. 2018 Sep 17;84(19):e01139-18. doi: 10.1128/AEM.01139-18

TABLE 2.

Potential contributing factors associated with in vivo titrations

Variable	Mode of modulation
Protein concn	Removed in knockout E. coli strains; readded at tunable levels using the P_tet promoter and tetracycline in pKTS^a
	Minimized via use of SsrA degradation tag in pKTS^a
	Proteins can be stabilized or destabilized by interaction with osmolytes^b and/or crowders.^c
	Enzyme activity can be modulated by interactions with neighboring macromolecules.^d
	Altered protein expression levels possible due to osmotic stress^e
	May be affected by folding mechanism associated with oligomerization; e.g., if monomer can fold coming off the ribosome, it may be more stable than if an unfolded monomer needs another monomer to be synthesized to form a dimer
Enzyme activity	k_on and k_off rates as well as k_cat/K_m can be altered by interaction with crowders as well as increased viscosity upon addition of osmolytes and/or crowders. At high substrate concn, if chemistry is rate determining, then viscosity will likely have minimal effects.^f
	Substrate capture (e.g., k_cat/K_m) can be rate determining at low substrate concn.^g
	If enzyme activity is inhibited by osmotic stress, the substrate concn will rise.
Substrate concn in cell	Weak interaction of ligands with osmolytes, for example, folate interaction with trehalose and betaine, can lower the concn of free ligand.^h
Cofactor concn in cell	Weak interaction of ligands with macromolecular surfaces; for example, folate interaction with lysozyme, BSA, and others can lower the concn of free ligand.ⁱ
	Gene knockout results in buildup of the substrate concn for the encoded enzyme.
	No metabolomics information on folate redox state concn or NADPH in E. coli under osmotic stress^j
	Exclusion of osmolytes from NADPH can tighten binding.^k
Domino effects	Buildup in substrate concn of inhibited enzymes can inhibit other enzymes in pathway. For example, inhibition of DHFR by trimethoprim results in buildup of DHF concn, which, in turn, inhibits folyl-polyglutamate synthase.^l
	Inhibition of one enzyme will deplete the concn of its product, which, as the substrate of another enzyme, will reduce this rate as well. For example, a mathematical model of folate liver metabolism predicts that >90% inhibition of DHFR is necessary to affect thymidylate synthase activity.^m
Identity of osmolytes in E. coli	Normally trehalose is produced as an osmoprotectant against low-water conditions.ⁿ Addition of betaine to the media results in its uptake and higher growth rates due to the preferential exclusion mechanism. Cells cannot grow when no free water remains (∼1.9 osmol).^o
	Addition of betaine switches E. coli to fermentative pathways at high NaCl concn.^p

From reference 27.

From references 64 and 65.

From references 21, 66, and 67.

From references 21, 98, and 99.

From references 48 and 100 to 104.

From references 75 to 80.

From references 105 to 107.

From references 8 to 11 and 13.

ⁱ

From unpublished data and references 11 and 71. BSA, bovine serum albumin.

From reference 108.

From references 8 to 10.

From reference 72.

From reference 109.

ⁿ

From reference 110.

From reference 7.

From reference 100.