The Mechanism of Bidirectional pH Taxis in Bacillus subtilis

ABSTRACT We investigated pH taxis in Bacillus subtilis. This bacterium was found to perform bidirectional taxis in response to external pH gradients, enabling it to preferentially migrate to neutral environments. We next investigated the chemoreceptors involved in sensing pH gradients. We identified four chemoreceptors involved in sensing pH: McpA and TlpA for sensing acidic environments and McpB and TlpB for sensing alkaline ones. In addition, TlpA was found to also weakly sense alkaline environments. By analyzing chimeras between McpA and TlpB, the principal acid- and base-sensing chemoreceptors, we identified four critical amino acid residues—Thr199, Gln200, His273, and Glu274 on McpA and Lys199, Glu200, Gln273, and Asp274 on TlpB—involved in sensing pH. Swapping these four residues between McpA and TlpB converted the former into a base receptor and the latter into an acid receptor. Based on the results, we propose that disruption of hydrogen bonding between the adjacent residues upon pH changes induces signaling. Collectively, our results further our understanding of chemotaxis in B. subtilis and provide a new model for pH sensing in bacteria. IMPORTANCE Many bacteria can sense the pH in their environment and then use this information to direct their movement toward more favorable locations. In this study, we investigated the pH sensing mechanism in Bacillus subtilis. This bacterium preferentially migrates to neutral environments. It employs four chemoreceptors to sense pH. Two are involved in sensing acidic environments, and two are involved in sensing alkaline ones. To identify the mechanism for pH sensing, we constructed receptor chimeras of acid- and base-sensing chemoreceptors. By analyzing the responses of these chimeric receptors, we were able to identify four critical amino acid residues involved in pH sensing and propose a model for the pH sensing mechanism in B. subtilis.

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Four chemoreceptors govern bidirectional pH taxis in Bacillus subtilis

10.1101/715946 ◽

2019 ◽

Author(s):

Payman Tohidifar ◽

Matthew J. Plutz ◽

George W. Ordal ◽

Christopher V. Rao

Keyword(s):

Bacillus Subtilis ◽

Amino Acid ◽

Ph Sensing ◽

Amino Acid Residues ◽

Ph Gradients ◽

Sensing Mechanism ◽

Critical Amino Acid ◽

Alkaline Environments ◽

Acid And Base ◽

Acidic Environments

ABSTRACTWe investigated pH taxis in Bacillus subtilis. This bacterium was found to perform bidirectional taxis in response to external pH gradients, enabling it to preferentially migrate to neutral environments. We next investigated the chemoreceptors involved in sensing pH gradients. We found that four chemoreceptors are involved in sensing pH: McpA and TlpA for sensing acidic environments and McpB and TlpB for alkaline ones. In addition, TlpA was found to also weakly sense alkaline environments. By analyzing chimeras between McpA and TlpB, the principal acid and base-sensing chemoreceptors, we identified four critical amino-acid residues – Thr199, Gln200, His273, and Glu274 on McpA and Lys199, Glu200, Gln273, and Asp274 on TlpB – involved in sensing pH. Swapping these four residues between McpA and TlpB converted the former into a base receptor and the latter into an acid receptor. Based on the results, we propose that disruption of hydrogen bonding between the adjacent residues upon pH changes induces signaling. Collectively, our results further our understanding of chemotaxis in B. subtilis and provide a new model for pH sensing in bacteria.IMPORTANCEMany bacteria can sense the pH in their environment and then use this information to direct their movement towards more favorable locations. In this study, we investigated the pH sensing mechanism in Bacillus subtilis. This bacterium preferentially migrates to neutral environments. It employs four chemoreceptors to sense pH. Two are involved in sensing acidic environments and two are involved in sensing alkaline ones. To identify the mechanism for pH sensing, we constructed receptor chimeras of acid and base sensing chemoreceptors. By analyzing the response of these chimeric receptors, we were able to identify four critical amino-acid residues involved in pH sensing and propose a model for the pH sensing mechanism in B. subtilis.

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Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony-stimulating factor and their involvement in species specificity

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)92772-9 ◽

1991 ◽

Vol 266 (21) ◽

pp. 13804-13810 ◽

Cited By ~ 1

Author(s):

A.B. Shanafelt ◽

K.E. Johnson ◽

R.A. Kastelein

Keyword(s):

Amino Acid ◽

Species Specificity ◽

Amino Acid Residues ◽

Colony Stimulating Factor ◽

Critical Amino Acid ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor ◽

Human And Mouse

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Direct demonstration of critical amino acid residues required for cytotoxic T-lymphocyte allorecognition of H-2 class I antigens.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.9.3085 ◽

1988 ◽

Vol 85 (9) ◽

pp. 3085-3089 ◽

Cited By ~ 6

Author(s):

E. McLaughlin-Taylor ◽

C. G. Miyada ◽

M. McMillan ◽

R. B. Wallace

Keyword(s):

Amino Acid ◽

T Lymphocyte ◽

Cytotoxic T Lymphocyte ◽

Class I ◽

Amino Acid Residues ◽

Critical Amino Acid ◽

Direct Demonstration

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Identification of Critical Amino Acid Residues for Human iNOS Functional Activity

The Protein Journal ◽

10.1007/s10930-008-9139-y ◽

2008 ◽

Vol 27 (5) ◽

pp. 309-318 ◽

Cited By ~ 5

Author(s):

Saule Naureckiene ◽

Sreekumar R. Kodangattil ◽

Edward J. Kaftan ◽

Philip G. Jones ◽

Jeffrey D. Kennedy ◽

...

