developing culture-independent methods to isolate, clone and express environmental DNA. So far, metagenome-based approaches have led to the isolation of many novel biocatalysts and a variety of other molecules with a high potential for downstream applications.
The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B12) biosynthesis and B12-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B12 de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B12 biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B12-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B12-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B12-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B12-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B12-dependent enzyme, the methylcobalamin-dependent methionine synthase.
Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C(2) to C(4)) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000 Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive E. coli clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of E. coli were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (orf12, orf14, and orf22) encoded novel alcohol dehydrogenases of different types, four (orf5, sucB, fdhD, and yabF) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (glpK) encoded a putative glycerol kinase, and one (orf1) encoded a protein which showed no similarity to any other known gene product.
To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.
Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors.
A Gram-positive spore-forming thermophilic strict anaerobic bacterium, designated FH1, was isolated from enrichments at 65 degrees C with dextran as sole carbon and energy source. A sequence analysis of the 16S rRNA gene revealed 99.2% identity of FH1 to Thermoanaerobacterium thermosaccharolyticum. Furthermore, the substrate spectra of both organisms were similar. It was therefore concluded that FH1 represents a new strain within the species T. thermosaccharolyticum. The optimal growth temperature of strain FH1 was 68 degrees C. The isolated organism produced a thermostable and thermoactive dextranase with a native molecular mass of approximately 200,000 Da. The enzyme was concentrated from the cell-free culture supernatant by ammonium sulfate precipitation. The resulting crude dextranase exhibited optimal activity from 65 to 70 degrees C and a pH optimum of 5.5.