therefore desirable to ensure that cells with remodeled ribosomes are harvested. While remodeling generally occurs within
24–30 h of incubation under the described condition, the
timing can vary depending on the level of zinc contamination
in the inoculum and/or culture medium. We note that the
cultures described in this protocol were prepared in glass flasks.
Because glass containers leach trace amounts of zinc [25], use
of zinc-free plastic containers may also be considered to minimize zinc contaminants in the culture. These changes may
impact the timing of CÀ expression in the culture. If the
induction of CÀ proteins occurs outside 24–30 h period
described here, the timepoint for harvesting cells with
Mpy-bound ribosomes should also be adjusted accordingly.
5. Induction of ribosome hibernation occurs at a lower zinc concentration than remodeling [21], and therefore, extended
incubation of a batch culture is needed to deplete zinc beyond
the level necessary for ribosome remodeling. Note that the
optical density of the cultures increases during this time
(Fig. 1).
6. M. tuberculosis is more sensitive to zinc starvation than
M. smegmatis, and depletion of zinc with TPEN results in
severe growth arrest. To obtain optimum biomass necessary
for maximum yield of CÀ ribosomes, it is necessary to culture
the slow-growing species as pellicles in Sauton’s medium base
without any supplemental zinc.
7. While we established that Mpy recruitment in M. tuberculosis
reaches saturating levels by 7 weeks of growth in pellicles, we
have not yet determined the precise timings of ribosome remodeling and Mpy recruitment in this species. It is however
possible to obtain the earliest time of CÀ ribosomal protein
expression by RT-PCR. Ribosomes purified at this timepoint
would be expected to be remodeled, but not bound to Mpy.
8. Pellicles formed in this condition are similar in morphological
appearance and biomass to those cultured in low-zinc
condition.
9. Perforation of the caps is necessary to allow evaporation of
nitrogen gas, and thus to avoid explosion of tubes.
10. High-salt wash with this buffer reduces nonspecific association
of Mpy with ribosomes that may occur after lysis. Skipping this
step can potentially increase the background signal and may
confound comparative analysis of Mpy binding by
immunoblot.
11. Volume of gradients can be adjusted depending on the quantity of ribosomes. For example, ribosomes equivalent to
OD 260 ¼ 5 U are better resolved on a 15 mL sucrose gradient,
while 5 mL analytical gradient is better suited for ribosomes
Purification of Three Types of Ribosomes from Mycobacteria
163
24–30 h of incubation under the described condition, the
timing can vary depending on the level of zinc contamination
in the inoculum and/or culture medium. We note that the
cultures described in this protocol were prepared in glass flasks.
Because glass containers leach trace amounts of zinc [25], use
of zinc-free plastic containers may also be considered to minimize zinc contaminants in the culture. These changes may
impact the timing of CÀ expression in the culture. If the
induction of CÀ proteins occurs outside 24–30 h period
described here, the timepoint for harvesting cells with
Mpy-bound ribosomes should also be adjusted accordingly.
5. Induction of ribosome hibernation occurs at a lower zinc concentration than remodeling [21], and therefore, extended
incubation of a batch culture is needed to deplete zinc beyond
the level necessary for ribosome remodeling. Note that the
optical density of the cultures increases during this time
(Fig. 1).
6. M. tuberculosis is more sensitive to zinc starvation than
M. smegmatis, and depletion of zinc with TPEN results in
severe growth arrest. To obtain optimum biomass necessary
for maximum yield of CÀ ribosomes, it is necessary to culture
the slow-growing species as pellicles in Sauton’s medium base
without any supplemental zinc.
7. While we established that Mpy recruitment in M. tuberculosis
reaches saturating levels by 7 weeks of growth in pellicles, we
have not yet determined the precise timings of ribosome remodeling and Mpy recruitment in this species. It is however
possible to obtain the earliest time of CÀ ribosomal protein
expression by RT-PCR. Ribosomes purified at this timepoint
would be expected to be remodeled, but not bound to Mpy.
8. Pellicles formed in this condition are similar in morphological
appearance and biomass to those cultured in low-zinc
condition.
9. Perforation of the caps is necessary to allow evaporation of
nitrogen gas, and thus to avoid explosion of tubes.
10. High-salt wash with this buffer reduces nonspecific association
of Mpy with ribosomes that may occur after lysis. Skipping this
step can potentially increase the background signal and may
confound comparative analysis of Mpy binding by
immunoblot.
11. Volume of gradients can be adjusted depending on the quantity of ribosomes. For example, ribosomes equivalent to
OD 260 ¼ 5 U are better resolved on a 15 mL sucrose gradient,
while 5 mL analytical gradient is better suited for ribosomes
Purification of Three Types of Ribosomes from Mycobacteria
163