Keyword(s):

Amino Acid ◽

Functional Activity ◽

Amino Acid Residues ◽

Critical Amino Acid

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Mutational Screen Identifies Critical Amino Acid Residues of β-Actin Mediating Interaction between Its Folding Intermediates and Eukaryotic Cytosolic Chaperonin CCT

Journal of Structural Biology ◽

10.1006/jsbi.2001.4389 ◽

2001 ◽

Vol 135 (2) ◽

pp. 185-197 ◽

Cited By ~ 26

Author(s):

Elizabeth A. McCormack ◽

Mattias J. Rohman ◽

Keith R. Willison

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

Folding Intermediates ◽

Critical Amino Acid

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Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00124-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 2

Author(s):

Cierra A. Birch ◽

Madison J. Davis ◽

Lea Mbengi ◽

Peter Zuber

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Amino Acid ◽

Dna Binding ◽

Rna Polymerase ◽

Α Subunit ◽

Stress Induction ◽

Content Type ◽

Terminal Domain ◽

Codon Substitution

ABSTRACT Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where “Y263C” indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

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A703 CRITICAL AMINO ACID RESIDUES FOR THE ALLOSTERIC ACTIONS OF ISOFLURANE AND ENFLURANE IN THE HUMAN GABA sub A RECEPTOR

Anesthesiology ◽

10.1097/00000542-199709001-00703 ◽

1997 ◽

Vol 87 (Supplement) ◽

pp. 703A

Author(s):

N.L. Harrison ◽

&NA; Mihic ◽

Q. Ye ◽

V. Koltchine ◽

S. Finn ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

Critical Amino Acid

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Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.03499-16 ◽

2017 ◽

Vol 83 (9) ◽

Cited By ~ 14

Author(s):

M. Fata Moradali ◽

Shirin Ghods ◽

Bernd H. A. Rehm

Keyword(s):

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Structural Model ◽

Second Messenger ◽

Catalytic Site ◽

Activation Mechanism ◽

Amino Acid Residues ◽

Human Pathogen ◽

Content Type ◽

Opportunistic Human Pathogen

ABSTRACT The exopolysaccharide alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa, confers a survival advantage to the bacterium by contributing to the formation of characteristic biofilms during infection. Membrane-anchored proteins Alg8 (catalytic subunit) and Alg44 (copolymerase) constitute the alginate polymerase that is being activated by the second messenger molecule bis-(3′, 5′)-cyclic dimeric GMP (c-di-GMP), but the mechanism of activation remains elusive. To shed light on the c-di-GMP-mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops, including H323 (loop A) and T457 and E460 (loop B), surrounding the catalytic site in the predicted model. The activities of the respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels, while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 copolymerase constituted a stable dimer, with its periplasmic domains required for protein localization and alginate polymerization and modification. Superfolder green fluorescent protein (GFP) fusions of Alg8 and Alg44 showed a nonuniform, punctate, and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP-mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44, including their subcellular localization and distribution. IMPORTANCE The exopolysaccharide alginate is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principal cause of the mucoid phenotype that is the hallmark of chronic infections of cystic fibrosis patients. The production of alginate is mediated by interacting membrane proteins Alg8 and Alg44, while their activity is posttranslationally regulated by the second messenger c-di-GMP, a well-known regulator of the synthesis of a range of other exopolysaccharides in bacteria. This study provides new insights into the unknown activation mechanism of alginate polymerization by c-di-GMP. Experimental evidence that the activation of alginate polymerization requires the engagement of specific amino acid residues residing at the catalytic domain of Alg8 glycosyltransferase was obtained, and these residues are proposed to exert an allosteric effect on the PilZAlg44 domain upon c-di-GMP binding. This mechanism is dissimilar to the proposed mechanism of the autoinhibition of cellulose polymerization imposed by salt bridge formation between amino acid residues and released upon c-di-GMP binding, leading to activation of polymerization. On the other hand, conserved amino acid residues in the periplasmic domain of Alg44 were found to be involved in alginate polymerization as well as modification events, i.e., acetylation and epimerization. Due to the critical role of c-di-GMP in the regulation of many biological processes, particularly the motility-sessility switch and also the emergence of persisting mucoid phenotypes, these results aid to reach a better understanding of biofilm-associated regulatory networks and c-di-GMP signaling and might assist the development of inhibitory drugs.

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