Physiology, pathogenicity and immunogenicity of lon and/or cpxR deleted mutants of Salmonella Gallinarum as vaccine candidates for fowl typhoid

To construct a novel live vaccine candidate for fowl typhoid (FT) caused by Salmonella Gallinarum (SG), the lon and cpxR genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Δlon) and JOL916 (ΔlonΔcpxR) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (ΔcpxR), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD50 using 1-week-old chickens, were attenuated approximately at 101-, 101-, and > 103-fold, respectively. The oral inoculations of 2 × 107 cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT

Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity

Evidence suggesting that eukaryotes and archaea use reversible Nε-lysine (Nε-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of Nε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsBAc), demonstrating that Nε-Lys acetylation of RcsB is reversible. Analysis of RcsBAc and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible Nε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria.

The ccd locus contributes to the stability of plasmid F by post-segregational killing of plasmid-free bacteria. The ccdB gene product is a potent cell-killing protein and its activity is negatively regulated by the CcdA protein. In this paper, we show that the CcdA protein is unstable and that the degradation of CcdA is dependent on the Lon protease. Differences in the stability of the killer CcdB protein and its antidote CcdA are the key to post-segregational killing. Because the half-life of active CcdA protein is shorter than that of active CcdB protein, persistence of the CcdB protein leads to the death of plasmid-free bacterial segregants.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe