Genetic Manipulation of Pathogenicity Loci in Non-Typhimurium Salmonella

Abstract

The traditional genetic tools used in Salmonella enterica serovar Typhimurium rely heavily on a high-transducing mutant of bacteriophage P22. P22 recognizes its hosts by the structure of their O-antigens, which vary among serovars of Salmonella; therefore, it cannot be used in most non-Typhimurium Salmonella, including the majority of those causing food-borne illnesses in both humans and livestock. Bacteriophage P1 infects a variety of enteric bacteria, including galE mutants of serovar Typhimurium; however, the degree to which the presence of coimmune prophages, the lack of required attachment sites or the lack of host factors act as barriers to using phage P1 in natural isolates of Salmonella is unknown. Here, we show that recombineering can be used to make virtually any serovar of Salmonella susceptible to P1 infection; as a result, P1 can be utilized for facile genetic manipulation of non-Typhimurium Salmonella, including movement of very large pathogenicity islands. A toolkit for easy manipulation of non-Typhimurium serovars of Salmonella is described.

Characterization of the Prophage Repertoire of African Salmonella Typhimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP1

“…An aph (Km R ) cassette was amplified from pKD4 with primers NW_86 and NW_87 and was inserted by λ Red recombination into the intergenic region STM0777-galE496 of MA6684. Due to the defective O-antigen of the galE mutants, the resulting strain was grown in LB supplemented with 0.2% of glucose and 0.02% of galactose for the preparation of a P22 lysate ( Butela and Lawrence, 2012 ). The aph - galE496 module was subsequently transduced into D23580 derivative strains and transductants were screened by PCR with the primers NW_61 and NW_82 and checked by sequencing to confirm the presence of galE496 .…”

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

“…Each of these 12 genes is discussed in more detail below. Some of the LPS synthesis genes found to be under selection were previously known to be required for P22 adsorption; for example, several O-antigen synthesis (rfb) genes and the galE gene (which encodes an enzyme that catalyzes the production of UDP-galactose, a necessary precursor for O-antigen and LPS outer core production) are needed to adsorb P22 (Lindberg, 1973;Kong et al, 2011;Butela and Lawrence, 2012). Furthermore, a plasmid borne Salmonella rfb operon, whose products synthesize the O-antigen portion of LPS, is sufficient to allow P22 to adsorb to E. coli K12 cells (Neal et al, 1993).…”

DNA transduction inSodalisspecies: implications for the genetic modification of uncultured endosymbionts of insects

“…CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 1988; Wolf-Watz et al, 1985;Westwater et al, 2002;Butela and Lawrence, 2012). Here we establish that P1 is capable of infecting two members of the bacterial genus Sodalis,…”