TY  - JOUR
A1  - Barquist, Lars
A1  - Mayho, Matthew
A1  - Cummins, Carla
A1  - Cain, Amy K.
A1  - Boinett, Christine J.
A1  - Page, Andrew J.
A1  - Langridge, Gemma C.
A1  - Quail, Michael A.
A1  - Keane, Jacqueline A.
A1  - Parkhill, Julian
T1  - The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries
JF  - Bioinformatics
N2  - Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology.
KW  - mechanisms
KW  - Transposon insertion sequencing
KW  - sequencing protocol
KW  - TraDIS
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189667
VL  - 32
IS  - 7
ER  - 
TY  - JOUR
A1  - Hickey, Scott F.
A1  - Sridhar, Malathy
A1  - Westermann, Alexander J.
A1  - Qin, Qian
A1  - Vijayendra, Pooja
A1  - Liou, Geoffrey
A1  - Hammond, Ming C.
T1  - Transgene regulation in plants by alternative splicing of a suicide exon
JF  - Nucleic Acids Research
N2  - Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that splicing-regulated transgenes have not been shown to be reliably and simply designed. Here, we demonstrate that a cassette bearing a suicide exon can be inserted into a variety of open reading frames (ORFs), generating transgenes whose expression is activated by exon skipping in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln or Lys. Furthermore, a single copy of the splicing cassette was optimized by rational design to confer robust gene activation with no background expression in plants. Thus, conditional splicing has the potential to be generally useful for transgene regulation.
KW  - kingdom
KW  - pre-messenger RNA
KW  - gene expression
KW  - elements
KW  - decay
KW  - arabidopsis
KW  - eukaryotes
KW  - mechanisms
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134724
VL  - 40
IS  - 10
ER  - 
TY  - JOUR
A1  - Nguyen, Minh Thu
A1  - Kraft, Beatrice
A1  - Yu, Wenqi
A1  - Demicrioglu, Dogan Doruk
A1  - Hertlein, Tobias
A1  - Burian, Marc
A1  - Schmaler, Mathias
A1  - Boller, Klaus
A1  - Bekeredjian-Ding, Isabelle
A1  - Ohlsen, Knut
A1  - Schittek, Birgit
A1  - Götz, Friedrich
T1  - The vSa\(\alpha\) Specific Lipoprotein Like Cluster (lpl) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells
JF  - PLoS Pathogens
N2  - All Staphylococcus aureus genomes contain a genomic island, which is termed vSa\(\alpha\) and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the vSa\(\alpha\) islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I vSa\(\alpha\) island. Since the contribution of the lpl gene cluster encoded in the vSa\(\alpha\) island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the vSa\(\alpha\) encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes high-lights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor.
KW  - resistant Staphylococcus-aureus
KW  - bacterial lipoproteins
KW  - internalization
KW  - evolution
KW  - fibronectin-binding protein
KW  - toll-like receptor 2
KW  - epithelial cells
KW  - genome sequence
KW  - activation
KW  - mechanisms
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151856
VL  - 11
IS  - 6
ER  -