1887

Abstract

We purified osmoregulated periplasmic glucans (OPGs) from serovar Typhimurium and found them to be composed of 100 % glucose with 2-linked glucose as the most abundant residue, with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structural genes for OPG biosynthesis, and , form a bicistronic operon, and insertion of a kanamycin resistance gene cassette into this operon resulted in a strain devoid of OPGs. The mutant strain was impaired in motility and growth under low osmolarity conditions. The mutation also resulted in a 2 log increase in the LD in mice compared to the wild-type strain SL1344. Inability to synthesize OPGs had no significant impact on the organism's lipopolysaccharide pattern or its ability to survive antimicrobial peptides-, detergent-, pH- and nutrient-stress conditions. We observed that the -defective strain respired at a reduced rate under acidic growth conditions (pH 5.0) and had lower ATP levels compared to the wild-type strain. These data indicate that OPGs of Typhimurium contribute towards mouse virulence as well as growth and motility under low osmolarity growth conditions.

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2009-01-01
2019-08-25
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Metabolic panel PM10, and IF-0 and IF-10 media (Biolog) were used in this study. Typhimurium strains SL1344 and SG111 were assayed phenotypically using a patented tetrazolium dye oxidation-reduction growth system (Bochner , 2001; Bochner, 2003), engineered by Biolog. Incubation and recording of phenotypic data were performed on an Omnilog instrument, which captured a digital image of the microarray panel over time and stored quantitative changes as turbidity/redox dye colour values. Twenty-hour-old colonies from an LB agar plate (or LB with kanamycin) were suspended in 15 ml IF-0 medium to obtain a cell suspension with 42% transmittance. This was inoculated into 75 ml complete IF-0 medium (60 ml 1.2x IF-0, 14.1 ml sterile distilled water, 0.9 ml 100x Dye mix D), corresponding to a cell suspension with 85% transmittance. A sample (750 7mu;l) of the IF-0 culture was used to inoculate a bottle of complete IF-10 medium (125 ml IF-10, 22.75 ml distilled water, 1.5 ml 100x Dye mix D). This culture was used to inoculate PM plate 10. All of the plates were incubated for 48 h at 37 °C, and turbidity/redox dye colour values were recorded simultaneously every 15 min by using the Omnilog instrument.

IMAGE

Characterization of the mutants of Typhimurium. (a) locus. Dotted lines represent where was deleted and the kanamycin resistance gene was inserted. H, dIII; S, I. (b) Southern blot hybridization analysis of dIII- (lanes 1, 3, 5, 7 and 9) and dIII/ I-digested (lanes 2, 4, 6, 8 and 10) chromosomal DNA from FIRN, SL1344, FG111 and SG111 using a 1.9 kbp probe (pOPGG1).

IMAGE

Stress-tolerance phenotypes of Typhimurium strains. (a) Cells were grown to stationary growth phase (except as noted) and subjected to specific stresses: (1) pH 3 for 2 h; (2) pH 9.8 for 2 h; (3) 58 °C for 5 min; (4) 15% ox bile for 24 h; (5) 1 μg polymyxin B for 1 h (using exponential-phase cells); (6) 20 mM H O for 2 h. Error bars indicate SEM(not shown when smaller than the symbol). Filled bars, strain SL1344; open bars, strain SG111. (b) Effect of detergent (5% SDS) on growth in LB medium. SDS (5%, w/v) was added (vertical arrow) 3 h after inoculation (SL1344: open circle; SG111, open square) or continued without addition of SDS (SL1344: filled circle; SG111, filled square).

IMAGE

Phenotypic array analysis of SL1344 and SG111 with metabolic panel PM10.

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