Nucleotide sequences of the spacer-1, spacer-2 and trailer regions of the rrn operons and secondary structures of precursor 23S rRNAs and precursor 5S rRNAs of slow-growing mycobacteria.

The single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 16S, 23S and 5S rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 16S rRNA and 23S rRNA genes and the spacer-2 region separating the 23S rRNA and 5S rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, 'Mycobacterium lufu' and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 5S rRNA gene, the trailer region and the downstream region of the rrn operon of Mycobacterium tuberculosis were cloned and sequenced. These data, together with those obtained previously for Mycobacterium leprae, were used to identify putative antitermination signals and RNase III processing sites within the spacer-1 region. Notable features include two adjacent potential Box B elements and a Box A element. The latter is located within a sequence of 46 nucleotides which is very highly conserved among the slow-growers which were examined. The conserved sequence has the capacity to interact through base-pairing with part of the spacer-2 region. Secondary structures for mycobacterial precursor 23S rRNA and for precursor 5S rRNA were devised, based on sequence homologies and homologous nucleotide substitutions. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. A putative motif for the intrinsic termination of transcription was identified approximately 33 bp downstream from the 3'-end of the 5S rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species.

Nucleotide sequence and secondary structures of precursor 16S rRNA of slow-growing mycobacteria.

Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 16S, 23S and 5s rRNA being present in that order. The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli. PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-1 region separating the 16S rRNA and 23S rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu', Mycobacterium simiae and Mycobacterium marinum. The amplified DNA was sequenced. The sequence data, together with those obtained previously for Mycobacterium leprae and M. tuberculosis, were used to identify putative antitermination signals and RNase III processing sites within the leader region. Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized. A secondary structure for mycobacterial precursor-16S rRNA was devised, based on sequence homologies and homologous nucleotide substitutions. The 18 nucleotides at the 5'-end of spacer-1 have the capacity of binding sequences close to the 5'- and 3'-ends of mature 16S rRNA, suggesting that secondary structure is important to the maturation process. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. The scheme proposed for M. tuberculosis is a variant of the main theme. The leader and spacer sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species. 'M. lufu' appears to be a close relative of M. intracellulare.

The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae precursor rRNA.

Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tuberculosis and M. leprae; although the principal features are common to both species there are notable differences.

The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction.

Nucleotide sequence data for bacterial 16S ribosomal RNA was used to identify oligodeoxyribonucleotide primers suitable for probing the rRNA gene of mycobacteria and related organisms, with the polymerase chain reaction. The method enabled us to distinguish mycobacteria from other closely related genera, and to differentiate between slow- and fast-growing mycobacteria. Mycobacterium leprae fell within the slow-growing group of mycobacteria but there are significant differences between the sequence of the M. leprae 16S rRNA gene and that of other slow-growing mycobacteria. These differences were used to devise a rapid, non-radioactive method for detecting M. leprae in infected tissue.

Determination and evolutionary significance of nucleotide sequences near to the 3'-end of 16S ribosomal RNA of mycobacteria.

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.

Partial nucleotide sequence of 16S ribosomal RNA isolated from armadillo-grown Mycobacterium leprae.

Ribosomal RNA (rRNA) was isolated from Mycobacterium leprae recovered from infected tissue of the Nine-banded Armadillo, and nucleotide sequences near the 3' end of the 16S species were determined by primer extension in the presence of dideoxynucleotides. Previously published data for bacterial 16S rRNAs show a pattern of conserved and non-conserved sequences that fit a common secondary structure. Our data for M. leprae fits this general pattern.

Step-wise isolation of RNA and DNA from mycobacteria.

Mycobacterium phlei and M. smegmatis were lysed at -15 degrees C in the presence of guanidinium salts and poly(A)+-RNA. Nonpolyadenylated RNA (mainly rRNA) and DNA were isolated in successive steps from the same lysate. DNA isolated by this procedure was sufficient to yield distinct bands on treatment with restriction endonucleases as shown by hybridization to 125I-labeled rRNA. The successive isolation of poly(A)+-RNA, nonpolyadenylated RNA, and high molecular weight DNA from the same lysate by this procedure, which is being reported for the first time, provides a very economical approach for isolation and purification of nucleic acids from mycobacteria and other organisms. This could be of special value for various genetic recombination studies, particularly in the case of mycobacteria which are available in very limited quantities, e.g., noncultivable or difficult-to-grow mycobacteria, especially M. leprae.