Genetic Dissection of the Francisella novicida Restriction Barrier

Abstract

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10 6 -fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.Restriction-modification (R-M) systems in bacteria limit the acquisition of foreign genes that enter the cell by infecting phage, conjugal transfer systems, or transformation. Incoming DNA is cleaved (restricted) if it has not been modified by methylation at specific sequences (24,35). Almost all bacterial species carry R-M systems, with some species predicted to harbor dozens of R-M genes (11). There are four types of R-M systems (types I to IV), classified according to mechanism of action and the distribution of restriction, modification, and specificity functions among enzyme subunits (24,27).Francisella tularensis is an important human and zoonotic pathogen whose ecology and natural reservoirs are only partially understood. There are at least four subspecies which differ in their infectivity. Two of them, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, the causative agents of tularemia, are among the most infectious pathogens known and are classified as category A select agents due to their high infectivity, ease of dissemination, and severity of disease (5). Two others, "F. tularensis subsp. novicida" (Francisella novicida) and F. tularensis subsp. mediasiatica, do not generally cause disease in humans. The genomes of Francisella species are small (Ͻ2.0 Mbp), yet the bacteria are able to colonize hundreds of different animal and insect species, effectively evade host immune responses, and cause serious disease at e...

Membrane Proteases and Aminoglycoside Antibiotic Resistance

“…To test this possibility, we examined the phenotypes of mutants lacking multiple functions. Double, triple, and quadruple insertion mutants were generated using an iterative technique involving phage lambda red recombination (Materials and Methods) (10), and their tobramycin sensitivities were determined ( Table 2 and Fig. 1).…”

“…Mutants carrying multiple transposon insertions were generated using a technique analogous to that described previously for Francisella novicida (10). Transposon insertions (ISphoA/hah and ISlacZ/ hah) were converted into 189-bp insertions by Cre recombination at loxP sequences at the ends of the transposons (2).…”

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

“…To create the ilvC::Tnflp panG::Tn mutant ("flp" signifies a cured Km cassette) we performed Flp-mediated recombination of ilvC::Tn bearing the T20 insertion by introducing pFFlp-hyg, a temperature-sensitive plasmid, by electroporation and isolated Km-sensitive colonies grown at 30°C (25). Removal of the Km cassette left an 80-bp insertion with the expected single FLP recombination target (FRT) site remaining.…”

“…Once the Km resistance marker was excised, pFFlp-hyg was cured by growing the culture overnight at 37°C. The panG mutation was introduced by transformation of genomic DNA from the panG::Tn strain into the ilvC::Tnflp mutant and selecting for Km-resistant colonies, creating the double mutant (25). For genetic complementation, mutant strains were transformed with their respective plasmids (29).…”

Working toward the Future: Insights intoFrancisella tularensisPathogenesis and Vaccine Development

“…Additionally, this system was used to identify FevR, a novel regulator of iglB (22). Finally, useful genetic surrogates of F. novicida which lack all of the resident restriction-modification systems that otherwise act as a barrier to gene transfer have now been developed (75). Clearly, the ongoing genetic work with Francisella is rapidly expanding our understanding of Francisella pathogenesis, and it will no doubt lead to identification of key virulence mediators that can be exploited for the development of potential vaccines and therapeutics.…”