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Updated: Aug 14, 2026
Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses
Published on: February 22, 2019
Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial
Galina V Mukamolova1, Alexey G Murzin, Elena G Salina
1Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK.
Abstract:
The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.
Insights
Resuscitation-promoting factors (Rpfs) are muralytic enzymes that break down bacterial cell walls. Their enzymatic activity is crucial for reviving dormant bacteria, demonstrating their role in bacterial culturability.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Bacterial culturability is often limited by dormancy.
- Resuscitation-promoting factors (Rpfs) are proteins known to enhance the revival of non-culturable bacteria.
- Rpfs share a conserved domain with a lysozyme-like fold and a catalytic glutamate residue.
Purpose of the Study:
- To investigate the enzymatic activity of Rpf from Micrococcus luteus.
- To determine the role of conserved residues (glutamate and cysteines) in Rpf's muralytic and resuscitation-promoting functions.
Main Methods:
- Zymography using Micrococcus luteus cell walls.
- Assays measuring lysis of Escherichia coli.
- Hydrolysis of a fluorescently labeled peptidoglycan substrate (4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside).
- Site-directed mutagenesis of conserved glutamate and cysteine residues.
Main Results:
- Rpf from Micrococcus luteus exhibits muralytic activity against bacterial cell walls.
- Mutations in the catalytic glutamate residue reduced but did not abolish muralytic activity.
- Alterations in cysteine residues also impaired but did not abolish activity.
- Mutant Rpf activities correlated with their ability to stimulate bacterial resuscitation.
Conclusions:
- Resuscitation-promoting factors (Rpfs) are muralytic enzymes that cleave bacterial peptidoglycan.
- The catalytic glutamate and cysteine residues are important, but not solely essential, for Rpf's muralytic function.
- The muralytic activity of Rpfs is directly linked to their ability to promote bacterial culturability and resuscitation.
