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A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

Grau RR, de Oña P, Kunert M, Leñini C, Gallegos-Monterrosa R, Mhatre E, Vileta D, Donato V, Hölscher T, Boland W, Kuipers OP, Kovács ÁT - MBio (2015)

Bottom Line: Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host.Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question.The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas (FCByF), Universidad Nacional de Rosario (UNR)-CONICET, Argentina robertograu@fulbrightmail.org akos-tibor.kovacs@uni-jena.de.

No MeSH data available.


Related in: MedlinePlus

KinB harbors a cytosolic selectivity filter motif responsive to potassium ions that specifically allows sliding proficiency. (A) Amino acid sequence of KinB. The six continuous underlines indicate the six transmembrane domains of KinB. The histidine highlighted in green corresponds to the residue of autophosphorylation; the blue amino acid triplets represent the top and bottom sites of the ATP-binding domain of the kinase. The yellow box highlights the cytosolic sequence in KinB with homology to the potassium selectivity filter sequence present in potassium channels. (B to D) Sporulation and sliding proficiencies are separable KinB functions. KinB mutant strains affected in the integrity of the selective filter sequence were able to restore full sporulation proficiency of a Spo0 kinA kinB double mutant strain (A−B− in panel B) but did not restore KinB-dependent sliding activity in that A−B− background (B), either in kinB (B−) (C)- or in kinB kinC (B−C−) (D)-deficient mutant strains. Bk+→A and BΔk+ indicate KinB proteins with Ala-exchanged and Ala-deletion K+-filter domains, respectively. (E) Mutation of the potassium selectivity sequence in KinB abolished the ability of B. subtilis to slide in response to potassium addition. Sliding and sporulation proficiencies were measured as indicated in Materials and Methods. Results presented in panels B to E are representative of four experiments performed separately after 40 h of incubation.
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fig7: KinB harbors a cytosolic selectivity filter motif responsive to potassium ions that specifically allows sliding proficiency. (A) Amino acid sequence of KinB. The six continuous underlines indicate the six transmembrane domains of KinB. The histidine highlighted in green corresponds to the residue of autophosphorylation; the blue amino acid triplets represent the top and bottom sites of the ATP-binding domain of the kinase. The yellow box highlights the cytosolic sequence in KinB with homology to the potassium selectivity filter sequence present in potassium channels. (B to D) Sporulation and sliding proficiencies are separable KinB functions. KinB mutant strains affected in the integrity of the selective filter sequence were able to restore full sporulation proficiency of a Spo0 kinA kinB double mutant strain (A−B− in panel B) but did not restore KinB-dependent sliding activity in that A−B− background (B), either in kinB (B−) (C)- or in kinB kinC (B−C−) (D)-deficient mutant strains. Bk+→A and BΔk+ indicate KinB proteins with Ala-exchanged and Ala-deletion K+-filter domains, respectively. (E) Mutation of the potassium selectivity sequence in KinB abolished the ability of B. subtilis to slide in response to potassium addition. Sliding and sporulation proficiencies were measured as indicated in Materials and Methods. Results presented in panels B to E are representative of four experiments performed separately after 40 h of incubation.

Mentions: Does potassium represent a direct or an indirect signal to activate KinB? Searching for conserved domains and sequence motifs present in KinB that might be involved in the potassium response, we discovered a disregarded sequence (SLKTNGTG) residing on the ATP-binding region of KinB that is absent in the sequences of the other four phosphorelay sensor kinases (56) (Fig. 7A). This sequence possesses a significant homology to the highly conserved K+-filter (selectivity) sequence of the pore loop domain (P-domain) of potassium channels (T/S-x-x-T-x-G-x-G consensus sequence) (57, 58). Despite the many protein motifs and domains present in different types of potassium channels (58), the KinB K+-selectivity-like sequence (here called K* for simplicity) is the only common element related to K+ channels. While active KinB is a dimer (59), typical potassium channels are tetramers made up of predominantly identical subunits clustered to form the ion permeation pathway across the membrane (60–62). In addition to the absence of the pore motifs that surround the selectivity filter (Fig. 7B), KinB also lacks the different domains that have been described in different potassium channels (58, 60, 61). Furthermore, the K* resides in the cytosolic region of the kinase, while in all known (eukaryotic and prokaryotic) potassium channels the K+-filter resides in transmembrane domains. While these topological features exclude KinB as a potassium channel, they open the possibility that the kinase might sense the intracellular concentration of the ion throughout its cytosolic K*. Therefore, we tested if the K* plays a role or not in sliding motility. To this end, we constructed two types of kinB mutant strains harboring specific mutations in the K* (see Fig. S6 in the supplemental material). In one case, three out of the four conserved amino acids of the consensus K* were replaced by alanines (the fourth conserved amino acid, G, of the consensus sequence was not altered as it overlaps with a predicted ATPase motif of the kinase [Fig. 7A]) to give rise to the mutant KinBK*→A (see Fig. S6). In the second constructed kinB mutant strain, 7 out of the 8 amino acids of the K* were deleted (mutant strain KinBΔK* [see Fig. S6]). One important consideration for both mutant strains before analysis of their roles in sliding is that they must be functional (i.e., promote spore formation). In this sense, the sliding-promoting activity of KinB should be separable from its biofilm/sporulation-promoting activities, a scenario that would explain why KinB-dependent sliding proficiency and KinB-dependent biofilm/spore formation are not simultaneously activated (see below).


