. Author manuscript; available in PMC: 2022 Jul 27.

Published in final edited form as: J Mol Biol. 2022 May 18;434(13):167641. doi: 10.1016/j.jmb.2022.167641

Table 1.

Sporulation frequencies of C. difficile Spo0A site-directed mutants

C. difficile Spo0A allele	Spo0A region	Corresponding B. subtilis Spo0A residue	Corresponding B. subtilis Spo0F residue	Spo0A function in B. subtilis	Spo0F function in B. subtilis	Average sporulation frequency (%)^a,c	Mutant phenotype in B. subtilis^b
Wildtype	-	-	-	Interaction with Spo0B, Spo0E	Interaction with KinA, KinB, KinC, KinD, and KinE, interaction with Spo0B	12.1±1.0	-
spo0A::erm	-	-	-	-	-	0.00
D10A	β1-α1	D10	D10	Forms aspartyl pocket, Spo0A dimerization, divalent cation binding, predicted Spo0E interaction [4, 25, 36, 68]	Forms aspartyl pocket, divalent cation binding [57]	0.0002±0.0001^**	n.d.
D11A	β1-α1	D11	D11	Forms aspartyl pocket, Spo0A dimerization, divalent cation binding, predicted Spo0E interaction [4, 25, 36, 68]	Forms aspartyl pocket, divalent cation binding [57]	0.00055±0.0004^**	n.d.
N12A	β1-α1	N12	Q12	Interaction with Spo0E, N12K is resistant to hyperactive Spo0E, predicted Spo0B interaction [12, 28, 33–35, 68]	Interaction with Spo0B, inferred interaction with KinA [12, 13, 39, 69]	14.8±1.86	Spo0A gain-of-function
K13A	α1	R13	Y13	n.d.	Interaction with RapA and RapB, Y13S is resistant to RapB [70–73]	19.1±3	Spo0F gain-of-function
D14A	α1	E14	G14	E14A allows direct interaction with KinC, resistant to hyperactive Spo0E [28, 33]	Interaction with Spo0B, and RapB [12, 71–73]	49.7±6.5^*	Spo0A gain-of-function
F15A	α1	L15	I15	n.d.	Interaction with KinA, Spo0B, and RapB [12, 13, 71–73]	23.1±1.7	Spo0F decreased sporulation
C16A	α1	V16	R16	n.d.	Interaction with KinA, Spo0B, and RapB [12, 13, 71]	0.7±0.3^*	Spo0F decreased sporulation
Q17A	α1	S17	I17	n.d.	Interaction with RapB [13, 69, 71–73]	<LOD ^**	Spo0F gain-of-function
V18A	α1	L18	L18	n.d.	Interaction with KinA, Spo0B, RapB [12, 13, 71–73]	<LOD ^**	Spo0F decreased sporulation
L19A	α1	L19	L19	n.d.	Inferred structural importance [13]	6.4±1.5	Spo0F decreased sporulation
E21A	α1	E21	E21	Inferred to interact with Spo0B [12]	Interaction with Spo0B, inferred interaction with KinA [12, 39]	0.01±0.01^**	n.d.
A35S	β2	A35	A33	n.d.	n.d.	0.15±0.1^*	n.d.
K36A	β2-α2	Y36	A34		Interaction with Spo0B [12]	26.4±5.7	n.d.
D56A	β3	D56	D54	Site of phosphorylation by Spo0B, forms aspartyl pocket, Spo0A dimerization, predicted to interact with Spo0E [4, 25, 36, 68]	Site of phosphorylation by KinA, KinB, KinC, KinD, and KinE,[27], forms aspartyl pocket [4, 25, 36, 57]	0.008±0.001^**	Spo0A
I58A	β3-α3	I58	K56	Stabilizes aspartyl pocket, Spo0A dimerization [25]	Interaction with KinA, Spo0B, and RapB, stabilizes aspartyl pocket [12, 13, 57, 71]	2±0.9	Spo0F decreased sporulation
M59A	β3-α3	M59	I57	n.d.	Interaction with KinA [13]	0.006±0.001^**	Spo0F decreased sporulation
P60A	β3-α3	P60	P58	Interaction with Spo0E, P60S is resistant to hyperactive Spo0E; active without phosphorelay [28, 33]	Interaction with Spo0B [12]	<LOD ^**	Spo0A gain-of-function
H61A	β3-α3	H61	G59	n.d.	Interaction with Spo0B [12]	0.6±0.2^*	n.d.
L62A	β3-α3	L62	M60	Interaction with Spo0E, L62P is resistant to hyperactive Spo0E [28]	M60A results in reduced spoIIG transcription [12]	11.5±1.8	Spo0A gain-of-function
S86A	β4	T86	T82	Stabilizes phosphorylation of active site [47]	Interaction with KinA, interaction with RapH, interaction with Spo0B [13, 38, 39, 74]	0.1±0.1^*	Spo0F decreased sporulation
A87S	β4	A87	A83	n.d.	Interaction with Spo0B [12]	0.01±0.01^**	n.d.
V88A	β4-α4	F88	Y84	Interaction with Spo0E; F88L is resistant to hyperactive Spo0E [28]	Interaction with Spo0B, KinA, RapH, Y84A is resistant to RapB, RapH [13, 38, 71]	17.9±1.0	Spo0F reduced sporulation
G89A	β4-α4	G89	G85	Inferred to interact with Spo0B [12]	Interaction with Spo0B [12]	5.6±2.1	n.d.
Q90A	β4-α4	Q90	E86	Q90R allows for direct interaction with KinC, and resistant to hyperactive Spo0E [34, 35]	Interaction with Spo0B, interaction with KinA [12, 13]	59.3±10.7^*	Spo0F reduced sporulation, Spo0A gain-of-function
D91A	α4	E91	L87	n.d.	Interaction with Spo0B, interaction with KinA [12, 13]	37.8±12.5	Spo0F reduced sporulation
K92A	α4	D92	D88	D92Y is resistant to hyperactive Spo0E and is functional without phosphorelay [27, 33]	n.d.	33±2.7^*	Spo0A gain-of-function
K108A	β5	K108	K104	Stabilizes aspartyl pocket, Spo0A dimerization, predicted Spo0E and Spo0B interaction [12, 25, 68]	Interaction with Spo0B, stabilizes aspartyl pocket, inferred interaction with KinA [12, 39, 57]	0.6±0.2^*	n.d.
P109A	β5-α5	P109	P105	Spo0E and Spo0B interaction [12, 68]	Interaction with Spo0B [12]	76.9±9.3^*	n.d.
F110A	β5-α5	F110	F106	Predicted Spo0E interaction [68]	Interaction with Spo0B [12]	17.9±2.1	Spo0F reduced sporulation
D111A	β5-α5	D111	D107	Predicted Spo0E interaction [68]	Interaction with Spo0B, inferred interaction with KinA [12, 39]	7.6±0.9	n.d.

^{a *}

, P = > .05

^**

, P = > .01

B. subtilis phenotype that differs from C. difficile phenotype noted in bold

Spo0A site-directed mutant sporulation frequency where protein was undetectable by western blot is underlined

n.d., not determined