Viable cells of Micrococcus luteus secrete a factor, which promotes the resuscitation and growth of dormant, nongrowing cells of the same organism. The resuscitation-promoting 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.

Summary: Mycobacterium 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. Affinity‐purified 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.

Rationale: Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. Although mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesized that tuberculous sputum might include a population of Rpf-dependent Mtb cells. Objectives: To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population. Methods: In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay with that determined by limiting dilution, most-probable number assay in the presence or absence of Rpf preparations. Measurements and Main Results: In 20 of 25 prechemotherapy samples from separate patients, 80–99.99% of the cells demonstrated by cultivation could be detected only with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf dependency was lost after primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population. Conclusions: Smear-positive sputum samples are dominated by a population of Mtb cells that can be grown only in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.

Summary: 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‐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.

After growth of Rhodococcus rhodochrous in Sauton’s medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3–4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of ‘non-culturable’ cells of the ‘Academia’ strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton’s medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4–5 months post-inoculation, of a homogeneous population of ostensibly ‘non-culturable’ cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 105 organisms ml−1, and this value was further increased by one log using supernatant from an actively growing culture. Populations of ‘non-culturable’ cells of Mycobacterium tuberculosis were also obtained by the filtration of ‘clumpy’ cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ‘non-culturable’ cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ‘non-culturable’ bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

Summary: Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non‐productively at the cell surface, as a truncated form of the protein, encompassing only the ‘Rpf domain’ is fully active. The C‐terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti‐Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid‐located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf‐like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.