Genetic evidence for signal transduction within the Bacillus subtilis GerA germinant receptor

Dominant Negative ◽

Bacterial Spores ◽

Wild Type ◽

Enrichment Strategy ◽

Functional Receptor

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to L-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB-E105K) reduces the GerA complex's ability to respond to L-alanine, while another (gerAB-F259S) disrupts the germinant signal downstream of L-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway.

Genetic evidence for signal transduction within the Bacillus subtilis GerA germinant receptor

10.1128/jb.00470-21 ◽

2021 ◽

Author(s):

Jeremy D. Amon ◽

Lior Artzi ◽

David Z. Rudner

Keyword(s):

Signal Transduction ◽

Bacillus Subtilis ◽

Dominant Negative ◽

Transduction Pathway ◽

Sense And Respond ◽

Enrichment Strategy ◽

Functional Receptor

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to L-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA , gerAB , and gerAC . The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA* ) that carries a mutation in gerAA and show it constitutively activates germination even in the presence of a wild-type copy of gerA . Using an enrichment strategy to screen for suppressors of gerA* , we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one ( gerAB[E105K]) reduces the GerA complex's ability to respond to L-alanine, while another ( gerAB[F259S] ) disrupts the germinant signal downstream of L-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway. Importance Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid L-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

AJP Cell Physiology ◽

10.1152/ajpcell.00313.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. C423-C431 ◽

Cited By ~ 24

Author(s):

Li Liu ◽

Yukisato Ishida ◽

Gbolahan Okunade ◽

Gail J. Pyne-Geithman ◽

Gary E. Shull ◽

...

Keyword(s):

Signal Transduction ◽

Smooth Muscle ◽

Plasma Membrane ◽

Contractile Response ◽

Transduction Pathway ◽

Wild Type ◽

Bladder Smooth Muscle

We previously showed that plasma membrane Ca2+-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle ( 8 ). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca2+ ([Ca2+]i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1+/−, Pmca4+/−, Pmca4−/−, and Pmca1+/− Pmca4−/− mice. There were no differences in basal [Ca2+]i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca2+]i (130–180%) in smooth muscle from Pmca1+/− and Pmca1+/− Pmca4−/− bladders than those in WT or Pmca4−/−. The responses to carbachol (CCh: 10 μM) were also greater in Pmca1+/− (120–150%) than in WT bladders. In contrast, the responses in Pmca4−/− and Pmca1+/− Pmca4−/− bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca2+]i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4−/− (130–190%) and Pmca1+/− Pmca4−/− (120–250%) bladders, but not in Pmca1+/− bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca2+ clearance, while PMCA4 is essential for the [Ca2+]i increase and contractile response to the CCh receptor-mediated signal transduction pathway.

Role of Ger Proteins in Nutrient and Nonnutrient Triggering of Spore Germination in Bacillus subtilis

10.1128/jb.182.9.2513-2519.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2513-2519 ◽

Cited By ~ 198

Author(s):

Madan Paidhungat ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Mutant Strain ◽

Spore Germination ◽

Dipicolinic Acid ◽

Family Members ◽

Wild Type ◽

Receptor Proteins ◽

Rich Media

ABSTRACT Dormant Bacillus subtilis spores germinate in the presence of particular nutrients called germinants. The spores are thought to recognize germinants through receptor proteins encoded by the gerA family of operons, which includesgerA, gerB, and gerK. We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known gerA-like loci. As expected, the mutant spores germinated very poorly in a variety of rich media. In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca2+ and dipicolinic acid (DPA). These observations showed that proteins encoded bygerA family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants. Further characterization of Ca2+–DPA-induced germination showed that the effect of Ca2+–DPA on spore germination was saturated at 60 mM and had a Km of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca2+–DPA but not in nutrient germinants, indicating that Ca2+–DPA and nutrient germinants probably act through parallel arms of the germination pathway.

