Resuscitation factors from mycobacteria: homologs of Micrococcus luteus proteins.
METHODS
Resuscitation promoting factors (Rpfs) are proteins, originally identified in Micrococcus luteus, that promote recovery of bacteria from a viable but non-replicating phase (e.g., stationary phase or latency) to a replicating phase. Purified M. luteus Rpf can stimulate growth and increase recovery of M. luteus bacteria as well as Mycobacterium tuberculosis bacteria from prolonged stationary cultures.


OBJECTIVE
To clone and characterize Rpfs from mycobacteria.


METHODS
We cloned one M. avium subsp. paratuberculosis rpf gene and one M. tuberculosis rpf gene into the pET19b or pET21a vector for expression in Escherichia coli. The His-tag recombinant proteins were purified and characterized.


RESULTS
When the purified recombinant proteins were added to Sauton medium (a relatively minimal medium) at 100-500 pM, lag phase for mycobacteria from non-replicating cultures was shortened and there was a 10- to 100-fold increase in colony-forming units compared with control samples. In most probable number assays, the mycobacterial Rpfs increased recovery of mycobacteria from late stationary culture by about 10-fold. The Rpfs also promoted recovery of extensively washed Mycobacterium smegmatis bacteria inoculated into Sauton medium. Rpfs had only minor effects on growth of M. tuberculosis in BACTEC 12B broth, a rich medium.


CONCLUSIONS
The mycobacterial Rpfs demonstrate resuscitation activities similar to those of the M. luteus Rpf.
Zhu
Wenming
W
Tuberculosis/Mycobacteriology Branch, Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Atlanta, GA 30333, USA.
Plikaytis
Bonnie B
BB
Shinnick
Thomas M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D015169:Colony Count, Microbial; D016207:Cytokines / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D016982:Micrococcus luteus / Q000737:chemistry*; D009161:Mycobacterium / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology
D001426:Bacterial Proteins; D016207:Cytokines; D011993:Recombinant Fusion Proteins; C113704:resuscitation-promoting factor, bacteria