Global expression profiling of Bacillus subtilis cells during industrial‐close fed‐batch fermentations with different nitrogen sources

Abstract

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation.

A Comprehensive Proteomics and Transcriptomics Analysis ofBacillus subtilisSalt Stress Adaptation

“…The quality of RNA preparations was controlled using the Agilent 2100 Bioanalyzer according to the manufacturer's instructions (Agilent Technologies, Waldbronn, Germany). Generation of the Cy3/Cy5-labeled cDNAs and hybridization to wholegenome DNA microarrays (Eurogentec, Cologne, Germany) containing DNA fragments originating from 4,022 B. subtilis genes as duplicate spots were performed as described by Jürgen et al (35). The slides were scanned with a ScanArray Express scanner (PerkinElmer Life and Analytical Sciences, Monza, Italy), and signal intensities of the individual spots were quantified with the ScanArray Express image analysis software (PerkinElmer Life and Analytical Sciences).…”

The Paralogous MarR/DUF24-Family Repressors YodB and CatR Control Expression of the Catechol Dioxygenase CatE inBacillus subtilis

“…Total RNA was isolated by the acid phenol method as described previously (19). Generation of fluorescence-labeled cDNA and hybridization with B. subtilis whole-genome microarrays (Eurogentec) was performed as described previously (11). Two independent hybridization experiments were performed using RNAs from two independent experiments.…”

In-Depth Profiling of the LiaR Response of Bacillus subtilis

“…The RNA samples obtained from three independent cultivations were used for independent cDNA synthesis and DNA microarray hybridization. The generation of the Cy3/Cy5-labeled cDNAs and hybridization to B. subtilis whole-genome DNA microarrays (Eurogentec) were performed as described previously (32). The slides were scanned with a ScanArray Express scanner (PerkinElmer).…”