Figure 3: The effects of ptsN on the expression of genes involved in propionate catabolism, vitamin B₁₂ synthesis, and 1,2-PDL utilization.

(a) Heat-map analysis of the expression ratios of the prp, cob-cbi, and pdu genes between the ΔptsN mutant and wild-type strains (log₂[ΔptsN mutant/wild-type]). (b) Evaluation of mRNA levels of the cob-cbi, pdu, and prp genes in strains of wild-type, ΔptsN, and ΔptsN containing pPtsN. Every mRNA level was normalized using that of gyrB in each strain and its relative expression was estimated comparing with the mRNA level of ptsN in wild-type containing pACYC184, which was set at 1.0. The pPtsN plasmid has ptsN and its putative promoter sequences on the backbone of pACYC184. Ado-B₁₂ (20 nM) and 1,2-PDL (12.5 mM) were added to the culture to stimulate the prp operon under aerobic conditions. (c) Medium pH was assayed to compare the activity of 1,2-PDL catabolism in wild-type SL1344 containing pACYC184 and the ΔptsN mutant strain harboring pPtsN or pACYC184. Neutral-red (0.0033%) was added to the culture as a pH indicator and changed its color to dark red in proportion to the level of propionate, a byproduct of the 1,2-PDL pathway. All cultures were supplemented with Ado-B₁₂ (20 nM), and 1,2-PDL (12.5 mM) was added as a substrate for the 1,2-PDL pathway. The result shown is representative of three independent tests. (d) The mRNA levels of prp genes were measured in a similar manner as conducted in (b) with normalization to the mRNA level of ptsN in the wild-type containing pACYC184, but propionate (10 mM) was added as an alternative carbon source instead of 1,2-PDL and Ado-B₁₂.