Mapping of rRNA genes with integrable plasmids in Bacillus subtilis

Abstract

Integrable plasmids pGR102 and pWR103 containing ribosomal sequences from within the transcriptional units for 16S and 23S were used to transform Bacillus subtilis. To date, these plasmids integrated into 7 of 10 known rrn operons. Two such events occurred at unassigned operons, revealing a close linkage of the CAT gene of the plasmid to pha-1 situated between dal-) and purB33 for rrnE and to thiA78 situated between glyB133 and tre-12 for rrnD. All seven integration events that led to the loss of unique ribosomal BclI fragments can now be assigned to known rrn operons.In the genome of Bacillus subtilis there are as many as 10 rRNA gene sets clustered in three groups (5). A major group is composed of rrnO and rrnA located at the replication origin (24,26,35) and the closely situated (5, 26) repeats (rrnI, rrnH, rrnG) found near the attachment site of phage SP02 (5). Minor clusters containing rrnB and rrnC have been mapped in the region between thr-S and aroG (5), and one cistron is believed to be located at the ilvBC-leu region (7,11,31). Strains of B. subtilis have been shown to contain only nine operons because of a deletion (11,18) or to exhibit rearrangements involving ribosomal sequences (3,11). Each transcriptional unit is arranged in the order 16S, 23S, and 5S rRNA genes, with two to three operons (including rrnO and rrnA) containing tRNAAla and tRNAlie genes in the abutment region between the 16S and 23S rDNA sequences (18, 33). We employed the integrative mapping method of Haldenwang et al. (13) to ascertain whether integrations can occur in operons, to locate various unmapped rrn genes on the chromosome, and to determine the physiological effects on growth and sporulation of such events in B. subtilis. In this study, the bifunctional plasmid pJH101 was used (10). The plasmid cannot replicate in B. subtilis because it does not contain an origin of replication for B. subtilis. If a region of homology with the B. subtilis chromosome is inserted in pJH101, the resultant plasmid gains the ability to transform competent cells and also, on occasion, integrates by Campbell-like recombination (10). We subcloned two similar but not identical ribosomal fragments from within the transcriptional unit for 16S and 23S which originated from the operons rrnO and rrnI. The resultant plasmids pGR102 and pWR103 can theoretically integrate into any of the 10 ribosomal operons by a Campbell-like insertion, leaving the entire plasmid with its antibiotic marker adjacent to the homologous region. We report here seven different integration events by these plasmids, two of which occur at unassigned operons (rrnD and rrnE), increasing the number from seven to nine known genomic locations in B. subtilis. containing integrated plasmids were constructed by competent cell transformation, verified for integration by hybridization, and checked for site of integration by PBS1 transduction mapping. We designated the various transformants as the B. subtilis strain Ql plasmids, with fl denoting plasmid integration. The plasmids with and wi...

Physical mapping of stable RNA genes in Bacillus subtilis using polymerase chain reaction amplification from a yeast artificial chromosome library

“…First, amplification from 16SD-23SU revealed a ribosomal region without internal-tRNA genes, as expected for nnE. Second, the overlap of the two clones is mapped to a position to which rniE has been mapped previously by genetic means (28 (19,32), the catalytic RNA component has escaped localization. Because this gene has been sequenced (6,34), primers P-RNAD and P-RNAU (Table 1) were able to be designed.…”

Two tRNA gene clusters associated with rRNA operons rrnD and rrnE in Bacillus subtilis

“…Some of the B. subtilis strains and plasmids were described previously (9,11,26). Plasmids pYR104, pGR111, and pWR103 are derivatives of plasmid pJH101 (3), containing a selectable Cmr gene and a 2.3-kb EcoRI-HindIII 23S-5S fragment from rrnA (pYR104), a 1.5-kb EcoRI-SmaI 23S fragment (pGR111), or a 1.0-kb fragment of EcoRI-PstI 16S-155 bp of the 23S sequence (pWR103) (9,11). The B. subtilis strains are listed in Table 1 as parental and integrant strains.…”

“…In our search for additional tRNA gene clusters, we cloned and sequenced DNA downstream of operon rnE located at 44°on the chromosomal map (9,11). A small cluster containing tRNAM"t and tRNA P was found 14 bases downstream of the 5S gene of rrnE.…”

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome

“…It can first be said that their members have acquired increased rRNA gene dosage. In general, the number of rrn operons on the chromosomes of diverse bacterial species and strains varies, irrespective of their whole-genome sizes; for example, the Bradyrhizobium diazoefficiens genome (9.1 Mb) contains only one copy (24), whereas the genomes of Escherichia coli (4.6 Mb) and Bacillus subtilis (4.2 Mb) contain seven and 10 copies, respectively (25,26). In E. coli, the presence of multiple rrn operons facilitates a sudden increase in the rate of rRNA synthesis, enabling rapid adaptation to nutritional upshift or favorable temperature shift (27).…”