Phosphorylation of Spo0A by the histidine kinase KinD requires the lipoprotein med in Bacillus subtilis

Abstract

The response regulatory protein Spo0A of Bacillus subtilis is activated by phosphorylation by multiple histidine kinases via a multicomponent phosphorelay. Here we present evidence that the activity of one of the kinases, KinD, depends on the lipoprotein Med, a mutant of which has been known to cause a cannibalism phenotype. We show that the absence of Med impaired and the overproduction of Med stimulated the transcription of two operons (sdp and skf) involved in cannibalism whose transcription is known to depend on Spo0A in its phosphorylated state (Spo0A∼P). Further, these effects of Med were dependent on KinD but not on kinases KinA, KinB, and KinC. Additionally, we show that deletion or overproduction of Med impaired or enhanced, respectively, biofilm formation and that these effects, too, depended specifically on KinD. Finally, we report that overproduction of Med bypassed the dominant negative effect on transcription of sdp of a truncated KinD retaining the transmembrane segments but lacking the kinase domain. We propose that Med directly or indirectly interacts with KinD in the cytoplasmic membrane and that this interaction is required for KinD-dependent phosphorylation of Spo0A.

Fig. 1.

Med is required for the maximal expression of the sdpABC and skfABCDE operons. Strains harbored either amyE::P_sdp-lacZ (top) or amyE::P_skf-lacZ (bottom) and were wild type (WT; RL4264), carried a med mutation (AB332), or harbored an overexpression construct, P_hyperspank-med (AB333). Cells were grown in liquid DSM; hour 0 of stationary phase was the end of the exponential phase of growth. Expression of P_hyperspank-med was induced by addition of 1 mM (final concentration) IPTG to the medium. The experiment was repeated a minimum of three times. Shown are the results of a representative experiment.

Fig. 2.

Med acts independently of KinC and in the same pathway as KinD to promote expression of sdp. (A) Strains harbored amyE::P_sdp-lacZ and were wild type (RL4264) or harbored a med mutation (AB332), P_hyperspank-med (▵; AB333), a kinC mutation (AB341), a med kinC mutation (AB342), or a kinC mutation and P_hyperspank-med (AB343). (B) Strains harbored amyE::P_sdp-lacZ and were either wild type (RL4264) or harbored a med mutation (AB332), P_hyperspank-med (AB333), a kinD mutation (AB337), a med kinD mutation (AB338), or a kinD mutation and P_hyperspank-med (AB339). Cells were grown in liquid DSM; hour 0 of stationary phase was the end of the exponential phase of growth. Expression of P_hyperspank-med was induced by addition of 1 mM (final concentration) IPTG to the medium. The experiment was repeated a minimum of three times. Shown are the results of a representative experiment.

Fig. 3.

Med acts independently of KinA and KinB to promote expression of sdp. (A) Strains harbored amyE::P_sdp-lacZ and were either wild type (RL4264) or harbored a med mutation (AB332), P_hyperspank-med (AB333), a kinA mutation (AB481), a med kinA mutation (AB482), or a kinA mutation and P_hyperspank-med (AB483). (B) Strains harbored amyE::P_sdp-lacZ and were either wild type (RL4264) or harbored a med mutation (AB332), P_hyperspank-med (AB333), a kinB mutation (AB484), a med kinB mutation (AB485), or a kinB mutation and P_hyperspank-med (AB486). Cells were grown in liquid DSM; hour 0 of stationary phase was the end of the exponential phase of growth. Expression of P_hyperspank-med was induced by addition of 1 mM (final concentration) IPTG to the medium. The experiment was repeated a minimum of three times. Shown are the results of a representative experiment.

Fig. 4.

Med acts independently of KinC and in the same pathway as KinD to influence biofilm formation. Shown are biofilms formed by strains grown on solid MSgg medium that were either wild type (3610) or harbored a med mutation (AB349), a kinC mutation (AB399), a kinD mutation (RL4552), a kinC kinD mutation (AB401), a med kinC mutation (AB400), a med kinD mutation (AB351), P_hyperspank-med (AB396), a kinC mutation and P_hyperspank-med (AB402), or a kinC mutation and P_hyperspank-med (AB398). Expression of P_hyperspank-med was induced by addition of 1 mM (final concentration) IPTG to the medium.

Fig. 5.

Overexpression of truncated KinD has a dominant-negative effect on sdp expression that is rescued by overproduction of Med. Strains harbored amyE::P_sdp-lacZ and were either wild type (RL2886) or harbored a med mutation (AB307), P_hyperspank-med (▵; AB430), a construct that overexpressed truncated KinD (amino acids 1 to 294) (P_xylA-kinD*) (AB432), a med mutation and P_xylA-kinD* (AB433), or P_hyperspank-med and P_xylA-kinD* (AB434). Cells were grown in liquid DSM; hour 0 of sporulation was the end of the exponential phase of growth. Expression of P_hyperspank-med was induced by addition of 1 mM (final concentration) IPTG to the medium. Expression of P_xylA-kinD* was induced by addition of 20 mM (final concentration) xylose to the medium. The experiment was repeated a minimum of three times. Shown are results of a representative experiment.