Figure 1.
Determination of curli production by Congo red binding.
Phenotypes on CR medium of MG1655 (wild type strain), AM70 (csgA deletion mutant, unable to produce curli), MG1655carB::Tn5kan, MG1655carB::Tn5kan ΔcsgA::cat and MG1655ΔcarB::cat. Strains were grown either at 30°C (for 24 hours) or at 37°C (for 18 hours). Plates were incubated for 48 hours at 4°C to enhance Congo red binding.
Figure 2.
UMP biosynthetic pathways in Escherichia coli.
Adapted from Ecocyc (http://ecocyc.org/).
Figure 3.
Congo red binding by E. coli strains deficient in UMP biosynthesis.
The MG1655 strain and isogenic mutants deficient in UMP biosynthetic genes were spotted on either CR medium or CR(ura) medium (CR medium supplemented with 0.25 mM uracil) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding.
Table 1.
Determination of gene expression levels.
Figure 4.
Congo red binding by E. coli strains deficient in pyrimidine sensing (cytR and rutR mutants) and purine biosynthesis (purH mutant).
4A. The MG1655 strain and its isogenic mutants in the purH, cytR and rutR genes were spotted either on CR medium (left panel) or on CR(ura) medium (right panel) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding. Determination of transcript levels. 4B. Relative expression of either the csgD gene (left panel) or the udp gene (right panel) was determined by Real-Time PCR on RNA extracted from overnight cultures of MG1655 and of its isogenic purH and cytR mutants. 16S RNA transcript was used as reference gene. ΔCt values between the genes of interest and 16S RNA were set at 1 for MG1655 in LB1/4 medium, and transcript levels in other strains and/or growth conditions are expressed as relative values. Experiments were repeated at least three times, each time in duplicate; standard deviations were always lower than 5%.
Figure 5.
Effect of cellulose production on Congo red binding.
Phenotypes on CR medium of MG1655, MG1655ΔcarB::cat, MG1655ΔcarB::cat ΔbcsA::kan, MG1655ΔcarB::cat ΔyedQ::kan, MG1655ΔcarB::cat ΔadrA::kan. Strains were spotted on either CR medium or CR(ura) medium (CR medium supplemented with 0.25 mM uracil) and grown for 24 hours at 30°C. Plates were incubated for 48 hours at 4°C to enhance Congo red binding.
Figure 6.
Determination of cellulose amounts.
Strains MG1655, MG1655ΔcarB::cat, and MG1655ΔpyrC::tet were grown 48 hours at 30°C on either LB1/4 agar (no added uracil, Cellulose extraction and determination was performed as described [35]. Data shown are the average of two independent experiments giving very similar results. For strains MG1655ΔcarB::cat and MG1655ΔpyrC::tet grown on LB1/4 agar no glucose was detectable in the assays. A value of 0.5 nmol glucose, corresponding to the lowest detectable concentration in the assay, as determined by a glucose standard curve, was thus arbitrarily assigned to these strains.
Table 2.
Escherichia coli strains and plasmids used in this work.