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Mycoplasma pneumoniae (M129; ATCC 29342), a member of the bacterial class Mollicutes, possesses one of the smallest genomes of all known self-replicating organisms, with a size of only 816 kb (8). Based on the complete genome sequence, 677 open reading frames (ORFs) and 39 genes coding for various untranslated RNA species were predicted. These RNAs include 35 tRNA genes (17; J. Brosius and K. Lühn, personal communication), one set of rRNAs (5S, 16S, and 23S), the 4.5S RNA, the 10Sa RNA, and the RNase P RNA (8).

A recent publication on the identification of several small RNAs in Mycoplasma capricolum (19) prompted us to search for similar molecules in M. pneumoniae. Here, we report the isolation and characterization of a hitherto unknown, 205- to 210-base-long small RNA from M. pneumoniae, which is also conserved in a shorter, 170- to 180-base-long version in Mycoplasma genitalium. The detailed description of our laboratory procedures can be found on our website (http://www.zmbh.uni-heidelberg.de/M_pneumoniae/MP200).

Identification of a highly abundant RNA and mapping on the genome. M. pneumoniae cells grown under standard conditions at 37°C (15) were used for the isolation of total RNA. The RNA was extracted by treating the bacteria with a solution of guanidinium thiocyanate followed by phenol-chloroform extraction and precipitation of the RNA with isopropanol. Separation of total RNA on an 8% polyacrylamide gel containing 7 M urea revealed a reproducible pattern of bands (Fig. 1) after silver staining (2). Several known species of small RNAs, like tRNAs, 4.5S RNA, and 5S rRNA, could be assigned to individual bands. In addition, two RNAs were migrating closely together, but clearly separated just above the 200-base RNA size marker. Therefore, we estimated their sizes to be 205 and 210 bases (Fig. 1). Treatment of a gel after the run with RNase in the presence of 25 mM EDTA confirmed the RNA character, since it caused the disappearance of these two bands and all of the other ones. According to their size, the two newly identified RNA species were named MP200 RNA. To map the MP200 RNA on the genome, the two RNAs were isolated from a denaturing 8% polyacrylamide gel, radioactively labeled at their 5' ends by phage T4 polynucleotide kinase in the presence of [-³²P]ATP, and then used as probes in Southern blot and dot blot experiments (16). The MP200 RNA could be first localized on an EcoRI fragment by Southern blotting against a cosmid gene bank covering the complete genome of M. pneumoniae (23) and then mapped more precisely within the pyruvate dehydrogenase gene (pdh) cluster between pdhB and pdhC. This cluster consists of the four genes pdhA, pdhB, pdhC, and pdhD, which are organized in this order and all transcribed in the same direction (8). Screening with strand-specific oligonucleotides showed that the MP200 RNA was also transcribed in the same direction as these four genes. Primer extension analysis with the oligonucleotides 5'-TTT CTT GTC TTC CAT CTT-3' and 5'-AAG ATG GAA GAC AAG AAA TGC-3' allowed us to map the 5' ends of both RNAs to the same adenine at nucleotide position 553,546 of the M. pneumoniae genome (8). Based on these mapping results, the boundaries of the coding region of the MP200 RNA could be predicted in the 319-bp-long intergenic region between pdhB and pdhC. Upstream of the experimentally defined 5' end of the MP200 RNA, a putative promoter 10 region (TAGAAT) is present, but a conserved 35 region similar to the corresponding regions from Escherichia coli and Bacillus subtilis has not been found, which is consistent with observations for other M. pneumoniae genes (6, 11, 20).

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Separation of different total RNA preparations on an 8% polyacrylamide gel with 7 M urea. The RNA was silver stained as described previously (2). Lanes: A and B, different M. genitalium preparations; 3 to 7, M. pneumoniae preparations from the 3rd through 7th days of culture growth, respectively; M, RNA size marker (Novagen). Sizes are shown to the right (bases [b]).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Comparison of the sequences encoded by MP200 RNA in M. pneumoniae and MG170 RNA in M. genitalium. The ORFs coding for the cysteine-rich peptides are boxed. The first nucleotide (numbered 1 at the top) at the 5' end corresponds to genome positions 553546 in M. pneumoniae and 331214 in M. genitalium. Proposed 3' ends are indicated by solid triangles, and the sites of transposon insertion are indicated by open triangles.

