43NISR RESEARCH GRANT
2008 Research Grant

Development of a translation system highly expressing thetarget gene in B. subtilis
Fujio KAWAMURA
College of Science, Rikkyo University

Research aims

Methods

It is an important problem in industrial fermentation
technology to develop a system which is able to highly produce useful substances such as antibiotics and enzymes.
One of the solutions is to develop a constitutively active
translation system, which translates only the target genes,by improving the ribosome. In order to develop such a highand specific translation system, it is necessary to understand the regulatory mechanisms for translation specificity
and for metabolism of ribosomes, as well as the synthesis
and degradation of ribosomes, in greater detail. The bacterial ribosome is composed of over 50 kinds of ribosomal
proteins and three kinds of ribosomal RNAs, 23S, 16S and
5S rRNA. Since rRNAs play a key role in translation, we
studied the transcription activity of individual rRNA operons and the intracellular levels of ribosomes in Bacillus
subtilis mutants deficient in (p)ppGpp synthetase genes,relA, yjbM, and ywaC and compared them with those in thewild type strain1).

All Bacillus subtilis strains used in this study were isogenic with B. subtilis strain 168. We constructed strains carrying a promoter- and terminator-less cat gene downstreamof the promoter of each rRNA operon (rrnO, -A, -J, -I, -E, D, or -B) (Fig. 1). Disruption of the relA gene in strain
RIK350-6 was achieved by transformation of chromosomal
DNA extracted from strain RIK900 (trpC2 relA::erm) followed by selection of erythromycin-resistant transformants.
In a similar manner, strain RIK908 (trpC2 ywaC::spc) andRIK1000 (trpC2 ΔyjbM) with a catpt1-tagged rrn operon(conferring chloramphenicol resistance) yielded double
deletion mutants (trpC2 ywaC::spc relA::erm rrn::catpt1)and (trpC2 ΔyjbM relA::erm rrn::catpt1), respectively, by
subsequent disruption of the relA locus by transformationof the RIK900 chromosomal DNA. Triple deletion mutants(trpC2 ΔyjbM ywaC::spc relA::erm) with rrn operons containing catpt1 were constructed analogously from RIK1002(trpC2 ΔyjbM ywaC::spc).
Cells grown in LB medium to an early exponential phaseFig. 1.

Sequence alignment of the promoter region of seven rrn operons in B. subtilis.

44(optical density at 600 nm0.2) at 37°C with shaking, werecollected and then disrupted by passage through a Frenchpressure cell (Aminco) at 8,000 p.s.i., after which cell debris was removed by centrifugation as previously described2). Supernatants were used as crude cell extracts.
Aliquots of extract equivalent to 3.09 of OD600 units ofthe culture were layered onto 10–40% sucrose density
gradients and centrifuged at 4°C for 17.5 h at 65,000 g(HITACHI P40ST rotor).
Results
Isolation and identification of two types of suppressor
mutations from the relA null mutant. During the courseof characterizing a relA null mutant of B. subtilis, we foundthat this mutant strain grew more slowly than wild-type
cells in LB medium1). This growth-defect could be suppressed by introduction of the deletion(s) of yjbM and/orywaC, both encoding (p)ppGpp synthetase1). Interestingly,
during cultivation of the relA null mutant on LB agar plates
we frequently observed the appearance of two types oflarger colonies distinguishable from one another by colonymorphology and growth characteristics on LB agar. We isolated 35 spontaneous suppressor mutants. Of the 35 suppressor mutants we characterized, 16 had mutations in yjbM
and other 19 mapped within ywaC. A variety of mutations
including point mutation, deletion and addition were observed among the suppressor mutations although deletion
mutations were found only in yjbM and addition mutations
in ywaC. More interestingly, neither suppressor completelyrestored the growth defect of the relA null mutant, which isin good agreement with our previous work3). Furthermore,as these were the only suppressors found, it is most likelythat there are no (p)ppGpp synthetases other than RelA,
YjbM and YwaC in B. subtilis. Srivatsan and co-workers
have recently reached a conclusion similar to ours regarding the identification of the suppressor mutations in the relAmutant4).
Effects of relA and its suppressor mutations on 70S ribosome formation. To explore the possibility that thegrowth defect of the relA null mutant could be due to poortranscription from promoters in rrn operons, we examined
the formation of 70S ribosomes in relA null strains carrying
a deletion of either or both of yjbM and ywaC genes by a
10%–40% sucrose density gradient centrifugation and compared them with that in the wild-type strain (Fig. 2). The
amount of 70S ribosomes formed in the relA mutant wasapparently low compared with that in the wild-type cells

Fig. 2.

