Residues in the N-Terminal Domain of MutL Required for Mismatch Repair in Bacillus subtilis

ABSTRACT The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.

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Single-Molecule Fluorescence Imaging Reveals Mismatch Repair Dynamics in Live Bacillus Subtilis

Biophysical Journal ◽

10.1016/j.bpj.2013.11.2450 ◽

2014 ◽

Vol 106 (2) ◽

pp. 435a

Author(s):

Yi Liao ◽

Jeremy W. Schroeder ◽

Lyle A. Simmons ◽

Julie S. Biteen

Keyword(s):

Bacillus Subtilis ◽

Mismatch Repair ◽

Fluorescence Imaging ◽

Single Molecule ◽

Single Molecule Fluorescence

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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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Crystal structure of the in vivo-assembled Bacillus subtilis Spx/RNA polymerase α subunit C-terminal domain complex

Journal of Structural Biology ◽

10.1016/j.jsb.2009.07.001 ◽

2009 ◽

Vol 168 (2) ◽

pp. 352-356 ◽

Cited By ~ 17

Author(s):

Valerie Lamour ◽

Lars F. Westblade ◽

Elizabeth A. Campbell ◽

Seth A. Darst

Keyword(s):

Crystal Structure ◽

Bacillus Subtilis ◽

Rna Polymerase ◽

Α Subunit ◽

Subunit C ◽

Terminal Domain

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Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

Journal of Bacteriology ◽

10.1128/jb.00257-15 ◽

2015 ◽

Vol 197 (16) ◽

pp. 2675-2684 ◽

Cited By ~ 4

Author(s):

Seram Nganbiton Devi ◽

Brittany Kiehler ◽

Lindsey Haggett ◽

Masaya Fujita

Keyword(s):

Bacillus Subtilis ◽

Mutant Protein ◽

Phosphoryl Group ◽

Atp Binding ◽

Histidine Kinases ◽

Content Type ◽

Point Mutant ◽

Terminal Domain

ABSTRACTEntry into sporulation inBacillus subtilisis governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur intrans. To test this hypothesis, we generated a series ofB. subtilisstrains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in atransfashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressedin vivo. Taken together, thesein vitroandin vivoresults reinforce the evidence that KinA autophosphorylation is able to occur in atransfashion.IMPORTANCEAutophosphorylation of histidine kinases is known to occur by either thecis(one subunit of kinase phosphorylating itself within the multimer) or thetrans(one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided directin vivoandin vitroevidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur intranswithin the homotetramer complex. While the physiological and mechanistic significance of thetransautophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.

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