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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.

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.

Elongation factors G, Tu, and related proteins (including LepA) form a distinct subgroup within the GTPase superfamily. This observation is based primarily upon amino acid comparisons of the effector region (G2) of the GTP-binding domain. To examine the functional importance of the highly conserved elongation factor G2 domain a series of chimeric proteins were constructed between Escherichia coli EF-G and Micrococcus luteus EF-G, and between E. coli EF-G and LepA (a protein of unknown function). The M. luteus EF-G/E. coli EF-G hybrid, M. luteus EF-G, and E. coli EF-G efficiently complemented EF-G function in an E. coli strain (PEM101) harbouring a temperature-sensitive mutation in fusA (the gene encoding EF-G). A comparison of the amino acid sequences of the M. luteus EF-G and E. coli EF-G indicated that groups of divergent amino acid residues (amino acids 1-9 and 72-80) were not important for function. LepA and LepA/EF-G chimeric proteins were tested for the ability to complement EF-G function in vivo, for cross-linking to 8-azido-[gamma-32P]-GTP in vitro and for fusidic acid-dependent co-sedimentation with 70S ribosomes. With one exception, all chimeras could be readily cross-linked to azido-GTP in an EF-G-like manner, indicating that hybrid protein construction did not generally result in improperly folded GTP-binding domains. However, the inability of such chimeras to complement EF-G function in vivo indicates that the effector domains are not functionally interchangeable. All LepA/EF-G chimeric proteins were severely defective in fusidic acid-dependent complex formation with 70S ribosomes. A comparison of the amino acid sequences of all three proteins suggests that residues 30-33, 43-48, and 63-66 of E. coli EF-G are important for EF-G specific ribosome-associated function.

We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1-depleted E. coli ribosomes were similar to each other and differed from non-depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)