Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitationpromoting factor (Rpf) is a protein, which has been purified to homogeneity. In picomolar concentrations, it increases the viable cell count of dormant M. luteus cultures at least 100-fold and can also stimulate the growth of viable cells. Rpf also stimulates the growth of several other high G؉C Gram-positive organisms, including Mycobacterium avium, Mycobacterium bovis (BCG), Mycobacterium kansasii, Mycobacterium smegmatis, and Mycobacterium tuberculosis. Similar genes are widely distributed among high G؉C Gram-positive bacteria; genome sequencing has uncovered examples in Mycobacterium leprae and Mb. tuberculosis and others have been detected by hybridization in Mb. smegmatis, Corynebacterium glutamicum, and Streptomyces spp. The mycobacterial gene products may provide different targets for the detection and control of these important pathogens. This report is thus a description of a proteinaceous autocrine or paracrine bacterial growth factor or cytokine.The essential role of cytokines in controlling the activation, growth, and proliferation of eukaryotic cells is now widely recognized (1). These proteinaceous cell-signaling molecules include many growth factors that are widely distributed among vertebrates, and probably also invertebrates (2). Similar growth factors have also recently been found in unicellular organisms such as ciliates (3-6). In prokaryotic organisms, intercellular signaling usually involves small metabolites (e.g., N-acyl homoserine lactones) or peptides (7-16). Although specific interactions have been documented between vertebrate cytokines and prokaryotes (17-20), there are no known examples of autocrine or paracrine growth factors produced by prokaryotic microorganisms.The number of observable microbial cells in a natural sample often exceeds the number that can be cultured therefrom by orders of magnitude (21-23). It is not known in general whether such nonculturable (and often noncultured) cells are dead, are killed by our media, or are in a dormant state from which we could, in principle, resuscitate them with appropriate growth factors (24). After growth to stationary phase and starvation in spent growth medium, cells of the nonsporulating, Gram-positive bacterium Micrococcus luteus can enter a dormant state in which they can persist for at least 7 months. Whereas exponentially growing cultures have a viability of Ϸ100%, as estimated by comparing colony forming units (cfu) on agar plates with the total cell count determined microscopically, such dormant cultures can exhibit a viability of less than 10 Ϫ4 (25). The viable count of this type of culture as measured by the Most Probable Number (MPN) method also corresponds to the number of cfu. However, in the presence of sterile (filtered) culture supernatant from the late logarithmic phase of batch growth, resuscitation occurs and the viable count by MPN increases ...

SummaryMycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus . Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus , Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinitypurified antibodies inhibit bacterial growth in vitro , suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo . The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

SummaryMycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

A.S. KAPRELYANTS AND D.B. KELL. 1992. T h e fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler-sensitive fashion. Steady-state fluorescence measurements with Micrococcus Iuteus indicated that provided the added dye concentration is below approximately 1 mmol/l, uptake is fully uncoupler-sensitive and is not accompanied by significant self-quenching of the fluorescence of accumulated dye molecules. 'Viable' and 'non-viable' cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. T h e viability of a very slowly growing chemostat culture of Mic. Iuteus is apparently only about 4&50%, as judged by plate counts, but most of the 'non-viable' cells can be resuscitated by incubation of the culture in nutrient medium before plating. T h e extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture ('non-viable', 'viable' and 'non-viable-but-resuscitable'). Gram-negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.

SummaryThe 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-β β β β -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.