Novel tRNA gene organization in the 16S-23S intergenic spacer of the Streptococcus pneumoniae rRNA gene cluster

Abstract

Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA gene clusters. Southern hybridization analysis and DNA sequence analysis demonstrated a novel gene organization for an rRNA gene cluster on the Streptococcus pneumoniae chromosome. A sequence specifying an alanine tRNA was found within the intergenic spacer, but no sequence specifying an isoleucine tRNA was found there. Southern hybridization analysis indicated that the location of the isoleucine tRNA gene was near the 5S rRNA gene in two of four rRNA gene clusters.Typically, the gene organization of eubacterial rRNA gene clusters is 5'-16S-23S-5S-3' (2 and references within; 20), although several exceptions have been observed (12,21,23 Southern hybridization analysis of rRNA and spacer tRNA genes. Total DNA was extracted (11) and purified from late-log-phase cultures of S. pneumoniae (ATCC 33400). The concentration of the DNA preparation was determined by the diphenylamine assay as previously described (6). In separate reactions, 1 ,ug of total DNA was digested with either HindlIl or PstI. Conditions for the digestions were those specified by the supplier of the restriction endonucleases (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). The restriction digests and lambda HindIll fragments were electrophoresed in a 1% agarose gel. The fractionated DNA was transferred to a solid support membrane (Nytran; Schleicher & Schuell, Inc., Keene, N.H.) as previously described (19). The membrane-bound DNA fragments were sequentially probed with 5'-end-labeled [y-32P] ATP oligodeoxyribonucleotides specific for the three rRNA genes and the tRNAIle and tRNAAla genes. Probes are described in Table 1. The 5S rDNA probe was mixed, in that the sixth nucleotide was synthesized with equimolar ratios of all four deoxyribonucleotides, because of the variability at that position in eubacterial 5S rRNAs. Conditions for prehybridizations, hybridizations, and probe removal were those specified by the supplier of the hybridization membrane (Schleicher & Schuell). Figure 1 shows the results of the hybridization experiments. Estimated sizes of the DNA fragments that hybridized with the probes are presented in Most striking of our observations was the lack of similarity between the hybridization patterns generated from the tDNAIIe and tDNAAla probes. Two signals corresponding to 6.1 and 3.5 kb in the HindIII-digested DNA were observed from the hybridization with the tDNAI'e probe. Four signals corresponding to 6.1, 3.5, 3.2, and 2.5 kb were observed when the 5S rDNA probe was hybridized against the HindIII-digested DNA. Two signals corresponding to 8.9 and 7.1 kb in the PstI-digested DNA were observed from the hybridization with the tDNAIle probe. Four signals corresponding to 20.0, 8.9, 7.8, and 7.1 kb were observed following hybridization of the 5S rDNA probe to PstI-digested DNA. Thus, the two signals from the tDNAIle hybridization correspond to two of the four signals from the 5S rDNA hybridization (HindlIl-or PstI-di...

Primary and secondary structures of rRNA spacer regions in enterococci

“…Until now, such a feature had been found only in streptococci (Bacot & Reeves, 1991 ;Chiaruttini & Milet, 1993;Nour et al, 1995), lactobacilli (Pittet & Hottinger, 1989) and some archaeobacteria (reviewed by Bacot & Reeves, 1991 …”

“…PCR amplifications were performed as described by Nour et al (1995). The DNA fragments encoding the 16s-23s rRNA spacer region (spacer region 1) were amplified using primer combination 1 : primer 514, 5'-TGGATCACCTCCTTTC-TA-3' (sequence of the 18 nt at the 3'-end of Streptococcus pneumoniae 16s rRNA; Bacot & Reeves, 1991) and primer 554, 5'-ACCGTGTACGCTTAGTCGCTTAA-3', which is complementary to a highly conserved 23s rRNA sequence (positions 1-25 of the Esch. coli 23s rRNA; Branlant et al, 1981).…”

Heterogeneity among 16S-23S rRNA intergenic spacers of species within the ‘Streptococcus milleri group’

“…The 16S-23s spacers were determined to be either 270 bp (short) or 391/392 bp (long) in length for S. intermedius, 319 bp for S. constellatus and either 388/391 bp (long) or 295 bp (short) for S. anginosus. All sequences contained a single tRNA,,, gene as described previously for S. pneumoniae (Bacot & Reeves, 1991).…”

“…Within the rRNA operon (rrn), the intergenic spacer separating the 16s and 23s rRNA genes has been suggested as a candidate region from which DNA probes for identification and strain typing may be derived (Barry e t al., 1991;Uemori et a/., 1992;Kostman e t al., 1992;Gurtler, 1993). This region is involved in the processing of pre-RNA (Young & Steitz, 1978) and may contain coding sequences for tRNA (Morgan et al , 1977 ;Young e t al., 1979;Bacot & Reeves, 1991) but is otherwise considered to be largely non-functional, under minimal selective pressure and therefore likely to contain sequence variations from which taxonomically useful information might be obtained (Barry et al, 1991). Indeed, sequence divergence between different species within a genus has already been reported (Barry etbl., 1991 ;Frothingham & Wilson, 1994;Uemori e t al., 1992).…”

16S-23S rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous

“…oenos and Lezlc. mesenteroides were aligned with those of four closely related bacteria (Chiaruttini & Milet, 1993;Bacot & Reeves, 1991;Hall, 1994;Sechi & DaneoMoore, 1993) using the CLUSTAL method of the PC/GENE program in an IBM computer, and sequence similarity percentages were calculated. (1) and Bgll + Hinfl-cleaved pBR328 (7).…”