Introduction

Bacillus anthracis is a gram+ve bacterium and exists mostly in spore form. The spores will come in contact with the wounds or food of the animals and will enter into the animal. The entered spores will further germinate to a vegetative organism. Most of the infections or 95% of the infections in animals occurs through cutaneous entry while rarely infections occur through pulmonary or gastrointestinal paths. If no antibiotic is used for its treatment, 20 percent of cases with disease incidence through cutaneous exposure and 100 percent through pulmonary exposure are considered fatal. The bacterial host defenses, septicemia of vegetative cells and bacterial toxins are reasons for fatality.

The macrophages surround the spores and they are taken to the lymph nodes where the spores germinate to form vegetative cells. These cells spread through the blood and lymph creating septicemia and toxemia. The vegetative cells express 3 monomeric proteins called protective antigen or PA, edema factor or EF and lethal factor or LF. When PA binds to the surface receptors CMG2 and TEM8 of human cells, it gets cleaved into octameric and heptameric pores. The binding of PA to the receptors initiates the translocation of EF and LF to the cytoplasm. After the formation of binary complex, EF and LF are termed as ET and LT respectively.

ET is adenyl cyclase which disturbs water homeostasis and increases cAMP production resulting in kidney failure and death. LT is zinc protease, which can split mitogen activated protein kinase kinases, induce apoptosis of cells and induces inflammatory cytokines. The antibodies raised against PA can be used as vaccines for treatment.

As the transformation efficiency of Bacillus anthracis, the genetic marker is inserted into a plasmid along with the selection marker to integrate the desired gene into the chromosome by low frequency homologous recombination. The physical screening of the colonies is done for screening individual desired colonies. Gene deletions are made by using loxP sites flanking site-specific recombinase Cre gene.

The group II intron called L1.LtrB is a naturally mobile intron, which can be used in targeting gene technology. The engineered group II intron, inserts with high efficiency at the programmed insertion sites allowing gene disruption in the genome. The insertion allows the creation of a new sequence with multiple knockouts at various chromosomal sites. This study made use of L1.LtrB intron insertion technology applied to Bacillus anthracis, so that gene activation is achieved through intron insertion

Results of the study

Group II introns are autocatalytic RNA molecules. L1.LtrB intron inactivating relaxase of Lactococcus lactis, got separated from its flanking exons by RNA catalysis supported by an intron encoded protein called as LtrA. LtrA not only helps in efficient RNA led catalysis, it codes for reverse transcriptase and DNA endonuclease that are utilized to integrate the intron into the target DNA. The L1.LtrB intron that was spliced combine with LtrA to generate a RNP particle.
The RNP has intron and RNA components. The intron component can identify the exon regions of DNA by exon recognition features like EBS1/ᵟ and EBS2 present in the intron. As the suitable target is reached, the RNA component of RNP can carry out reverse splicing and integrate the RNA of intron into DNA. The reverse transcriptase activity of LtrA protein cleaves the other DNA strand and allows the copying of intron into DNA.

Group II intron gene inactivation using a vector

For achieving targeted gene disruption in Bacillus anthracis, L1.LtrB intron is cloned into a vector along with LtrA protein accompanied by cadmium inducible promoter. The vector pRB373 is used as the backbone utilizing pUB110 as replication origin for gram positives and ColE1 as replication origin for gram negatives. The orfB of IS605 sequence which has 3 copies in B.anthracis genome was recognized by the intron sequences. Plasmid was introduced into Bacillus anthracis by electroporation and the kanamycin resistant colonies were identified. The desired colonies were screened using PCR and the sequencing of PCR product showed that intron got integrated at the targeted region successfully. This intron is highly efficient in targeted insertion.

Genetic selection for detecting the insertion of group II intron

To encourage intron insertions by less efficient introns, a vector was engineered with retrotransposition accompanied by kanamycin resistance marker according to a research study. Kanamycin resistance aids in selecting the insertion events of the intron. Eythromycin resistant marker was used in a shuttle vector pRB373. The expression of group II intron was done by using Ntr promoter. The engineered plasmid vectors were introduced into Bacillus anthracis by electroporation followed by erythromycin selection. Later, Kanamycin resistant colonies are screened to check for successful insertion of intron by using PCR. The intron was inserted between 343 and 344 nucleotides of BAS4553 gene as well as between 1794 and 1795 nucleotides of BAS4597 gene. The Kanamycin resistant colonies for BAS4553 had a PCR product which is similar to the group II intron insertion. The PCR products indicate the exact integration of intron at the targeted locus.

Reference:

Roland J Saldanha, Adin Pemberton, Patrick Shiglett, jiri Perutka, Jacob T Whitt, Andrew Ellington, Alan M Lambowitz, ryan Kramer, Deborah Taylor and Thomas J Lamkin. Rapid targeted gene disruption in Bacillus anthracis. BMC Biotechnology 2013, 13:72.


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