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. Author manuscript; available in PMC: 2023 Sep 27.
Published in final edited form as: Mol Microbiol. 2023 Jul 14;120(3):351–383. doi: 10.1111/mmi.15122

Figure 10. Tn-seq demonstrates the essentiality of StkP(Spn) and GpsB(Spn) is suppressed by ΔkhpB in cells growing exponentially in BHI broth in 5% CO2.

Figure 10.

(A) Top: Predicted 3D structure of StkP(Spn) generated using the AlphaFold v2.0 webserver. P1, P2, P3 and P4 with indicated amino acid numbers are predicted extracellular PASTA domains. Bottom: Mini-Mariner Malgellan6 Tn-Seq transposon insertion profile for the genome region covering sun, phpP, stkP, and spd_1541 in the genomes of the unencapsulated WT parent (D39 Δcps rpsL1, IU1824) or ΔkhpB (IU10592) strain growing exponentially in BHI broth in 5% CO2. The same WT Tn-seq insertion profile was obtained for encapsulated D39 strain IU1781 grown in BHI broth or IU1824 grown in C+Y, pH 6.9 medium in 5% CO2 (data not shown). In vitro transposition reactions containing purified genomic DNA, Magellan6 plasmid DNA, and purified MarC9 mariner transposase, transformation, harvesting of transposon-inserted mutants, growth of pooled insertion libraries exponentially in BHI broth or C+Y, pH 6.9 medium, NextSeq 75 high-output sequencing, and analysis were performed as described in Experimental procedures based on (Lamanna et al., 2022). Sortable data for the profile shown are contained in Appendix A, Tabs C and D. Tn-insertions were recovered for the WT strains in the regions encoding P3 and P4, but not in other regions of stkP. The first TA insertion occurs in the WT strain at a TAT (Y515) codon, where the Tn insertion creates a TAA stop codon, while there is no insertion at the upstream TTA (L512) codon, indicating that StkP(M1-L512) is essential for viability. (B) Tn-Seq transposon insertion profiles for the genome region covering recU, spd_0338, gpsB, and rnpB of in the genomes of the WT parent (D39 Δcps rpsL1, IU1824) or ΔkhpB (IU10592) strain. (C) Representative growth curves of the WT parent (IU1824), ΔkhpA (IU9036), ΔkhpA ΔgpsB (IU16196) and ΔkhpA ΔstkP (IU16910) strains. Similar growth results were obtained with ΔkhpB (IU10592), ΔkhpB ΔgpsB (IU12977), and ΔkhpB ΔstkP (IU16912) strains compared to the strains of ΔkhpA background. The growths of merodiploid strains ΔgpsB//PZn-gpsB+ (IU16370) and ΔstkP::Pc-erm//PZn-stkP+ (IU16933) grown under conditions that result in depletion of GpsB or StkP were shown for comparison.