Funktionelle Analyse RNA-basierter Regulation des zentralen Energiestoffwechsels in Bacillus licheniformis

Functional analysis of RNA-based regulation in the central energy metabolism of Bacillus licheniformis

by Robert Hertel
Doctoral thesis
Date of Examination:2015-02-27
Date of issue:2015-12-17
Advisor:Prof. Dr. Rolf Daniel
Referee:Prof. Dr. Rolf Daniel
Referee:PD Dr. Michael Hoppert
Persistent Address: http://dx.doi.org/10.53846/goediss-5438

 

 

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Abstract

English

This present dissertation focused on the identification, analysis and genetic manipulation of RNA-based regulators for strain optimization purposes. The source material of this work was transcriptome data from industrial subtilisin fermentation of a B. licheniformis DSM13 derivate. The development of molecular tools for regulator analysis was successful, and the resultant vectors have already been used in experiments. The main result of this subproject was the construction of an E. coli - Bacillus shuttle vector pV2. Its modular structure allows experiment-specific extensions and as an example, reporter plasmids were constructed. The main project was the investigation of the antisense RNA, AprAs. This sRNA is complementary to the mRNA of the apr gene in B. licheniformis DSM13, which codes for the subtilisin protease. The removal of this sRNA led to a four-fold increase in native exoprotease production, and demonstrates the repressor nature of this regulator. This knowledge is useful for strain optimization and a contribution to the overall project. Furthermore, a sorting method was developed to reduce the large amount of identified RNA elements to a number of interesting candidates for new cis-regulatory elements. With that, it was possible to reduce the 1380 identified 5’UTR-elements to 3.7%, containing 86.4% of all known cis-regulatory RNAs. Of the 14 new candidates for cis-regulatory elements, 13 could be confirmed by covariance models. Of the sRNA BLi_r2291, 16 instances could be identified in the B. licheniformis DSM13 genome. Its distribution correlates with the phylogenetic relationship of B. licheniformis strains. It is possibly the first known sRNA with multiple instances in the Bacillus genus. Additionally a NGS- and genome-based approach was developed for the evaluation of the active prophage potential of a bacterial strain. The efficiency of the method was proven by the identification of a third active prophage of B. licheniformis DSM13, which was previously not known to be active.
Keywords: subtilisin; apr; prophage identification; pV2; Bacillus licheniformis DSM13
 
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