Identification of “Noncontiguous Operons” in the S. aureus Genome.

We screened genome-wide the transcriptome data obtained from the clinical isolates S. aureus 15981 (21) and S. aureus MW2 to identify genes cotranscribed together despite being separated by a gene transcribed in the opposite direction. We found six examples that fit the predicted model (Fig. 1 and SI Appendix, Figs. S1 and S2). RNA sequencing data of published results from different laboratories (rnamaps.unavarra.es/) confirmed the existence of identical transcriptional organizations in five other genetically unrelated S. aureus strains (Fig. 1 and SI Appendix, Figs. S1 and S2). The function of most of the proteins encoded by such operons is unknown. CoaD, which is part of the CoA biosynthesis pathway, MenADB and MenEC, required for menaquinone synthesis, and MoaABCED, required for molybdate transport, are among the proteins with known functions.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Analysis of the noncontiguous operon architecture. JBrowser software images showing RNA-seq or TSS-seq mapped reads distribution in the region comprising menE-menC-MW1733-ytkD-MW1731 genes of seven unrelated S. aureus strains. The scale (log₂ or ×10³) indicates the number of mapped reads per nucleotide position. A schematic representation of the structure under study is shown in the middle of the scheme. ORFs are represented as orange arrows for the genes that constitute the menE-menC-ytkD-MW1731 operon and as a blue arrow for the MW1733 gene. Promoters are shown as green triangles and transcriptional terminators as red rectangles. The transcript generated from the menE-menC-ytkD-MW1731 operon is represented as a dashed orange arrow, while the transcript generated from MW1733 is presented as a dashed blue arrow. The top line denotes the position in base pairs of the S. aureus MW2 genome. All genetic information about the start and the end of transcription was obtained from a previous study (21). RNA-seq data were obtained from 15981 (21), MW2 (this study), UAMS-1 (41), HG001 (42), WCH-SK2 (43), Homeland (24), and USA300-P23 (44).

To explore the significance of this transcriptional organization, we chose the region comprising menE, menC, MW1733, ytkD, and MW1731 genes based on the size of the transcripts and the relevance of menaquinone synthesis during S. aureus infections (22) (Fig. 1). menE-menC and ytkD-MW1731 are listed as two independent operons in the prokaryotic operons database (csbl.bmb.uga.edu/DOOR/) (23). However, transcriptome data indicated that both operons are transcribed as a single transcriptional unit (Fig. 1). These results agreed with published results obtained by mapping of transcriptional start sites (TSS) by differential RNA-seq that revealed a unique TSS upstream the menE gene (24) (Fig. 1). To experimentally confirm the transcriptome results and because the environmental conditions controlling the expression of menE remain unknown, we first generated two derivatives of the wild-type strain in which the promoter region upstream the menE gene was deleted (ΔPmen strain) or replaced by the constitutive blaZ promoter (PblaZ-men strain) (SI Appendix, Fig. S3). For each of these strains, we generated derivatives in which the chromosomal copy of either menC or MW1731 genes was tagged with the 3xFLAG sequence (SI Appendix, Fig. S3) and then examined MenC and MW1731 protein levels by Western blotting. Consistent with the transcriptome results, the ΔPmen mutation correlated with inhibition of both MenC and MW1731 proteins expression. Note that the ΔPmen deletion did not completely abolish protein production. On the contrary, the strains containing the constitutive blaZ promoter produced considerably higher levels of MenC and MW1731 compared with the wild-type strain (Fig. 2A). Thus, these findings suggested that expression of ytkD-MW1731 depends on the promoter region upstream the menE gene.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Experimental evidence showing that the region comprising menE-menC-MW1733-ytkD-MW1731 genes exhibits an architecture characteristic of a noncontiguous operon. (A) Western blots showing MenC, MW1731, or MW1733 protein levels in the WT and the following derivative strains: ΔPmen, PblaZ-men, ΔPMW1733, and PblaZ-MW1733. The 3xFLAG-tagged proteins were detected with commercial anti-3xFLAG antibodies. Coomassie stained or stain-free gel portions are shown as a loading control. (B) Northern blot analysis of RNA harvested from the strains described in A. Blots were probed with specific riboprobes for menEC, ytkD-MW1731, and MW1733 regions. The positions of RNA standards are indicated. Lower shows 16S and 23S ribosome bands stained with ethidium bromide as loading control. The strains used in this figure are depicted in SI Appendix, Fig. S3.

To further validate the cotranscription of menE-menC-ytkD-MW1731 genes, we performed Northern blot analysis using strand-specific riboprobes corresponding to menE-menC (probe A) and ytkD-MW1731 (probe B) coding regions with total RNA from exponentially growing cells of the wild-type strain, and its two isogenic derivatives, ΔPmen and PblaZ-men. Results showed that the mRNA expression levels are very low in the wild-type strain, because neither probe was able to detect the mRNA (Fig. 2B). In contrast, both probe A and B hybridizations with PblaZ-men RNA revealed an increased accumulation of a fuzzy band of ∼4 kb that was compatible with cotranscription of menE-menC with the downstream genes ytkD-MW1731 (Fig. 2B). Note that probe B also clearly detects an additional processing band (∼1.2 kb). Together, these results strongly suggest that menE-menC and ytkD-MW1731 expression depends on the promoter located upstream menE, which is consistent with transcriptome data indicating that menE-menC-ytkD-MW1731 genes comprise a single transcriptional unit.

