Structure of the stationary phase survival protein YuiC from B.subtilis

Quay, Doris H.X.; Cole, A.R.; Cryar, Adam; Thalassinos, Konstantinos; Williams, Mark A.; Bhakta, Sanjib; Keep, Nicholas H.

doi:10.1186/s12900-015-0039-z

Cited by 8 publications

(20 citation statements)

References 39 publications

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“…A truncated yuiC construct, R52-E218-pNIC28-BSA4 plasmid was used (Quay et al, 2015). Point mutations D129A, D151A, D162A and K102A were selected based on the identification of three active site aspartic acid residues (D129, D151 and D162) from the NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) using the yuiC amino acid sequence.…”

Section: Site-directed Ligase-independent Mutagenesis Polymerase Chamentioning

confidence: 99%

“…The starter cultures were incubated overnight at 37 °C with shaking at 180 rpm. Protein expression was carried out according to the methods described by Quay et al (2015). Starter cultures were inoculated into each 500 mL Terrific Broth (TB) supplemented with Kan (50 µg/mL) and Cam (34 µg/mL) to achieve an OD600 of 0.02.…”

Section: Cell Growth and Protein Expressionmentioning

confidence: 99%

“…Cells lysis and protein purifications were performed according to the methods described by Quay et al (2015). Briefly, the harvested cells were mixed with lysozyme (1 mg/mL) and treated by sonication (Sonics, Vibra Cell).…”

Section: Protein Purificationsmentioning

confidence: 99%

“…The dormant cells of Staphylococcus aureus have also been shown to be revived by addition of spent culture supernatant from cultures of the same organism (Pascoe et al, 2014). The structure of B. subtilis YuiC has been solved at 1.2 Å resolution and showed that it is a minimal version of the membrane bound lytic transglycosylase A (MltA) of Escherichia coli (Quay et al, 2015). Structural comparison of the E. coli MltA Domain A and the ordered β-barrel domain of YuiC (P73-E218) matches only to the Domain A of E. coli MltA and the core domain of YuiC is 33 residues shorter than the E. coli MltA (Quay et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…The structure of B. subtilis YuiC has been solved at 1.2 Å resolution and showed that it is a minimal version of the membrane bound lytic transglycosylase A (MltA) of Escherichia coli (Quay et al, 2015). Structural comparison of the E. coli MltA Domain A and the ordered β-barrel domain of YuiC (P73-E218) matches only to the Domain A of E. coli MltA and the core domain of YuiC is 33 residues shorter than the E. coli MltA (Quay et al, 2015). In this paper, some of the conserved residues in Sps domains were mutated to study the key residues involved in muralytic catalysis.…”

Section: Introductionmentioning

confidence: 99%

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Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Quay¹,

Qureshi²,

Bhakta³

et al. 2018

MJM

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Aims:YuiC is a stationary phase survival (Sps) protein from the Firmicute Bacillus subtilis that possesses muralytic activity to cleave bacterial cell-wall peptidoglycan. It has a small lytic transglycosylase (MltA) fold analogous to the resuscitation promoting factors (Rpfs) of Actinobacteria which have a hybrid of a mini lysozyme and soluble lytic transglycosylase (Slt35/70) fold. The present study aimed at identifying key residues of YuiC/Sps that are catalytically active and studying the effect of B. subtilis cell growth upon sps/yuiC deletion. Methodology and results: Four forms of mutated yuiC were created through Site-directed, Ligase-Independent Mutagenesis Polymerase Chain Reaction (SLIM PCR) that include the substitutions of D129A, D151A, D162A and K102A. These individual mutated yuiC genes were cloned and expressed in the Escherichia coli BL21 (DE3) expression system and subsequently purified to homogeneity using affinity, cation exchange and size exclusion chromatography. The D129A variant was shown to be insoluble, indicating its role in maintaining the right protein folding of YuiC. The remaining three variants resulted in soluble proteins but were inactive on zymograms indicating that they may be responsible for catalysis. B. subtilis cells harbouring individual sps genes (yuiC, yabE, yocH and yorM) knocked out showed stationary phase defects and altered colony morphologies compared to the wild type. Conclusion, significance and impact of study: This study has identified the key residues involved in catalysis of YuiC, which are the D151, D162 and K102. These are conserved in Sps domains. The catalytic mechanism of YuiC is similar to the mechanism reported for Neisseria gonorrhoeae MltA. sps/yuiC knock outs have implied that each sps/yuiC has a significant role on B. subtilis late growth stage. The B. subtilis YuiC/Sps model has given an insight into Sps functions in the final growth stage of the Firmicutes, which members include etiologic agents of anthrax, botulism and listeriosis. Inhibition of Sps protein may inactivate pathogen replication and facilitate entrance into a non-contagious dormant sporulation stage.

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Section: Site-directed Ligase-independent Mutagenesis Polymerase Chamentioning

confidence: 99%

Section: Cell Growth and Protein Expressionmentioning

confidence: 99%

Section: Protein Purificationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Quay¹,

Qureshi²,

Bhakta³

et al. 2018

MJM

View full text Add to dashboard Cite

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Exploring the structure and dynamics of a fluorescent schiff base (1E,1′E)-1,1′-(1,4-phenylene) bis(N-(4-chlorophenyl) methamine: Synthesis, spectroscopic analysis, thermal, electronic and crystallographic study with biological applications

Rajimon,

Alzahrani,

Nair

et al. 2024

Journal of Molecular Structure

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Lytic transglycosylase MltG cleaves in nascent peptidoglycan and produces short glycan strands

et al. 2021

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Bacteria encase their cytoplasmic membrane with peptidoglycan (PG) to maintain the shape of the cell and protect it from bursting. The enlargement of the PG layer is facilitated by the coordinated activities of PG synthesising and -cleaving enzymes. In Escherichia coli , the cytoplasmic membrane-bound lytic transglycosylase MltG associates with PG synthases and was suggested to terminate the polymerisation of PG glycan strands. Using pull-down and surface plasmon resonance, we detected interactions between MltG from Bacillus subtilis and two PG synthases; the class A PBP1 and the class B PBP2B. Using in vitro PG synthesis assays with radio-labelled or fluorophore-labelled B. subtilis -type and/or E. coli -type lipid II, we showed that both, Bs MltG and Ec MltG, are lytic tranglycosylases and that their activity is higher during ongoing glycan strand polymerisation. MltG competed with the transpeptidase activity of class A PBPs, but had no effect on their glycosyltransferase activity, and produced glycan strands with a length of 7 disaccharide units from cleavage in the nascent strands. We hypothesize that MltG cleaves the nascent strands to produce short glycan strands that are used in the cell for a yet unknown process.

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Structure of the stationary phase survival protein YuiC from B.subtilis

Cited by 8 publications

References 39 publications

Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Mutation studies of the gene encoding YuiC, a stationary phase survival protein in Bacillus subtilis

Exploring the structure and dynamics of a fluorescent schiff base (1E,1′E)-1,1′-(1,4-phenylene) bis(N-(4-chlorophenyl) methamine: Synthesis, spectroscopic analysis, thermal, electronic and crystallographic study with biological applications

Lytic transglycosylase MltG cleaves in nascent peptidoglycan and produces short glycan strands

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