Allan Kent Nielsen

Global Transcriptional Analysis of Bacillus licheniformis Reveals an Overlap between Heat Shock and Iron Limitation Stimulon

In this study, we characterized the heat shock stimulon of the important industrial microorganism Bacillus licheniformis using DNA microarrays. While sharing a high degree of homology with the closely related model organism Bacillus subtilis, the heat shock stimulon of B. licheniformis exhibited several novel and unexpected features. Most notably, heat shock in B. licheniformis resulted in decreased amounts of mRNA from the ytrABCEF operon, encoding a putative acetoin uptake system, and stimulated the transcription of purine biosynthesis and iron uptake genes. Unexpectedly, deletion of the ytrEF genes did not affect acetoin uptake, but increased heat sensitivity. To investigate the connection between heat stress and iron uptake further, we analyzed the iron limitation response of B. licheniformis by DNA microarrays and concluded that the response mostly involves the genes related to iron uptake and metabolism, while the only heat shock gene affected by iron limitation was clpE. We also attempted to delete the fur gene (encoding the ferric uptake repressor), but unexpectedly found it to be essential in B. licheniformis. Using the fluorescent protein-encoding reporter gene under control of the dhb promoter, which responded to both heat shock and iron-starvation, we confirmed the overlap between these responses.

Contributions of the Pre- and Pro-Regions of a Staphylococcus hyicus Lipase to Secretion of a Heterologous Protein by Bacillus subtilis

ABSTRACT Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (prelip) and pro (prolip) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the prolip-peptide, in combination with the lipase signal peptide (prelip), contributes significantly to the efficient secretion of PhoA by B. subtilis and that prelip directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

Transcriptional Profile of Escherichia coli in Response to Novispirin G10

Using a novel methodology, we have investigated the transcriptional response of Escherichia coli to novispirin G10, an α-helical cationic antimicrobial peptide. We show that novispirin G10 induces an exceptionally coherent transcriptional response in E. coli, resulting in upregulation of genes involved in response to osmotic stress, acid shock, phage shock, and antimicrobial peptides, and down-regulation of the heat shock response genes, e.g., dnaJK, GroES, and GroEL. This transcriptional pattern indicates that novispirin G10 acts by compromising the bacterial membrane and possibly also by targeting the heat shock response. The impact of novispirin G10 on E. coli cells was monitored directly using the fluorescent LIVE/DEAD assay verifying that the peptide, indeed, targets bacterial membranes. Furthermore, in agreement with the observed heat shock transcriptional response, we show that overexpression of the heat shock transcription factor in E. coli, σ32, leads to a significant decrease in sensitivity towards novispirin G10.