Mapping of the 3' end. Since the 5' ends of the two RNAs were identical, it seemed very likely that the size differences derived from a heterogeneity of the 3' end. Therefore, we attempted to map the 3' ends from both isolated RNAs by nuclease protection assay and two-dimensional RNA mobility shift (3). Both methods failed to define a unique 3' end; instead, we observed a terminal heterogeneity in both cases. The mobility shift and end group determination assay allowed us to define a mixture of A and U for the smaller RNA and U as the last base for the larger one (data not shown).

Identification of an MP200 RNA homolog in M. genitalium named MG170 RNA. Database searches with the program BLASTN (1) showed no significant DNA sequence similarity to any known DNA sequence except to a sequence from the genome of M. genitalium (Fig. 2). This sequence was also located in the pdh gene cluster in the 253-bp-long intergenic region between the genes pdhB and pdhC (7). Repetition of the analyses, which has been done for the characterization of the MP200 RNA, revealed that the homologous RNA from M. genitalium was about 30 nucleotides shorter than, but otherwise with very similar features to, the corresponding RNA in M. pneumoniae. For example, the RNA could be separated by polyacrylamide gel electrophoresis into two bands with sizes of 170 and 180 nucleotides (Fig. 1); therefore, it was named MG170 RNA. The 5' ends of the RNA from both bands were identical at genome position 331214, as determined by primer extension, with the possible 10 region (TAAAAT) of a promoter (Fig. 2). Three different complementary oligonucleotides, each of which shared at least 15 contiguous nucleotides, cross-reacted in Northern blotting analyses with the RNAs from both M. genitalium and M. pneumoniae and therefore stressed their sequence similarities. Comparative analyses of pdh clusters of other sequenced bacterial genomes, such as those of B. subtilis (12), Acholeplasma laidlawii (21), Escherichia coli (4), and Mycobacterium tuberculosis (5), showed that none had an intergenic space long enough to code for a 200-base-long RNA.

Secondary structure analysis. The RNAs from both Mycoplasma species contain palindromic sequences; therefore, we examined possible secondary structures by using the mfold program (version 3.1) (10). In either case, several structures have been found, with minimal free energy values (G) of 77 and 55.3 kcal/mol, respectively (Fig. 3). All structures share two similar stems at the 5' end and two more in the 3' region; furthermore, a stem-loop structure is conserved between bases 80 and 100 throughout all secondary structures examined (Fig. 3). The shorter RNAs (205 and 170 bases) have similar structures, except for slight differences in the 3' terminating stem. The U/A-rich terminating palindromic sequences are characteristic of many transcription terminator sequences (24).

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Prediction of secondary structures. The predicted structures and their respective free energy values (G) were determined with the mfold program.

Is the MP200 RNA an mRNA? Translation of the MP200 RNA into all three possible reading frames revealed that it has the potential to code for a 29-amino-acid-long peptide with 9 cysteine residues (Fig. 2). Strikingly, 8 of those 9 cysteines were conserved in a smaller 23-amino-acid-long protein, which could be translated from the homologous RNA in M. genitalium. Both RNAs lack a ribosomal binding site or a downstream box, as described for E. coli (18, 14) or M. genitalium (13), but this does not a priori exclude translation, since the absence of these translation signals has been also observed for other genes in M. pneumoniae and M. genitalium, which are known to be translated (8; unpublished data). So far, several attempts have failed to prove that the 29-amino-acid-long peptide is synthesized in M. pneumoniae. In vivo labeling of the bacterial proteins with [³⁵S]cysteine and subsequent electrophoretic analysis (Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis) did not reveal a peptide of the expected size. Similarly, an immunological approach to detect the peptide by enzyme-linked immunosorbent assay or Western blotting with monospecific antibodies produced in rabbits which were directed against the chemically synthesized peptide was unsuccessful (data not shown). The failure to detect the peptide in protein extracts of M. pneumoniae could also mean that the RNA is synthesized constitutively, but translation is induced only under certain growth conditions, or that the peptide is exported into the medium.

A possible function for the MP200 RNA. There are two reasons to assume that the identified small RNAs are functional. (i) Both small RNAs identified are conserved in both Mycoplasma species, although they are of different lengths. (ii) The RNAs are present in high copy numbers.