Development and Properties of Francisella tularensis Subsp. holarctica 15 NIIEG Vaccine Strain without the recD Gene

The genomic analysis of all subspecies F. tularensis, as found in Gen Bank NCBI, reveals the presence of genes encoding proteins like to the multifunctional RecBCD enzyme complex in E. coli and other bacteria. To date, the role of the recD gene in F. tularensis, which encodes the alpha chain of exonuclease V, in DNA metabolism processes, has not been studied either in vitro or in vivo. F. tularensis subsp. holarctica 15 NIIEG, a vaccine strain, served as the basis to create the F. tularensis 15D strain with recD deletion. The lack of the recD gene suppresses the integration of suicide plasmids with F. tularensis genome fragments into the chromosome. The modified strain showed reduced growth in vitro and in vivo. This study shows that such deletion significantly reduces the virulence of the strain in BALB/c mice.

Biochemical Characterization of RecBCD Enzyme from An AntarcticPseudomonasSpecies and Identification of Its Cognate Chi (χ) Sequence

ABSTRACTPseudomonas syringaeLz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential forP. syringaegrowth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPsenzyme displays distinct biochemical behaviors. UnlikeE. coliRecBCD enzyme, the RecD subunit is indispensable for RecBCDPsfunction. The RecD motor activity is essential for the Chi-like fragments production inP. syringae, highlighting a distinct role forP. syringaeRecD subunit in DNA repair and recombination process. Further, the ssDNA-dependent endonuclease activity is notably absent in RecBCDPsenzyme. Here, we demonstrate that the RecBCDPsenzyme recognizes a unique octameric DNA sequence, 5′-GCTGGCGC-3′ (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3′-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity inP. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.Abbreviations:ATPAdenosine triphosphateDSBdouble-strand break‘ChiCrossover hotspot instigatorNi-NTANitrio tri-acetic acidTLCthin layer chromatographyMMCmitomycin CUV lightUltra violetABMAntarctic bacterial mediumLBLuria-Bertani medium