Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation.
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.
Mukamolova
Galina V
GV
Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK.
Murzin
Alexey G
AG
Salina
Elena G
EG
Demina
Galina R
GR
Kell
Douglas B
DB
Kaprelyants
Arseny S
AS
Young
Michael
M
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001433:Bacteriolysis; D002473:Cell Wall / Q000378:metabolism*; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005450:Fluorescamine / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D051379:Mice; D016982:Micrococcus luteus / Q000378:metabolism*; D008958:Models, Molecular; D009113:Muramidase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D016207:Cytokines; D007202:Indicators and Reagents; D011994:Recombinant Proteins; C113704:resuscitation-promoting factor, bacteria; D005450:Fluorescamine; D018698:Glutamic Acid; D009113:Muramidase