A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

Grau RR, de Oña P, Kunert M, Leñini C, Gallegos-Monterrosa R, Mhatre E, Vileta D, Donato V, Hölscher T, Boland W, Kuipers OP, Kovács ÁT - MBio (2015)

KinB harbors a cytosolic selectivity filter motif responsive to potassium ions that specifically allows sliding proficiency. (A) Amino acid sequence of KinB. The six continuous underlines indicate the six transmembrane domains of KinB. The histidine highlighted in green corresponds to the residue of autophosphorylation; the blue amino acid triplets represent the top and bottom sites of the ATP-binding domain of the kinase. The yellow box highlights the cytosolic sequence in KinB with homology to the potassium selectivity filter sequence present in potassium channels. (B to D) Sporulation and sliding proficiencies are separable KinB functions. KinB mutant strains affected in the integrity of the selective filter sequence were able to restore full sporulation proficiency of a Spo0 kinA kinB double mutant strain (A−B− in panel B) but did not restore KinB-dependent sliding activity in that A−B− background (B), either in kinB (B−) (C)- or in kinB kinC (B−C−) (D)-deficient mutant strains. Bk+→A and BΔk+ indicate KinB proteins with Ala-exchanged and Ala-deletion K+-filter domains, respectively. (E) Mutation of the potassium selectivity sequence in KinB abolished the ability of B. subtilis to slide in response to potassium addition. Sliding and sporulation proficiencies were measured as indicated in Materials and Methods. Results presented in panels B to E are representative of four experiments performed separately after 40 h of incubation.
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Related In: Results  -  Collection

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fig7: KinB harbors a cytosolic selectivity filter motif responsive to potassium ions that specifically allows sliding proficiency. (A) Amino acid sequence of KinB. The six continuous underlines indicate the six transmembrane domains of KinB. The histidine highlighted in green corresponds to the residue of autophosphorylation; the blue amino acid triplets represent the top and bottom sites of the ATP-binding domain of the kinase. The yellow box highlights the cytosolic sequence in KinB with homology to the potassium selectivity filter sequence present in potassium channels. (B to D) Sporulation and sliding proficiencies are separable KinB functions. KinB mutant strains affected in the integrity of the selective filter sequence were able to restore full sporulation proficiency of a Spo0 kinA kinB double mutant strain (A−B− in panel B) but did not restore KinB-dependent sliding activity in that A−B− background (B), either in kinB (B−) (C)- or in kinB kinC (B−C−) (D)-deficient mutant strains. Bk+→A and BΔk+ indicate KinB proteins with Ala-exchanged and Ala-deletion K+-filter domains, respectively. (E) Mutation of the potassium selectivity sequence in KinB abolished the ability of B. subtilis to slide in response to potassium addition. Sliding and sporulation proficiencies were measured as indicated in Materials and Methods. Results presented in panels B to E are representative of four experiments performed separately after 40 h of incubation.
Mentions: Does potassium represent a direct or an indirect signal to activate KinB? Searching for conserved domains and sequence motifs present in KinB that might be involved in the potassium response, we discovered a disregarded sequence (SLKTNGTG) residing on the ATP-binding region of KinB that is absent in the sequences of the other four phosphorelay sensor kinases (56) (Fig. 7A). This sequence possesses a significant homology to the highly conserved K+-filter (selectivity) sequence of the pore loop domain (P-domain) of potassium channels (T/S-x-x-T-x-G-x-G consensus sequence) (57, 58). Despite the many protein motifs and domains present in different types of potassium channels (58), the KinB K+-selectivity-like sequence (here called K* for simplicity) is the only common element related to K+ channels. While active KinB is a dimer (59), typical potassium channels are tetramers made up of predominantly identical subunits clustered to form the ion permeation pathway across the membrane (60–62). In addition to the absence of the pore motifs that surround the selectivity filter (Fig. 7B), KinB also lacks the different domains that have been described in different potassium channels (58, 60, 61). Furthermore, the K* resides in the cytosolic region of the kinase, while in all known (eukaryotic and prokaryotic) potassium channels the K+-filter resides in transmembrane domains. While these topological features exclude KinB as a potassium channel, they open the possibility that the kinase might sense the intracellular concentration of the ion throughout its cytosolic K*. Therefore, we tested if the K* plays a role or not in sliding motility. To this end, we constructed two types of kinB mutant strains harboring specific mutations in the K* (see Fig. S6 in the supplemental material). In one case, three out of the four conserved amino acids of the consensus K* were replaced by alanines (the fourth conserved amino acid, G, of the consensus sequence was not altered as it overlaps with a predicted ATPase motif of the kinase [Fig. 7A]) to give rise to the mutant KinBK*→A (see Fig. S6). In the second constructed kinB mutant strain, 7 out of the 8 amino acids of the K* were deleted (mutant strain KinBΔK* [see Fig. S6]). One important consideration for both mutant strains before analysis of their roles in sliding is that they must be functional (i.e., promote spore formation). In this sense, the sliding-promoting activity of KinB should be separable from its biofilm/sporulation-promoting activities, a scenario that would explain why KinB-dependent sliding proficiency and KinB-dependent biofilm/spore formation are not simultaneously activated (see below).

Bottom Line: Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host.Here, we use the model plant-beneficial bacterium Bacillus subtilis to answer this question.The spatiotemporal response of these kinases to variable potassium levels and the gradual increase in Spo0A~Pi levels that orchestrates the activation of sliding before biofilm formation shed light on how multicellular behaviors formerly believed to be antagonistic work together to benefit the population fitness.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas (FCByF), Universidad Nacional de Rosario (UNR)-CONICET, Argentina robertograu@fulbrightmail.org akos-tibor.kovacs@uni-jena.de.

No MeSH data available.


Related in: MedlinePlus