ABSTRACT
The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. While there are numerous ways of doing so on protein or mRNA level, our approach aims for the underlying number of coding sequences. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. Strains are transformed with a linearized, low copy number plasmid containing an antibiotic resistance marker and homology regions up- and downstream of the gene of interest. Said gene is copied onto the vector, rendering it circular and replicative and thus selectable. We could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful transformation. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher. A major benefit of this method is the ability to work with very large regions of interest, since all relevant steps are carried out in vivo and are thus far less prone to mechanical DNA damage.
ABSTRACT
Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species.
ABSTRACT
Natural transformation has been described in bacterial species spread through nearly all major taxonomic groups. However, the current understanding of the structural components and the regulation of competence development is derived from only a few model organisms. Although natural transformation was discovered in members of the Actinobacteria (high GC Gram-positive bacteria) more than four decades ago, the structural components or the regulation of the competence system have not been studied in any representative of the entire phylum. In this report we identify a new role for a distinct type of pilus biogenesis genes (tad genes, for tight adherence), which so far have been connected only with biofilm formation, adherence and virulence traits. The tad-like genes found in the genome of Micrococcus luteus were shown to be required for genetic transformation in this actinobacterial species. We generated and analyzed individual knockout mutants for every open reading frame of the two predicted tad gene clusters as well as for a potential prepilin processing peptidase and identified the major component of the putative pili. By expressing a tagged variant of the major prepilin subunit and immunofluorescence microscopy we visualized filamentous structures extending from the cell surface. Our data indicate that the two tad gene islands complementarily contribute to the formation of a functional competence pilus in this organism. It seems likely that the involvement of tad genes in natural transformation is not unique only for M. luteus but may also prove to be the case in other representatives of the Actinobacteria, which contains important medically and biotechnologically relevant species.
