High Prevalence of Diverse Insertion Sequences within the rRNA Operons of Mycoplasma bovis

Abstract

Insertion sequences (ISs) are widespread in the genome of Mycoplasma bovis strain PG45, but no ISs were identified within its two tandemly positioned rRNA operons (rrn1 and rrn2). However, characterization of the rrn locus in 70 M. bovis isolates revealed the presence of ISs related to the ISMbov1 (IS30 family) and ISMbov4 (IS4 family) isomers in 35 isolates. ISs were inserted into intergenic region 1 (IGR-1) or IGR-3, which are the putative promoter regions of rrn1 and rrn2, respectively, and into IGR-5, located downstream of the rrl2 gene. Seven different configurations (A to G) of the rrn locus with respect to ISs were identified, including those in five annotated genomes. The transcriptional start site for the single rrn operon in M. bovis strain 88127 was mapped within IGR-1, 60 bp upstream of the rrs gene. Notably, only 1 nucleotide separated the direct repeat (DR) for ISMbov1 and the promoter -35 element in configuration D, while in configuration F, the -35 motif was a part of the ISMbov1 DR. Relative quantitative real-time (qRT) PCR analysis and growth rate comparisons detected a significant increase (P < 0.05) in the expression of the rrs genes and in the number of viable cells during log phase growth (8, 12, and 16 h) in the strains with configuration F in comparison to strains with one or two rrn operons that did not have ISs. A high prevalence of IS elements within or close to the M. bovis rrn operon-promoter region may reflect their important role in regulation of both ribosome synthesis and function.

Importance: Data presented in this study show a high prevalence of diverse ISs within the M. bovis rrn locus resulting in intraspecies variability and diversity. Such abundance of IS elements near or within the rrn locus may offer a selective advantage to M. bovis Moreover, the fact that expression of the rrs genes as well as the number of viable cells increased in the group of strains with IS element insertion within a putative promoter -35 sequence (configuration F) in comparison to that in strains with one or two rrn operons that do not have ISs may serve as a basis for understanding the possible role of M. bovis IS elements in fundamental biological processes such as regulation of ribosome synthesis and function.

FIG 1

Schematic representation of the genomic regions containing the ribosomal genes and their flanking regions in different M. bovis strains. The rrn loci are represented for M. bovis strains PG45 (configuration A), HB0801 or NM-2012 (B), Hubei-1 or CQ-W70 (C), 88127 (C-a), 268B07 (D), 393B08 (E), 72242 (F), and 422 (G). The locations and directions of the genes as well as intergenic regions (IGR) are shown by arrows and spiral lines, respectively. Numbers below arrows correspond to gene names from M. bovis strain PG45 (NC_014760.1 [without the prefix MBOVPG45]).

FIG 2

Southern blot hybridization of representative M. bovis strains showing different configurations and numbers of rrn. Molecular size markers are indicated on the left of the panel. The M. bovis strains are as follows (by lane): 1, PG45; 2, 341B09 (equivalent of HB0801 strain for the rrn configuration); 3, 88127 (equivalent of Hubei-1 strain for the rrn configuration, but without ISMbov7 upstream of MBOV_0282); 4, 268B07; 5, 393B08; 6, 72242; and 7, 422.

FIG 3

M. bovis rrn promoter region and IS insertion. (A) The promoter region for the M. bovis 88127 rrn operon, as determined by 5′-RACE. Part of the nucleotide sequence of the rrs gene and its upstream region is shown. The position of the transcriptional start site (arrowhead) and the sequences corresponding to prokaryotic RpoD (σ^A)-dependent promoter −10 element and −35 element sequences are in boldface and underlined. The positions of the 5′ ends of potential processed and mature rrs products, identified by 5′ RACE, are shown in boldface and underlined. The same (TSS, −10, and −35) promoter elements were predicted in rrn1 and rrn2 of M. bovis PG45. In addition, the promoter region of the M. pneumoniae FH strain rrn operon as determined by Hyman et al. (43) is also shown. Identical nucleotides are marked by asterisks. (B) Analysis of IS insertion relative to the promoter region of the rrn2 operon. The nucleotide sequences of the rrn2 promoter region of M. bovis strains 393B08, 268B07, and 72242 are shown. The positions of the predicted transcriptional start site (arrowheads) and both promoter −10 and −35 sequence motifs are shown in boldface and underlined. The arrows show the orientations of the ISs. The ISMbov1 and ISMbov4 inverted repeats (IRs) are indicated by dots, and the target site duplications generated by IS transposition (direct repeats [DR]) are shaded in gray.

FIG 4

Relative expression of rrs genes (A) and growth rates (B) in M. bovis strains with different numbers and configurations of rrn loci. Strains with two rrn operons (configuration A [squares]), one rrn (configuration C-a [triangles]), with an insertion of ISMbov1 within IGR-1 (configuration G [diamonds]), or with the insertion of ISMbov1 and ISMbov4 within IGR-3 and ISMbov1 in IGR-5 (configuration F [circles]) at 8, 12, and 16 hpi are shown. The y axes indicate expression levels of rrs relative to control group (configuration A at each time point) (A) or growth rate expressed as log₁₀ CFU per microliter (B). The results of qRT-PCR were normalized against the GAPDH gene expression used as an internal control. Relative levels of rrs mRNA were analyzed by the 2^ΔΔCT method. The data presented are values from groups containing six or nine strains, with medians indicated as horizontal bars. Asterisks indicate significant differences (*, P ≤ 0.01; **, P ≤ 0.001; ***, P ≤ 0.0001) among strains in configurations C-a, F, and G versus the group of M. bovis strains in configuration A calculated by one-way ANOVA with Duncan's post hoc test.