Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation

Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK.
Molecular Microbiology (Impact Factor: 5.03). 02/2006; 59(1):84-98. DOI: 10.1111/j.1365-2958.2005.04930.x
Source: PubMed

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.

  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: PknB is a transmembrane Ser/Thr protein kinase that defines and belongs to an ultraconserved kinase subfamily found in Gram-positive bacteria. Essential for Mycobacterium tuberculosis growth, its close homolog in Bacillus subtilis has been linked to exit from dormancy. The kinase possesses an extracellular region composed of a repetition of PASTA domains, believed to bind peptidoglycan fragments that might act as a signaling molecule. We report here the first solution structure of this extracellular region. Small-angle X-ray scattering and nuclear magnetic resonance studies show that the four PASTA domains display an unexpected linear organization, contrary to what is observed in the distant protein PBP2x from Streptococccus pneumoniae where two PASTA domains fold over in a compact structure. We propose a model for PknB activation based on a ligand-dependent dimerization of the extracellular PASTA domains that initiates multiple signaling pathways.
    Structure 05/2010; 18(5):606-15. DOI:10.1016/j.str.2010.02.013 · 6.79 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Here, we report that the model Gram-positive organism, Bacillus subtilis, expresses and secretes a muralytic enzyme, YocH, in response to cell wall-derived muropeptides derived from growing cells but not lysed cells. This induction is dependent on PrkC, a membrane Ser/Thr kinase that binds to peptidoglycan and that belongs to a broadly conserved family including the essential PknB kinase of M. tuberculosis. YocH stimulates its own expression in a PrkC-dependent manner demonstrating the presence of an autoregulatory loop during growth. Cells lacking YocH display a survival defect in stationary phase but enzymes secreted by other cells in the culture rescue this defect. The essential translation factor EF-G is an in vivo substrate of PrkC and this phosphorylation occurs in response to muropeptides. Therefore, we hypothesize that YocH is used by the bacterium to digest peptidoglycan released by other bacteria in the milieu and that the presence of these fragments is detected by a membrane kinase that modifies a key regulator of translation as well as to stimulate its own expression.
    Molecular Microbiology 03/2010; 75(5):1232-43. DOI:10.1111/j.1365-2958.2010.07046.x · 5.03 Impact Factor


Available from