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

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

doi:10.1074/jbc.m103903200

Cited by 54 publications

(66 citation statements)

References 43 publications

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“…Our YqiI in silico model also highlights the position of residue E-148 (Fig. 3C), a residue that is critical for the biochemical functioning of N-acetylmuramoyl-Lalanine amidases (15,54). We note that apparently no cell wallbinding domain is present in the YqiI protein (Fig.…”

Section: Resultsmentioning

confidence: 95%

Activity of the Osmotically Regulated yqiHIK Promoter from Bacillus subtilis Is Controlled at a Distance

Fischer

Bremer

2012

J Bacteriol

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The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein (YqiH), a secreted N-acetylmuramoyl-L-alanine amidase (YqiI), and a cytoplasmic glycerophosphodiester phosphodiesterase (YqiK). Reverse transcriptase PCR (RT-PCR) analysis showed that the yqiHIK genes are transcribed as an operon. Consistent with the in silico prediction, we found that the purified YqiI protein exhibited hydrolytic activity toward peptidoglycan sacculi. Transcription studies with yqiHtreA reporter fusion strains revealed that the expression of yqiHIK is subjected to finely tuned osmotic control, but enhanced expression occurs only in severely osmotically stressed cells. Primer extension analysis pinpointed the osmotically responsive yqiHIK promoter, and site-directed mutagenesis was employed to assess functionally important sequences required for promoter activity and osmotic control. Promoter variants with constitutive activity were isolated. A deletion analysis of the yqiHIK regulatory region showed that a 53-bp AT-rich DNA segment positioned 180 bp upstream of the ؊35 sequence is critical for the activity and osmotic regulation of the yqiHIK promoter. Hence, the expression of yqiHIK is subjected to genetic control at a distance. Upon the onset of growth of cells of the B. subtilis wild-type strain in high-salinity medium (1.2 M NaCl), we observed gross morphological deformations of cells that were then reversed to a rod-shaped morphology again when the cells had adjusted to the high-salinity environment. The products of the yqiHIK gene cluster were not critical for reestablishing rod-shaped morphology, but the deletion of this operon yielded a B. subtilis mutant impaired in growth in a defined minimal medium and at high salinity.

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Section: Resultsmentioning

confidence: 95%

Activity of the Osmotically Regulated yqiHIK Promoter from Bacillus subtilis Is Controlled at a Distance

Fischer

Bremer

2012

J Bacteriol

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“…In addition to an N-terminal catalytic domain, the protein contains a C-terminal SPOR domain (Fig. 11A) that has affinity for amidase-digested B. subtilis murein, as inferred from nuclear magnetic resonance chemical shift analyses (44,51).…”

Section: Ftsn-dependent Targeting Of S Ftsn To Constriction Sites Whmentioning

confidence: 99%

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in Escherichia coli Cell Constriction

et al. 2009

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Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an ␣-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain ( E FtsN) of at most 35 residues that is centered about helix H2 in the periplasm. Bacterial cytokinesis is mediated by a ring-shaped apparatus. Assembly of this septal ring (SR; also called the divisome or septasome) begins at the future site of fission, well before cell constriction initiates, and it remains associated with the leading edge of the invaginating cell envelope until fission is completed. The mature ring in Escherichia coli is made up of at least 10 essential division proteins

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“…Moreover, although most of the SPOR domain proteins that have been studied are components of the septal ring that mediates cell division (14)(15)(16)19), there are exceptions. Indeed, the name "SPOR" domain arose because the founding member of the family, CwlC of Bacillus subtilis, is a cell wall amidase that helps degrade PG to release the spore from the mother cell (20)(21)(22). Another exception is a small Vibrio parahaemolyticus protein, designated VPA1294, that has been implicated in swarmer cell differentiation (23).…”

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confidence: 99%

Identification of SPOR Domain Amino Acids Important for Septal Localization, Peptidoglycan Binding, and a Disulfide Bond in the Cell Division Protein FtsN

Duncan¹,

Yahashiri²,

Arends³

et al. 2013

Journal of Bacteriology

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bSPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein ( TT GFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a ␤-sheet in the published solution structure of FtsN SPOR . Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share <20% amino acid identity. Taken together, these findings support the proposal that SPOR domains localize by binding to septal peptidoglycan and imply that the binding site is associated with the ␤-sheet. We also show that FtsN SPOR contains a disulfide bond between ␤-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.

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

Cited by 54 publications

References 43 publications

Activity of the Osmotically Regulated yqiHIK Promoter from Bacillus subtilis Is Controlled at a Distance

Activity of the Osmotically Regulated yqiHIK Promoter from Bacillus subtilis Is Controlled at a Distance

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in Escherichia coli Cell Constriction

Identification of SPOR Domain Amino Acids Important for Septal Localization, Peptidoglycan Binding, and a Disulfide Bond in the Cell Division Protein FtsN

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