Instability of rRNA operons in Bacillus subtilis

Abstract

Many laboratory strains ofBaciUus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnj-rrnW or rrnl-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-arol region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.The endospore-forming bacterium Bacillus subtilis has 10 rRNA gene sets, of which 9 are clustered between the origin and 75 degrees on the genomic map (1, 8) ( Fig. 1). In a separate study, we demonstrated by integrative mapping, Southern analysis, and transductional crosses two wellseparated sets of closely spaced rrn clusters in the region between cysA14 and aroI906 (Fig. 1). The first two, rrnJ and rrnW, are assigned to BclI homologs of 4.9 and 5.8 kilobases (kb), respectively, are located between cysAJ4 and rpsE2. The second set, rrnl, rrnH and rrnG, of which the latter two are assigned to BclI homologs of 5.5 and 4.8 kb, respectively, are located between attSPO2 and glpT6 (E. D. Jarvis, R. L. Widom, G. LaFauci, Y. Setoguchi, I. R. Richter, and R. Rudner, manuscript in preparation) (Fig. 1). Southern analysis showed that in certain common laboratory strains of B. subtilis, the number of BclI rrn homologs is 9 instead of 10, revealing the loss of either the 4.8-or the 5.8-kb fragments assigned to rrnG and rrnW, respectively. Our earlier studies with strains of the first group (6) and similar analyses in another laboratory (10) revealed the loss of a ribosomal homolog in other restriction enzyme digests of genomic DNA. During transformation with integrable plasmids containing rDNA, several strains were created which resembled the naturally occurring deletion strains with respect to their BclI and EcoRI Southern patterns.We report that rescue of DNA from the chromosomal region of rrn deletions indicates that the naturally occurring deletions arose by homologous recombination within the closely spaced operons rrnJ-rrnW and rrnI-rrnH-rrnG. Restriction analyses of rescued DNA from the region of the deletion provide additional evidence for the existence of rrnI, the third operon in the second cluster, whose location was predicted by the heteroduplex mapping of Chow and Davidson (2) but so far has had no plasmid insertions. MATERIALS AND METHODSBacterial strains and plasmids. Some of the B. subtilis strains and plasmids (pGR102 and pWR103) used in this study were described previously (8). Integrable plasmid pGR110, which contains a 0.5-kb PstI-SmaI 16S fragment,

Bacillus subtilis mutants harbouring a single copy of the rRNA operon exhibit severe defects in growth and sporulation

“…Some laboratory strains of B. subtilis contain only nine rrn operons due to spontaneous deletions within rrnW, H or G (Widom et al, 1988). This indicates that these operons are dispensable for growth in B. subtilis in the presence of the other rrn genes.…”

“…In general, organisms that possess multiple rrn operons are considered to be able to grow faster than those that possess one or two operons. However, it is possible to delete one rRNA operon in either E. coli or B. subtilis without having a major effect on cell growth rate or physiology (Ellwood & Nomura, 1980;Widom et al, 1988), which suggests that the full number of copies of the rRNA operon is not required for rapid growth in these two organisms. Instead, it is assumed that many prokaryotes evolved multiple rRNA operons to cope with a variety of environmental conditions (Condon et al, 1992(Condon et al, , 1995.…”

“…subtilis, which is the best-characterized Gram-positive bacterium, has been studied extensively as a model for unicellular differentiation because of its ability to form dormant spores (Schaeffer et al, 1965;Hoch, 1993;Grossman, 1995). Ten rrn operons (rrnO, A, J, W, I, H, G, E, D, B), which include the rrnJ-rrnW and rrnI-rrnHrrnG clusters, have been identified in the genome of B. subtilis (Widom et al, 1988;Kunst et al, 1997;Henkin, 2002) (Fig. 1a).…”

Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis

“…Deletion of a single rrn operon has been reported to have little effect on cell growth rates or physiological processes in either E. coli or B. subtilis (Ellwood & Nomura, 1980;Widom et al, 1988). Asai and co-workers observed reduced growth rates in E. coli strains in which the number of rrn operon copies was reduced from seven to five (Asai et al, 1999b).…”

Organization of the Bacterial Chromosome

“…Restriction map analysis of one such B. subtilis laboratory strain showed that homologous recombination between closely linked multiple rrn loci caused deletion of one of the loci and the loss of genetic material lying between the recombining rrn loci (375). A sequence of programmed deletions has been described for Anabaena heterocyst formation.…”