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Mukamolova, G. V. et al. Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Mol. Microbiol. 59, 84-98

Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK.
Molecular Microbiology (Impact Factor: 4.42). 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.

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    • "In addition , a previous study showed that neither pure Rpf protein from M. luteus culture supernatant nor recombinant Rpf could maintain its activity for over 1 week at 4 °C (Su et al. 2013b). What is more, several other functionally equivalent proteins were found in supernatant from M. luteus (Mukamolova et al. 2006). Therefore, it is more cost-effective and advantageous to resuscitate and stimulate VBNC or uncultured bacteria using the culture supernatant from M. luteus containing Rpf, also referred to as SRpf, than that using purified Rpf protein. "
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    ABSTRACT: A culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (SRpf) was used to enhance the biological nutrient removal of potentially functional bacteria. The obtained results suggest that SRpf accelerated the start-up process and significantly enhanced the biological nutrient removal in sequencing batch reactor (SBR). PO4 (3-)-P removal efficiency increased by over 12 % and total nitrogen removal efficiency increased by over 8 % in treatment reactor acclimated by SRpf compared with those without SRpf addition. The Illumina high-throughput sequencing analysis showed that SRpf played an essential role in shifts in the composition and diversity of bacterial community. The phyla of Proteobacteria and Actinobacteria, which were closely related to biological nutrient removal, were greatly abundant after SRpf addition. This study demonstrates that SRpf acclimation or addition might hold great potential as an efficient and cost-effective alternative for wastewater treatment plants (WWTPs) to meet more stringent operation conditions and legislations.
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    • "Of all the enzymes identified in this study, the Rpf family is the most extensively studied. This group of enzymes are of particular interest due to demonstrated importance for reactivation from dormancy and essentiality for growth in Micrococcus luteus[22,23]. Whilst Mi. luteus encodes a single, essential rpf gene, mycobacteria encode a multiplicity of rpf homologues and those present in M. tuberculosis, designated as rpfA-rpfE, encode closely related proteins all of which retain the Rpf domain [24-26], Figure  2. "
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    ABSTRACT: Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets. We conducted an in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival. PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt.
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    • "Similarity to c-type lysozyme led to suggestions that Rpfs targets peptidoglycan and this cleavage activates resuscitation, by some unknown mechanism (Cohen-Gonsuad et al., 2004). This prediction was supported by studies in which mutations of specific amino acids in the Rpf domains resulted in decreased cleavage of a synthetic lysozyme substrate and VBNC cell resuscitation (Mukamolova et al., 2006). "

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