Overexpression, Purification, and Characterization of<i>Bacillus subtilis N</i>-Acetylmuramoyl-<scp>L</scp>-alanine Amidase CwlC

Shida, Toshio; Hattori, Hirotsugu; Ise, Fuminori; Sekiguchi, Junichi

doi:10.1271/bbb.64.1522

Cited by 7 publications

(7 citation statements)

References 12 publications

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“…The sense primer was 5Ј-CCGCGGAATTCTATGGGCCTTCAGGAGAAAACG-3Ј (the cwlC sequence is italicized, the initiation codon is boldfaced, and the EcoRI site is underlined). As a result, Met was used as the substitution of Asn-20 for the first amino acid of D- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The antisense primer was 5Ј-GCTGCCTGCAGCTATGATTCTAGGATCACAATAGC-3Ј (the complimentary sequence of the cwlC sequence is italicized, the complimentary sequence of the TAG termination codon is boldfaced, and the PstI site is underlined).…”

Section: Methodsmentioning

confidence: 99%

“…In a previous study, we concluded that the CwlC amidase does not have catalytic action similar to that of serine protease as judged from the resistivity of the CwlC amidase to the serine protease inhibitor (16). The present study was undertaken to obtain new insights into the molecular mechanisms of cell wall lysis by the amidases.…”

mentioning

confidence: 87%

“…CwlC amidase and its mutant proteins (E24A and E141Q) were overexpressed and purified as described previously (16). The CwlV1 amidase and its mutant protein (E142Q) were overproduced in (17).…”

Section: Purification Of Cwlc and Cwlv1 Amidases And Their Mutant Promentioning

confidence: 99%

“…This showed that the tandem repeat of the C terminus of CwlC was not critical for activity. An N-terminal deletion mutant D- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) in which only the first 19 amino acids were deleted lost the cell wall lytic activity completely. These results are consistent with the prediction that the homologous domain is the catalytic domain of the amidases.…”

Section: Determination Of the Catalytic Domain By Deletion Mutagenesis-mentioning

confidence: 99%

“…In contrast, all of the mutations at E24 and E141 resulted in a loss of cell wall lytic activity. The mutants E24A and E141Q were overproduced in E. coli and purified as described previously (16). Fig.…”

Section: Determination Of the Catalytic Domain By Deletion Mutagenesis-mentioning

confidence: 99%

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Mutational Analysis of Catalytic Sites of the Cell Wall Lytic N-Acetylmuramoyl-l-alanine Amidases CwlC and CwlV

Shida

Hattori

Ise

et al. 2001

Journal of Biological Chemistry

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The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-L-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain. Sitedirected mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn 2؉ , Mn 2؉ , and Co 2؉ but not by the addition of Mg 2؉ and Ca 2؉ . These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 87%

Section: Purification Of Cwlc and Cwlv1 Amidases And Their Mutant Promentioning

confidence: 99%

Section: Determination Of the Catalytic Domain By Deletion Mutagenesis-mentioning

confidence: 99%

Section: Determination Of the Catalytic Domain By Deletion Mutagenesis-mentioning

confidence: 99%

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Mutational Analysis of Catalytic Sites of the Cell Wall Lytic N-Acetylmuramoyl-l-alanine Amidases CwlC and CwlV

Shida

Hattori

Ise

et al. 2001

Journal of Biological Chemistry

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Peptidoglycan Structure, Biosynthesis, and Dynamics During Bacterial Growth

Walter

Mayer

2019

Biologically-Inspired Systems

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CwlQ is required for swarming motility but not flagellar assembly inBacillus subtilis

Kearns

Sánchez

Dunn

2021

Preprint

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Hydrolytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan (PG) integrity must be unfailingly stable to preserve cell integrity but must also be dynamically remodeled for the cell grow, divide and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG and the insertion of which is aided by localized activity of a dedicated PG hydrolase in Gram-negative bacteria. To date, there is no known dedicated hydrolase in Gram-positive bacteria for insertion of flagella and here we take a reverse-genetic candidate-gene approach to find that cells mutated for the lytic transglycosylase CwlQ exhibited a severe defect in flagellar dependent swarming motility. We show that CwlQ required its active site to promote swarming, was expressed by the motility sigma factor SigD, and was secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells mutated for CwlQ remained proficient for flagellar biosynthesis even when mutated in combination with four other hydrolases related to motility (LytC, LytD, LytF, and CwlO). The PG hydrolase essential for flagellar synthesis in B. subtilis, if any, remains unknown.

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Overexpression, Purification, and Characterization ofBacillus subtilis N-Acetylmuramoyl-L-alanine Amidase CwlC

Cited by 7 publications

References 12 publications

Mutational Analysis of Catalytic Sites of the Cell Wall Lytic N-Acetylmuramoyl-l-alanine Amidases CwlC and CwlV

Mutational Analysis of Catalytic Sites of the Cell Wall Lytic N-Acetylmuramoyl-l-alanine Amidases CwlC and CwlV

Peptidoglycan Structure, Biosynthesis, and Dynamics During Bacterial Growth

CwlQ is required for swarming motility but not flagellar assembly inBacillus subtilis

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