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Microbiology vol. 155, part 1, pp. 229-237
Stress-tolerance experiments.
Stationary-phase cells grown in LB broth were subjected to various stress conditions. In order to avoid high cell density-dependent artefacts of acid (and other stress) survival phenotypes (Cui et al., 2001), initial cell density at time zero of the stress was maintained in the range of 1x107 to 4x107 cells ml_1. For acid and alkali shock, cells were diluted 200-fold in minimal E medium containing 0.4% glucose (EG medium), adjusted to pH 3.0 (with 6 M HCl) or pH 9.8 (with 10 M NaOH) at 37 °C (Bhagwat et al., 2006; Gawande & Bhagwat, 2002). Cell suspensions were incubated at 37 °C without shaking for 3 h, after which 10-fold serial dilutions were made in 50 mM PBS (pH 6.8) and viable counts were determined by plating aliquots on LB agar.
For heat-tolerance analysis, cells at the stationary growth phase were diluted 200-fold in EG medium (pH 6.8), pre-warmed to 58 °C and incubated for 5 min (without shaking) (Bhagwat et al., 2005). To determine viable cell counts, aliquots were withdrawn, diluted in PBS and immediately plated on LB agar.
For bile resistance analysis, stationary-phase cells were diluted (1:200) in 15% Millipore-filtered ox bile in EG medium and incubated at 37 °C without shaking. Viable cell counts were determined after 24 h by making 10-fold serial dilutions in PBS.
Oxidative stress was performed by diluting stationary-phase cells, grown in EG medium containing 20 mM H2O2, and after 2 h incubation at 37 °C, aliquots were withdrawn, diluted in PBS and immediately plated on LB agar.
Tolerance to detergent was determined by adding 5% SDS (w/v in LB broth) to freshly inoculated culture (3 h after 1:200 dilution of stationary-phase cells), and growth was monitored by a Klett turbidometer using a red transmission filter.
Phenotypic microarray analysis.
Metabolic panel PM10, and IF-0 and IF-10 media (Biolog) were used in this study. S. Typhimurium strains SL1344 and SG111 were assayed phenotypically using a patented tetrazolium dye oxidation-reduction growth system (Bochner et al., 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.
Fig. S1. Characterization of the opg mutants of S. Typhimurium. (a) opgGH locus. Dotted lines represent where opgG was deleted and the kanamycin resistance gene was inserted. H, HindIII; S, SmaI. (b) Southern blot hybridization analysis of HindIII- (lanes 1, 3, 5, 7 and 9) and HindIII/SmaI-digested (lanes 2, 4, 6, 8 and 10) chromosomal DNA from FIRN, SL1344, FG111 and SG111 using a 1.9 kbp opgG probe (pOPGG1).
Fig. S2. Stress-tolerance phenotypes of S. 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 H2O2 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).
Fig S3. Phenotypic array on plate PM-10 for S. Typhimurium strain SL 1344 (green) vs strain SG111 (red). The OmniLog-PM software generates time-course curves for respiration (tetrazolium colour formation) and plots the values for the wild-type (green) and mutant (red) cells; yellow indicates overlapping values. The units are arbitrary. Growth media for individual wells are as indicated in Table S1.
Table S1. Phenotypic array analysis of SL1344 and SG111 with metabolic panel PM10.
Bhagwat, A. A., Chan, L., Han, R., Tan, J., Kothary, M., Jean-Gilles, J. & Tall, B. D. (2005). Characterization of enterohaemorrhagic Escherichia coli strains based on acid resistance phenotypes. Infect Immun 73, 4993-5003. Medline
Bhagwat, A. A., Tan, J., Sharma, M., Kothary, M., Low, S., Tall, B. D. & Bhagwat, M. (2006). Functional heterogeneity of RpoS in stress tolerance of enterohaemorrhagic Escherichia coli strains. Appl Environ Microbiol 72, 4978-4986. Medline
Bochner, B. R. (2003). New technologies to assess genotype-phenotype relationships. Nat Rev Genet 4, 309-314. Medline
Bochner, B. R., Gadzinski, P. & Panomitros, E. (2001). Phenotype microarrays for high-throughput phenotypic testing and assay of gene function. Genome Res 11, 1246-1255. Medline
Cui, S., Meng, J. & Bhagwat, A. A. (2001). Availability of glutamate and arginine during acid challenge determines cell density-dependent survival phenotype of Escherichia coli strains. Appl Environ Microbiol 67, 4914-4918. Medline
Gawande, P. V. & Bhagwat, A. A. (2002). Inoculation onto solid surfaces protect Salmonella spp. during acid challenge: a model study using polyethersulfone membranes. Appl Environ Microbiol 68, 86-92. Medline
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