Organization and Characterization of the Promoter Elements of the rRNA Operons in the Slow-Growing Pathogen Mycobacterium kumamotonense

Abstract

The slow-growing, nontuberculous mycobacterium Mycobacterium kumamotonense possesses two rRNA operons, rrnA and rrnB, located downstream from the murA and tyrS genes, respectively. Here, we report the sequence and organization of the promoter regions of these two rrn operons. In the rrnA operon, transcription can be initiated from the two promoters, named P1 rrnA and PCL1, while in rrnB, transcription can only start from one, called P1 rrnB. Both rrn operons show a similar organization to the one described in Mycobacterium celatum and Mycobacterium smegmatis. Furthermore, by qRT-PCR analyses of the products generated from each promoter, we report that stress conditions such as starvation, hypoxia, and cellular infection affect the contribution of each operon to the synthesis of pre-rRNA. It was found that the products from the PCL1 promoter of rrnA play a pivotal role in rRNA synthesis during all stress conditions. Interestingly, the main participation of the products of transcription from the P1 promoter of rrnB was found during hypoxic conditions at the NRP1 phase. These results provide novel insights into pre-rRNA synthesis in mycobacteria, as well as the potential ability of M. kumamotonense to produce latent infections.

Keywords: Mycobacterium kumamotonense; rRNA operons; rrn promoters.

Figure 1

Organization of the rrnA operon of M. kumamotonense. (A) Schematic representation of the promoter region of the rrnA operon, which has two promoters, named P1 rrnA and PCL1, marked with red arrows. Promoter P1 rrnA was found downstream from the end of the murA coding region near the stop codon, whereas PCL1 was located within the HMPR. Horizontal arrows show the binding sites of the RAC1, RAC8, cR103, JY15, P1, and rrs primers. (B) Comparison of the nucleotide sequences of the HMPR regions of M. kumamotonense (M ku), M. celatum (M ce), M. smegmatis (M sm), and M. tuberculosis (M tb). The partial 3′-end of the murA gene is shown in lowercase letters. Putative-10 and -35 regions are shown within boxes in bold and underlined. The transcription starting point (tsp) of P1 rrnA is marked with an arrow and in bold located downstream from the end of murA near the stop codon. The tsp of PCL1 is marked with an arrow and in bold located near to the conserved leader sequence-1 (CL1 motif) shown in bold, in the same way as the conserved leader sequence-2 (CL2 motif). −, deletion. Both tsp sites were established by 5′-RACE experiments. The distance between tspP1 and tspPCL1 was found to be 103 bp. The HMPR organization in M. kumamotonense and M. tuberculosis showed 93% similarity, compared to 84% and 97% with M. celatum and M. smegmatis, respectively. See the Section 2 for details and primer sequences. GenBank accession numbers: KT878832.1, EF613279.1, X87943.1, and X58890.1 for M ku, M ce, M sm, and M tb, respectively.

Figure 2

Organization of the rrnB operon of M. kumamotonense. (A) Schematic representation of the promoter region of the rrnB operon, which has a single promoter, named P1 rrnB, marked with a red arrow. The promoter P1 rrnB was located within the HMPR downstream from the end of the tyrS gene. Horizontal arrows show the binding sites of the TYRS3, RAC8, cR103, JY15, P2, and rrs primers. (B) Comparison of the nucleotide sequences of the HMPR regions of M. kumamotonense (M ku), M. celatum (M ce), and M. smegmatis (M sm). The stop codon for the tyrS gene is shown in lowercase letters. N_n, specific sequence of each mycobacterium. Putative-10 and -35 regions are shown within boxes in bold and underlined. The transcription starting point (tsp) of P1 rrnB is marked with an arrow and in bold. The conserved leader sequence-2 (CL2 motif) is shown in bold. −, deletion. The tsp was established by 5′-RACE experiments. The HMPR organization in M. kumamotonense and M. celatum showed 78% similarity, while it was 83% similar to M. smegmatis. See the Section 2 for details and primer sequences. GenBank accession numbers: OQ344267, EF613280.1, and U09862.1 for M ku, M ce, and M sm, respectively.

Figure 3

Contribution of rrnA and rrnB promoters to rRNA synthesis in M. kumamotonense grown under different stress conditions. Each bar represents the absolute quantification of the amounts of pre-rrn products from each promoter PCL1, P1 rrnA, or P1 rrnB, as relates to their contribution to the expression of 16S RNA, under exponential, stationary, NRP1, NRP2, and alveolar cell infection. See Supplementary Table S1 for primers used. To obtain the specific number of copies of PCL1, the expression obtained from P1 rrnA and P1 rrnB was subtracted to the expression of 16S RNA (see the Section 2 for details). Exp., exponential; Stat., stationary; NRP1, non-replicative persistence 1; NRP2, non-replicative persistence 2; AC, alveolar cell infection. * p < 0.05; ** p < 0.005.