Purification and assay of Rpf (A) Elution profile of the resuscitation activity. Fractions eluted from the DEAE-Sepharose column (see Materials and Methods) with 0.25 M KCl were applied to a Mono Q column that was developed with a 20 ml linear gradient from 0.08 to 0.28 M KCl in 10 mM Tris⋅HCl buffer containing 10% glycerol, pH 7.4. The A₂₈₀ of the eluate was monitored continuously. (B) Resuscitation activity. 10 μl of a diluted suspension of starved cells (cfu 3 × 10⁶ cells/ml, total count of unstained cells 5 × 10⁹ cells/ml) was added to 200 μl of LMM supplemented with 0.5% wt/vol lactate and 0.05% yeast extract containing 2 μl of each fraction in 5–10 replicates in the Bioscreen instrument. For details see Materials and Methods. (C) Reduction of the apparent lag phase of viable cells. 10 μl of a diluted suspension of viable, stationary phase cells (viable count 20 cells) was added to 200 μl of LMM supplemented with 0.5% wt/vol l-lactate and containing 2 μl of each fraction (from a different experiment to that shown in A and B) in 5–10 replicates in the Bioscreen instrument. The apparent lag phase was estimated by extrapolating the exponential growth line to the abscissa. The detection limit of the instrument is approx. 10⁷ cells/ml. (D) SDS/PAGE profile of fractions after DEAE-cellulose and Mono Q chromatography. The molecular mass (kDa) markers (Pharmacia) were phosphorylase B (94), BSA (67), ovalbumin (43), carbonic anhydrase (30), soya bean trypsin inhibitor (20.1), and lactalbumin (14.4). Lanes: 1, markers; 2, fraction from DEAE-cellulose column; 3, purifed preparation (fraction number 8 from the Mono Q column).

Effect of purified Rpf on M. luteus. (A) Resuscitation of dormant cells with different concentrations of Rpf. 10 μl of a diluted suspension of starved cells (cfu 3 × 10⁶ cells/ml, total count 5 × 10⁹ cells/ml) was added to 200 μl of LMM supplemented with 0.5% wt/vol l-lactate and 0.05% yeast extract and Rpf at the concentrations shown in 5–10 replicates in the Bioscreen instrument. For details see Materials and Methods. (B) Growth of washed cells. Stationary phase cells of M. luteus grown in LMM were washed 5 times by suspension and centrifugation in LMM from which lactate had been omitted. Bacteria were finally suspended in the same medium by repeatedly passing them through a syringe, diluted, and inoculated into a 20 ml flask with LMM or LMM in the presence of Rpf (31 pmol/l). The initial cell density was ca. 10² viable cells per ml and incubation was at 30°C with intensive shaking. Growth was monitored by plating 0.1 ml samples on plates containing nutrient broth E solidified with agar.

Mb. tuberculosis and Mb. leprae contain genes whose products are similar to Rpf. Multiple sequence alignment of M. luteus Rpf (Z96935) with predicted gene products from Mb. tuberculosis g2052146 (MTCI237.26), g2791490 (MTV008.06c), g1655671 (MTCY253.32), g2225976 (MTCY180.34), e1254009 (MTV043.60c), and Mb. leprae g2440090 (MLCB57.05c), MSGB38COS (L01095, nucleotides 12,292–12,759). Conserved blocks are in uppercase, residues conserved or conservatively substituted in five or more sequences are in boldface and predicted signal sequences (35) are underlined.

Detection of genes similar to rpf in M. luteus, Mb. smegmatis, and S. rimosus. The 147-bp PCR product obtained with oligonucleotides A1 and A2 was used as hybridization probe. (A) Lane1, λPstI; lanes 2–8, M. luteus DNA digested with XhoI, StuI, SmaI, PvuII, PstI, KpnI, and BamHI, respectively, lane 9, λPvuII. (B) Lane 1, λPstI, lanes 2–7, S. rimosus DNA digested with XhoI, StuI, SmaI, PvuII, PstI, and BamHI; lanes 8–13, Mb. smegmatis DNA digested with XhoI, StuI, SmaI, PvuII, PstI, and BamHI; lane 14, λPvuII.

Purification and activity of recombinant Rpf. (A) Purification of his-tagged Rpf. Rpf was expressed in E. coli HSM174(DE3) and purified as described in Materials and Methods. SDS/PAGE profile of fractions after Ni²⁺-chelation chromatography. The molecular mass (kDa) markers (Sigma) were BSA (67), ovalbumin (43), glyceraldehyde 3-phosphate dehydrogenase (36), carbonic anhydrase (30), soya bean trypsin inhibitor (20.1), and lactalbumin (14.4). Lanes: 1, markers; 2, crude extract from E. coli containing pET19b vector; 3, crude extract from E. coli containing pRPF1; 4, purifed recombinant Rpf. (B) Reduction of the apparent lag phase of viable cells of M. luteus by purifed recombinant Rpf. For experimental details see the legend for Fig. 1C. A dilution factor of 10⁰ corresponds to 33 μg Rpf/ml. (C) Stimulation of the growth of washed cells. Stationary phase cells of M. luteus grown in LMM were washed 5 times by suspension and centrifugation in LMM from which lactate had been omitted. Bacteria were finally suspended in the same medium by repeatedly passing them through a syringe, diluted, and inoculated into a 20 ml flask with LMM or LMM in the presence of Rpf (230 pmol/liter). The initial cell density was ca. 10² viable cells per ml and incubation was at 30°C with intensive shaking. Growth was monitored by plating 0.1-ml samples on plates containing nutrient broth E solidified with agar.