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 postinoculation, of a homogeneous population of ostensibly ' non-culturable ' cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10 5 organisms ml N1 , 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.

SummaryMicrococcus 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.

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) Tuberculosis (TB) remains one of the most important causes of morbidity and mortality worldwide (8,10,38,39), and this situation dictates an urgent need for improved measures for controlling TB. The increasing numbers of multidrug-resistant TB cases (6, 37) suggest that the development of innovative vaccine strategies is, perhaps, a method of choice for controlling the spread of TB. Mycobacterium bovis BCG (attenuated M. bovis strain) represents the only vaccine available against TB as yet, although its efficacy in well-controlled clinical trials appears to be highly varied (9,13,14). Importantly, it is very likely that BCG vaccination does not protect against adult pulmonary TB in areas where TB is endemic (9, 19), i.e., the vaccine's effect is negligible exactly where it is most needed. An elegant, recent study of mice provided a rationale for the low efficacy of BCG in the regions where there is a high level of exposure to saprophytic mycobacteria (7). A varied BCG performance, as well as the obvious problem of using a live BCG vaccine in populations experiencing a substantial increase in the spread of human immunodeficiency virus (15, 33), validates the development of anti-TB vaccines whose efficacy is not dependent upon the persistence of live mycobacteria in the host.Among several strategies to replace BCG with novel TB vaccine candidates, e.g., vaccination with a subunit protein, naked DNA, and improved whole bacterial vaccines (for a review, see reference 20), vaccination with a subunit protein is the approach best characterized for animal models (12,17,29).

The dorsal lateral subnucleus of the rat pontine parabrachial nucleus is a major target for ascending nociceptive information from the spinal cord. With in situ hybridization histochemistry, using a radiolabelled cRNA probe, we demonstrate that neurones in and near the dorsal lateral subnucleus express preprodynorphin mRNA. The cRNA probe was constructed from a PCR product amplified from rat genomic DNA. Sequencing of the PCR product revealed that it corresponded to the sequence 466-1101 of the rat preprodynorphin gene exon 4. Tract tracing experiments, using injection of cholera toxin subunit B into the hypothalamic median preoptic nucleus, showed a retrograde labelling pattern of neurones in the parabrachial nucleus that was almost identical to that of the preprodynorphin mRNA expressing neurones. Double-labelling, combining immunohistochemical detection of tracer and in situ hybridization, revealed that the retrogradely labelled neurones expressed preprodynorphin mRNA. A similar double-labelling, combining in situ hybridization with immunohistochemical detection of noxious-evoked fos following formalin injection into one hindpaw of awake animals, showed that almost all fos-immunoreactive neurones in the dorsal lateral parabrachial subnucleus also expressed preprodynorphin mRNA. Quantitative analysis suggested that the evoked fos immunoreactivity was accompanied by an increased preprodynorphin mRNA expression. The findings provide evidence that neurones in the dorsal lateral subnucleus produce dynorphin and project to the median preoptic nucleus, and that noxious stimulation in awake animals synaptically activates the dynorphinergic neurones in this subnucleus. These observations are consistent with the idea of a functional and chemical heterogeneity among different parabrachial subnuclei that serves to produce specific homeostatic responses to stimuli that changes the physiological status of the organism, including tissue damage.

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A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leus]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.Sequence-specific DNA-binding factors serve as regulators of transcription, recombination, DNA replication and folding, and other processes as well. The target sequences for these factors are, in general, relatively short, 6-20 bp (1). In eukaryotic genes, DNA sequences regulating transcription are located upstream of the transcription initiation site in promoter or enhancer regions or downstream, in the 3'-untranslated region (see, for example, refs. 2 and 3). Such sequences have also been found in the first exon and first intron of several genes (4-6).Opioid peptides derive from three separate precursor proteins, prodynorphin (PD), proenkephalin (PE), and proopiomelanocortin (POMC) (reviewed in refs. 7 and 8). Opioid peptides have an N-terminal pentapeptide sequence in common, [Leu5]-or [Met5]enkephalin, which is essential for opioid activity. The enkephalin sequences are repetitive, with three copies in PD (7-9) and seven copies in PE (7,8,10), and are highly conserved between species (7-14).Regulation of PD gene transcription has not been studied extensively. It is known that the gene can be regulated by the cAMP-response element binding protein. Three cAMPresponse elements are clustered at unusually distant positions, and a fourth element is located in exon 1 (15-17). There are also several activator protein (AP-1)-like sequences, which could be potential targets for c-Fos-containing complexes in the promoter (18,19). It has been reported that both cAMPresponse element binding protein, by inhibiting, and AP-1, by activating, regulate PD gene transcription (15). There are also potential targets for transcript...

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