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. 2009 Jun 26;191(17):5358–5368. doi: 10.1128/JB.00503-09

FIG. 3.

FIG. 3.

Quantitative assay for detection of the enzymatic activities of a series of KinA mutants. Genes for full-length KinA and each truncated mutant protein were fused to Phyper-spank and introduced at the amyE locus of cells containing both kinA- and kinB-null mutants. To monitor Spo0A activity, a transcriptional reporter was constructed in which the gene encoding β-galactosidase (lacZ) was fused to the promoter region of the spoIIG operon (PspoIIG) and introduced at the thrC locus of each strain. The wild-type strain (wt; lacking the IPTG-inducible construct) was used as a control. For sporulation conditions (SM), cells in CH medium were grown to the mid-exponential phase (OD600, 0.5), collected by centrifugation, and resuspended in SM medium. Then IPTG was added to a final concentration of 10 μM. For nutrient-rich conditions (LB), cells in LB medium were grown to the mid-exponential phase (OD600, 0.5), collected by centrifugation, and resuspended in fresh LB medium. Then IPTG was added to a final concentration of 10 μM. (A) Sporulation efficiency, expressed in CFU per milliliter, was determined as described in Materials and Methods. (B) The accumulation of β-galactosidase from a PspoIIG-lacZ fusion was monitored at hour 3 after suspension as a measure of Spo0A activity. Data for β-galactosidase activity are expressed in Miner units and are averages of three independent results with standard deviations. Strains used are as follows: MF3366 (wt), MF3316 (full-length KinA), MF3317 (ΔPAS-A), MF3320 (ΔPAS-AB), MF3323 (KinAC), and MF3324 (KinAN). (C) Expression of full-length KinA or KinA mutant protein was examined by immunoblot analysis. Cell extracts of strains expressing GFP fusion proteins were prepared at hour 2 after suspension and were processed for immunoblotting with anti-GFP (α-GFP) antibodies. σA, a constitutively expressed protein, was used as a loading control. Strains used are as follows: MF3593 (wt) (lane 1), MF3352 (full-length KinA) (lane 2), MF3353 (ΔPAS-A) (lane 3), MF3356 (ΔPAS-AB) (lane 4), MF3359 (KinAC) (lane 5), and MF3360 (KinAN) (lane 6).