Table 2.

Analysis of spontaneous ΔgpsB suppressor mutations that arose in unencapsulated derivatives of strain D39 and Rx1^a

Strain number (suppressor designation)	Genetic background	Genotype	Large deletion/ duplication present?b	Phosphorylation phenotypec	Other mutations present
1. IU6442 (sup1)d	D39 Δcps ΔgpsB	phpP(G229D)	No	Normal	None detected; strain reconstructed (see Fig. 4)
2. IU5845 (sup2)e		Δ[spd_1026– spd_1037] Ω[spd_0889– spd_1026]	Yes	None	araD(ΔC at aa 123/224) zmpB(Q456P) miaA(N172K)
3. IU6441 (sup3)e		Δ[spd_1029– spd_1037] Ω[spd_0889– spd_1024]	Yes	None	None detected
4. IU9262 (sup4)f	Rx1 ΔgpsB	phpP(L148S)	No	Normal	stkP(I102T) Δ[spd_1037– spd_1038]
5. Additional classes of ΔgpsB suppressorsg

^aTransformations were performed as described in Experimental procedures. Control transformations with a ΔpurR<>aad9 amplicons gave >500 colonies in 24 h, whereas ΔgpsB<>aad9 transformations gave <10 colonies in 48 h (see Table 1, line 1). Mutations in the sup1–3 suppressors were located by whole-genome sequencing (see Experimental procedures) and are listed in columns 3 and 6. IU6442, IU5845, and IU6441 grew with similar doubling times (≈ 30 min) as the parent strain in BHI broth, but the growth yields of IU5845 and IU6441 were about 25% lower than those of IU6442 and the parent (Fig. 5A; data not shown).

^bChromosomal deletions/duplications Δ[spd_1026–spd_1037] (13 genes)/Ω[spd_0889–spd_1026] (134 genes) and Δ[spd_1029–spd_1037]/Ω[spd_0889–spd_1024] are depicted in Figure S3A and 3B.

^cDetection of proteins phosphorylated at Thr residues was performed by Western blotting using α-pThr antibody as described in Experimental procedures. See Results and Fig. 3 for details.

^dΔgpsB suppressor mutants containing phpP(G117D) (IU6444), phpP(T163P) (IU7736), or phpP(R125P) (IU11955) mutations were independently isolated and identified by conventional DNA sequencing of phpP-stkP, but were not further characterized in this study. These mutants contained wild-type stkP⁺ and lacked deletion of the spd_1034 region, as determined by PCR (Fig. S3).

^eDeletion in the spd_1034 region was detected by PCR in 15 additional, independently isolated ΔgpsB suppressor mutants that were not characterized further in this study.

^fsup4(phpP(L148S)) arose spontaneously in Rx1, which also contained the stkP(I102T) and Δ[spd_1037–spd_1038] mutations, which were confirmed by conventional DNA sequencing and PCR, respectively.

^g>5 independent, spontaneous ΔgpsB suppressors were isolated that are phpP⁺ stkP⁺ and lack deletion in the spd_1034 region of the chromosome. These suppressors were not further characterized in this study.