Friday, June 29, 2012

Transposon mutagenesis of an anaerobic bacterium

EZ-Tn5™ Transposomes are popular for generation of insertion and knockout mutants in microorganisms, 12 years after their introduction. A recent report by Veeranagouda et al. describes the generation of mutants in the anaerobe Bacteroides fragilis. The study is notable since anaerobes tend to be quite difficult to mutagenize through transposition, due to electroporation in the presence of oxygen that can poison the organism.

The experiments performed on B. fragilis used a custom transposome generated using the pMOD-3 [R6Kγ/MCS] vector. Screening/antibiotic resistance markers for erythromycin and kanamycin were introduced into the vector. As Bacteroides has a restriction system that requires bypassing, the pMOD3 construct (referred to as pVY02) was passaged through a strain of B. fragilis to methylate and protect the plasmid containing the transposon construct in a process called "transposon laundering" (Barry Hall, University of Rochester, personal communication). The transposon was released and purified from pYV02 using restriction digestion with Pvu II, gel-purified, and used to prepare the transposome complex with EZ-Tn5 Transposase. The transposome was electroporated into B. fragilis strain BF638 using standard methods and transposition mutants were recovered by screening on Brain/Heart Infusion (BHI) agar/erythromycin plates.

Results showed the transposon inserted in vivo at a relatively high frequency into BF638R, yielding 3.2 ± 0.35 x 10^3 CFU/µg. Transposome laundering resulted in a 6-fold increase in the number of transposome mutants over the nonprotected transposome controls. The use of the EZ-Tn5 pMOD-3 vector in the transposome construction process facilitated marker rescue by restriction digestion of DNA from the transposed host, recircularization, and transformation of the rescued DNA in E. coli strain EC100D (pir+). The transposome inserted into the Bacteroides genome in a single copy, demonstrated by Southern blotting and by single random-primer PCR (similar to the Random Amplification of Transposome Ends method). Further work by the researchers also demonstrated that the custom transposome was also useful in the mutagenesis of the bacterium Bacteroides thetaiotaomicron.

ResearchBlogging.orgVeeranagouda, Y et al. (2012). Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiology Letters PMID: 22639975

Thursday, June 21, 2012

rRNA removal from insect RNA

Genomic analysis has greatly increased our understanding of bacterial-insect interactions. Yet there remains a need for effective methods to remove insect rRNA to enable comprehensive, transcriptome-based studies. In a recent publication, Kumar et al. investigated the performance of the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) with insect samples. Eggs derived from a horizontal gene transfer of the Wolbachia bacterial genome into Drosophila ananassae were chosen as a model system. The authors performed paired-end RNA-Seq of untreated and Ribo-Zero-treated D. ananassae egg RNA using the Illumina® GAIIx system.

The authors conclude that the Ribo-Zero Kit (designed for human samples) efficiently removed >98% of D. ananassae rRNA from total RNA samples and that the Ribo-Zero-treated sample yielded a 6.2-fold increase in detection of mRNA transcripts. They further conclude that, based on their data, three times as many transcripts can be analyzed in a differential gene expression study that requires at least 100 reads per transcript. Finally, in D. ananassae, as in many insects, the 23S rRNA is naturally cleaved into two fragments. The Ribo-Zero treatment was found to efficiently remove both halves of the cleaved rRNA.

ResearchBlogging.orgKumar, N. et al. (2012). Efficient subtraction of insect rRNA prior to transcriptome analysis of
Wolbachia-Drosophila lateral gene transfer BMC Res Notes, 5 DOI: 10.1186/1756-0500-5-230

Monday, June 11, 2012

Development of a Tn-Seq method to study gene expression in Salmonella sp.

The EZ-Tn5™ R6Kγori/KAN-2 Transposome has been used for elucidation of bacterial gene structure and function for the last 12 years. In a recent publication by Khatiwara et al., it was used to mutagenize a strain of Salmonella enterica serotype Typhimurium to link specific gene expression information to the gene of interest. The mutagenesis work also demonstrates a new method of transposome insertion sequencing using Illumina technology instead of older Sanger sequencing techniques.

The researchers noted that the 19-base mosaic end (ME) of the transposome contains a DNA sequence similar to the recognition sequence of type II restriction enzyme BsmF I (recognition site 5'-GGGAC(N)10|/3'-CCCTG(N)14|5'), except for one nucleotide. Since BsmF I cuts the site 14 bp away from the recognition site, the researchers exploited this finding to extract 12-nucleotide sequences immediately adjacent to Tn5 insertion sites. These 12-bp transposon-junction sequences were selectively amplified from a mutant library and sequenced. The resulting profile provides information on both the identity and relative quantity of each insertion in the library. Next, the researchers tested whether any mutagenesis in the ME would negatively affect the activity of the transposome. The results suggested that a single nucleotide change in the ME would not affect the transposition reaction significantly, as determined by comparative numbers of Kan-resistant colonies after mutagenesis.

One of the MEs was mutated from A to G to create the BsmF I site. This change allowed the ligation of a "Tn-Seq" linker to the mutated ME, which provided an Illumina single-read sequencing priming site and index into the molecule. The transposome generated was electroporated into the Salmonella spp. bacterium and allowed to generate colonies as per the normal in vivo transposition protocol. Mutants were then screened for changes in activity of selected loci. Individual mutants with expression changes in the same locus were pooled, digested with BsmF I, ligated to the Tn-Seq linkers and amplified by PCR, using a short extension time to generate short amplicons for single-read Illumina sequencing with five separate indices.

Sequencing information permitted mapping of the transposition mutants and then mapping the transposon insertions that provided changes in gene expression activity. The study highlights the adaptations researchers are developing to enable deep sequencing applications for gene expression studies in bacteria.

ResearchBlogging.orgKhatiwara, A. et al. (2012). Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium Applied and Environmental Microbiology, 78 (9), 3098-3107 DOI: 10.1128/AEM.06865-11

Monday, June 4, 2012

Attending ASM 2012? Visit the Epicentre exhibit!

Visit Epicentre at the American Society for Microbiology General Meeting
San Francisco, CA, June 16-19, 2012, Booth 1617

Epicentre will showcase new products for RNA-Seq, transposon-based mutagenesis, and other technologies for microbial genomics. If you’re attending the conference, let us know on Twitter (@EpicentreBio) and stop by the booth to pick up a special Epicentre gift!