(Fig. 2). As the peaks corresponding to the 30S, 50S and70S particles were found in the profiles of the relA null mutant, it is most likely that the low amount of 70S ribosomesin the relA null mutant is not caused by the inhibition of thenormal processing pathways for ribosome formation, but by
the reduction of overall pre-rRNA synthesis, presumably
due to the poor transcription of rrn operons. In contrast, theamount of 70S ribosomes in the relA null mutant was partially restored by the introduction of the yjbM or ywaC mutation, and completely restored in the triple mutant (Fig. 2),
suggesting that the presence of yjbM and ywaC in the relA
null background has inhibitory effects on growth and on thetranscription activity of rrn operons.
Transcription activity of rrn operons from P1 promoters is abolished in the relA null mutant and can be restored by its suppressor mutations. We found that all promoters examined initiated transcription with GTP. Transcription activity from P1 promoters was drastically reduced in the relA null mutant. In addition, transcription
from P2 promoters in rrnA, D, E, and J operons was alsosignificantly decreased in the relA null mutant (Fig. 3). On
the other hand, transcription from P2 promoters in the otheroperons, as well as from P3 in rrnE, although slightly decreased compared to wild type (Fig. 3), provided the major
contribution to rRNA synthesis in relA null mutants. Incontrast, transcription activity from P1 promoters was improved to a higher degree in relA yjbM than in relA ywaC.
Moreover, in the triple disruption mutant, transcription activity from both P1 and P2 promoters was almost completely restored to wild-type levels for each rrn operon
compared to the relA null strain (Fig. 3).
Conclusion
We studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B)
individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites ofeach rrn operon (a G) and found that transcription of all rrn
operons from their P1 promoters was drastically reduced inthe relA mutant, while this was almost completely restored
in the relA yjbM ywaC triple mutant. Taken together withprevious results that the intracellular GTP concentration
was reduced in the relA mutant while it was restored in thetriple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.

Effects of the relA mutation, the relA yjbM and relA ywaC double mutation and the relA yjbM ywaC triple mutation on 70S ribosome formation.

45Fig. 3. Effects of the relA mutation, the relA yjbM and relA ywaC double mutation and the relA yjbM ywaC triple mutation on the transcription activity ofeach rrn operon.

References1) Natori, Y., Tagami, K., Murakami K., Yoshida S., Tanigawa O., Moh, Y.,
Masuda, K., Wada, T., Suzuki, S., Nanamiya, H., Tozawa, Y., andKawamura, F. Transcription activity of individual rrn operons inBacillus subtilis mutants deficient in (p)ppGpp synthetase genes,relA, yjbM, and ywaC. J. Bacteriol., 191: 4555–4561 (2009).
2) Natori, Y., Nanamiya, H., Akanuma, G., Kosono, S., Kudo, T., Ochi, K.,
and Kawamura, F. A fail-safe system for the ribosome under zinclimiting conditions in Bacillus subtilis. Mol. Microbiol. 63: 294–307(2007).
3) Nanamiya, H., Kasai, K., Nozawa, A., Yun, C. S., Narisawa, T., Murakami, K., Natori, Y., Kawamura, F., and Tozawa, Y. Identificationand functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis. Mol. Microbiol. 67: 291–304 (2008).
4) Srivatsan, A., Han, Y., Peng, J., Tehranchi, A. K., Gibbs, R., Wang, J.
D., and Chen, R. High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies. PLoS Genet. 4: e1000139(2008).