Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.

BACKGROUND: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. RESULTS: The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis. CONCLUSION: We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.

Salmonella-based vaccines for infectious diseases.

The use of Salmonella spp. as a delivery system for foreign antigens represents a unique opportunity for the development of ideal vaccines with unparalleled merits. Increased understanding of the mechanisms underlying Salmonella virulence and host immune response will continuously create novel strategies for more effective Salmonella-based vaccines. However, limitations in our capability to manipulate the genome of a vector strain efficiently have delayed the realization of vaccination ideas. Owing to the development of new technologies in recent years, it has now become feasible to rapidly construct Salmonella vaccine strains that carry precise modifications on the chromosomal DNA. This technical advancement will open a new avenue for the effective development of Salmonella-based vaccines for infectious diseases of both human and animal health importance.

Lepidopteran herbivory and oral factors induce transcripts encoding novel terpene synthases in Medicago truncatula.

Terpenes are an important class of defense compounds that accumulate in plants after pathogen infection or arthropod injury. Sequences predicted to encode terpene synthases were selected from an expressed sequence tag (EST) database of Medicago truncatula. Four putative terpene synthase clones (MtTps1-MtTps4), originating from a chewing insect-damaged M. truncatula leaf cDNA library, were isolated. Transcript levels of each gene examined increased in response to artificial wounding, Spodoptera exigua herbivory, and treatment with volatile methyl jasmonate (meJA). Addition of S. exigua regurgitant to wound sites triggered transcript accumulation of MtTps1 and levels increased with higher concentrations of regurgitant. Furthermore, induction of MtTps1 occurred after application of N-linolenoyl-glutamate or N-linoleoyl-glutamate, factors found in lepidopteran regurgitant. Genomic DNA blots indicate that each of the putative proteins is encoded by a single-copy gene or a small gene family. Proteins encoded by MtTps3 and MtTps4 are imported into the soluble fraction of chloroplasts in in vitro assays, whereas proteins encoded by MtTps1 and MtTps2 are not imported into chloroplasts. Combined with sequence comparisons of multiple plant terpene synthases, the import data indicate that MtTps1 and MtTps2 likely encode sesquiterpene synthases and that MtTps3 and MtTps4 encode mono- or di-terpene synthases. In addition to serving as a valuable model legume species for genomic studies, M. truncatula should prove a valuable source of novel terpene-producing enzymes. Induction of wound-responsive genes by insect oral factors suggests that M. truncatula senses biotic damage through the presence of elicitors originating in the herbivore.