A dye release assay for determination of lysostaphin activity

Zhou, Runqi; Chen, Shigen; Recsei, Paul A.

doi:10.1016/0003-2697(88)90134-0

Cited by 67 publications

(72 citation statements)

References 12 publications

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“…Gonococcal PG sacculi were incubated overnight at 37°C with 20 mM Remazol brilliant blue (RBB; Sigma) in 0.25 M NaOH as described previously (5,22). The reaction was neutralized by addition of an equal amount of 0.25 M HCl.…”

Section: Methodsmentioning

confidence: 99%

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

et al. 2016

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Key steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacterium Escherichia coli have revealed that this hydrolysis is highly regulated spatially and temporally. Neisseria gonorrhoeae is an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of the nlpD gene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-D-galactopyranoside (IPTG)-regulated nlpD complemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that the nlpD mutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate that N. gonorrhoeae NlpD is required for proper cell growth and division through its interactions with the amidase AmiC. IMPORTANCE N. gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, and N. gonorrhoeae has been classified as a "superbug" by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process of N. gonorrhoeae may point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcal nlpD gene and showed that it is essential for cell separation. In contrast to other pathogenic bacteria, the gonococcal system is streamlined and does not appear to have any redundancies.

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Section: Methodsmentioning

confidence: 99%

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

et al. 2016

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“…2B). Absorbance readings of the reaction supernatants after centrifugation serve as a semiquantitative measure of hydrolytic activity (Zhou et al 1988;T Uehara, T Dinh, and TG Bernhardt, in prep.). The extracellular domain of IIP was fused to a hexahistidine tag and purified from E. coli on Ni 2+ -agarose ( Fig.…”

Section: Iip Has Amidase and Endopeptidase Activitiesmentioning

confidence: 99%

A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis

Morlot¹,

Uehara²,

Marquis³

et al. 2010

Genes Dev.

146

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How proteins catalyze morphogenesis is an outstanding question in developmental biology. In bacteria, morphogenesis is intimately linked to remodeling the cell wall exoskeleton. Here, we investigate the mechanisms by which the mother cell engulfs the prospective spore during sporulation in Bacillus subtilis. A membraneanchored protein complex containing two cell wall hydrolases plays a central role in this morphological process. We demonstrate that one of the proteins (SpoIIP) has both amidase and endopeptidase activities, such that it removes the stem peptides from the cell wall and cleaves the cross-links between them. We further show that the other protein (SpoIID) is the founding member of a new family of lytic transglycosylases that degrades the glycan strands of the peptidoglycan into disaccharide units. Importantly, we show that SpoIID binds the cell wall, but will only cleave the glycan strands after the stem peptides have been removed. Finally, we demonstrate that SpoIID also functions as an enhancer of SpoIIP activity. Thus, this membrane-anchored enzyme complex is endowed with complementary, sequential, and stimulatory activities. These activities provide a mechanism for processive cell wall degradation, supporting a model in which circumferentially distributed degradation machines function as motors pulling the mother cell membranes around the forespore.[Keywords: Morphogenesis; cell shape; peptidoglycan; sporulation; amidase; lytic transglycosylase] Supplemental material is available at http://www.genesdev.org.

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“…Samples were taken every 24 h by centrifugation. The concentration of lysostaphin was measured by the method of Zhou et al (1988). The extraction of lysostaphin from cream was considered to be the maximum as a constant concentration of lysostaphin was achieved.…”

Section: Determination Of Lysostaphin Contentmentioning

confidence: 99%

“…Five hundred milligrams each of the 5 mg/g-B, 5 mg/g-C, 5 mg/g-D, 3.5 mg/g-C, 2 mg/g-C, the LPC cream, and Batch B, C, and D were loaded onto the membrane. Some (1 ml) receptor fluid was withdrawn at 0, 1, 2, 4, 6, and 23 h and concentrations of lysostaphin were then measured by the method based on Zhou et al (1988) basically. All these released lysostaphin were pooled.…”

Section: In Vitro Release Of Lysostaphin From the CCL Creammentioning

confidence: 99%

Extension of nasal anti-Staphylococcus aureus efficacy of lysostaphin by its incorporation into a chitosan-o/w cream

Cui

Li²,

Huang³

et al. 2010

Drug Delivery

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A dye release assay for determination of lysostaphin activity

Cited by 67 publications

References 12 publications

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis

Extension of nasal anti-Staphylococcus aureus efficacy of lysostaphin by its incorporation into a chitosan-o/w cream

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