Mutational Analysis and Membrane Topology of ComP, a Quorum-Sensing Histidine Kinase of Bacillus subtilis Controlling Competence Development

10.1128/jb.181.15.4540-4548.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4540-4548 ◽

Cited By ~ 50

Author(s):

Flavia Piazza ◽

Pablo Tortosa ◽

David Dubnau

Keyword(s):

Signal Transduction ◽

Bacillus Subtilis ◽

Histidine Kinase ◽

Mutational Analysis ◽

Membrane Topology ◽

Extracellular Loops ◽

Peptide Pheromone ◽

Membrane Spanning

ABSTRACT ComP is a sensor histidine kinase of Bacillus subtilisrequired for the signal transduction pathway that initiates the development of competence for genetic transformation. It is believed that ComP senses the presence of ComX, a modified extracellular peptide pheromone, and donates a phosphate to ComA, thereby activating this transcription factor for binding to the srfA promoter. In the present study, fusions to the Escherichia coli proteins PhoA and LacZ and analysis of its susceptibility to the protease kallikrein were used to probe the membrane topology of ComP. These data suggest that ComP contains six or eight membrane-spanning segments and two large extracytoplasmic loops in its N-terminal membrane-associated domain. Deletions were introduced involving the large extracellular loops to explore the role of the N-terminal domain of ComP in signal transduction. The absence of the second loop conferred a phenotype in which ComP was active in the absence of ComX. The implications of these data are discussed.

Mating in Saccharomyces cerevisiae: The Role of the Pheromone Signal Transduction Pathway in the Chemotropic Response to Pheromone

Genetics ◽

10.1093/genetics/147.1.19 ◽

1997 ◽

Vol 147 (1) ◽

pp. 19-32 ◽

Cited By ~ 5

Author(s):

Kathrin Schrick ◽

Barbara Garvik ◽

Leland H Hartwell

Keyword(s):

Signal Transduction ◽

Map Kinase ◽

Transcriptional Activation ◽

Transduction Pathway ◽

Wild Type ◽

Mating Partner ◽

Map Kinase Cascade ◽

Kinase Cascade ◽

Wild Type Control

Abstract The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the β or γ subunit (ste4 and stel8) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽

10.1093/genetics/151.1.107 ◽

1999 ◽

Vol 151 (1) ◽

pp. 107-117

Author(s):

Qi Yang ◽

Katherine A Borkovich

Keyword(s):

Signal Transduction ◽

Neurospora Crassa ◽

Sexual Cycle ◽

Intracellular Camp ◽

Wild Type ◽

Independent Functions ◽

Aerial Hyphae ◽

Mutational Activation ◽

Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

10.1128/jb.01613-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1565-1572 ◽

Cited By ~ 82

Author(s):

Venkata Ramana Vepachedu ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Small Molecules ◽

Spore Germination ◽

Dipicolinic Acid ◽

Temperature Sensitive ◽

Wild Type ◽

Ph Optimum ◽

Inner Membrane ◽

Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

Signal transduction and bacterial conjugation: characterization of the role of ArcA in regulating conjugative transfer of the resistance plasmid R1

Journal of Molecular Biology ◽

10.1006/jmbi.1997.1598 ◽

1998 ◽

Vol 277 (2) ◽

pp. 309-316 ◽

Cited By ~ 40

Author(s):

Heimo Strohmaier ◽

Rainer Noiges ◽

Sabine Kotschan ◽

Gary Sawers ◽

Gregor Högenauer ◽

...

Keyword(s):

Signal Transduction ◽

Conjugative Transfer ◽

Bacterial Conjugation ◽

Plasmid R1 ◽

Resistance Plasmid

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽

10.1093/genetics/158.3.1027 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1027-1036 ◽

Cited By ~ 2

Author(s):

Cletus A D'Souza ◽

Bee Na Lee ◽

Thomas H Adams

Keyword(s):

Aspergillus Nidulans ◽

Negative Influence ◽

Asexual Development ◽

Wild Type ◽

Significant Similarity ◽

Developmental Defects ◽

Asexual Sporulation ◽

Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.