Transcriptome data also indicated that the MW1733 gene (258 bp long) is transcribed at high levels with a short 5′ UTR of 26 nucleotides and a 3′ UTR of 60 nucleotides that overlaps the 3′ end of the menC coding sequence. To confirm transcriptome data, we generated two additional strains in which 27 nucleotides of the promoter region upstream the MW1733 gene were deleted (ΔPMW1733 strain) or replaced by the constitutive blaZ promoter (PblaZ-MW1733 strain) (SI Appendix, Fig. S3). For each of these strains, we generated a derivative in which the chromosomal copy of the MW1733 gene was tagged with the 3xFLAG sequence (SI Appendix, Fig. S3). As expected, analysis of MW1733 protein levels revealed that ΔPMW1733 mutation correlated with inhibition of MW1733 protein production whereas transcription from the constitutive blaZ promoter led to higher levels of MW1733 compared with those in the wild-type strain (Fig. 2A). Finally, we performed a Northern blot analysis using a strand-specific riboprobe corresponding to the MW1733 coding region (probe C) with total RNA from exponentially growing cells of the wild-type, ΔPMW1733 and PblaZ-MW1733 strains. Results showed the presence of a discrete band of ∼350 nucleotides in the wild-type strain (Fig. 2B). Hybridization with ΔPMW1733 and PblaZ-MW1733 RNA revealed a decreased and increased accumulation of the RNA product, respectively.

Overall, these results describe an operon organization that is different in the sense that all of the genes of the operon are not contiguous and, therefore, we refer to it as noncontiguous operon. The first two genes are followed by a gene transcribed from the opposite strand, and this gene is then followed by two other genes cotranscribed with the first two. In this spatial transcriptional organization, the noncoding region of the tetracistronic transcript completely overlaps the monocistronic unit.

The Expression of the menE-menC-ytkD-MW1731 Operon and the MW1733 Gene Is Reciprocally Regulated.

To determine whether transcriptional levels of menE-menC-ytkD-MW1731 have an effect on the amount of MW1733 mRNA, we compared by Northern blot the transcript levels of MW1733 in the wild-type, ΔPmen and PblaZ-men strains using probe C to detect MW1733 mRNA. Results showed that MW1733 transcript levels slightly increased when transcription of the operon was inhibited and, on the other hand, markedly decreased in PblaZ-men strain, which is under the presence of an excess of the overlapping tetracistronic transcript (Fig. 3A). To confirm the regulation of MW1733 expression at a protein level, we constructed derivatives of ΔPmen and PblaZ-men containing the chromosomal copy of the MW1733 gene tagged with a 3xFLAG epitope at the C terminus (SI Appendix, Fig. S4). Consistent with Northern blot results, MW1733 protein levels significantly decreased in PblaZ-men compared with those in the wild-type strain (Fig. 3A).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Mutual regulation of overlapping transcripts expression within the noncontiguous operon. (A, Upper) Northern blot showing MW1733 mRNA levels in the WT, ΔPmen, and PblaZ-men strains. A specific riboprobe (probe C) for MW1733 was used; Western blot showing MW1733 protein levels in the same strains producing a 3xFLAG-tagged MW1733 protein (A, Lower). (B, Upper) Northern blot showing menE-menC mRNA levels in the WT, ΔPMW1733, and PblaZ-MW1733 strains. A specific riboprobe (probe A) for menEC was used; Western blot showing MenC protein levels in the same strains producing a 3xFLAG-tagged MenC protein (B, Lower). (C, Upper) Northern blot showing ytkD-MW1731 mRNA levels in the strains described in B. A specific riboprobe (probe B) for ytkD-MW1731 was used; Western blot showing MW1731 protein levels in the same strains producing a 3xFLAG-tagged MW1731 protein (C, Lower). The positions of RNA standards are indicated and16S and 23S ribosome bands stained with ethidium bromide are shown as loading controls. The 3xFLAG-tagged proteins were detected with commercial anti-3xFLAG antibodies. Coomassie stained or stain-free gel portions are shown as a loading control. The strains used in this figure are depicted in SI Appendix, Fig. S4.

Next, we investigated the possibility of a reciprocal effect of MW1733 mRNA levels on the expression of the tetracistronic operon. To do so, we first analyzed by Northern blot, and with the use of probe A, menE-menC-ytkD-MW1731 mRNA levels in the wild-type, ΔPMW1733, and PblaZ-MW1733 strains. In agreement with the low level of expression of the tetracistronic mRNA in the wild-type strain (Fig. 2B), we could not find a significant difference in menE-menC-ytkD-MW1731 mRNA levels between strains when probe A was used (Fig. 3B). Thus, we repeated the Northern blot assay with the use of probe B, specific for ytkD-MW1731. Again, the ytkD-MW1731 transcript was undetectable in the wild-type and ΔPMW1733 strains. However, when MW1733 was overexpressed, a specific processing transcript was detected. The size of the discrete band (∼1.5 kb) is consistent with a transcript including ytkD-MW1731 that might be obtained upon processing of the menE-menC-ytkD-MW1731 mRNA (Fig. 3C). Next, we constructed derivatives of ΔPMW1733 and PblaZ-MW1733 harboring a chromosomal copy of either menC or MW1731 tagged with the 3xFLAG epitope in the carboxyl-terminal domain (SI Appendix, Fig. S4). Notably, constitutive expression of MW1733 caused a clear reduction in the levels of the MenC protein (Fig. 3B) and a significant accumulation of MW1731 protein levels in PblaZ-MW1733 compared with the wild-type strain (Fig. 3C).

Collectively, these results support the notion that in the noncontiguous operon, transcriptional units generated from opposite strands regulate each other’s expression. Thus, in the noncontiguous operon under study, an increase in tetracistronic operon transcription negatively regulates the expression of the interspersed MW1733 gene. Reciprocally, an increase in MW1733 mRNA discoordinates expression within the overlapped operon, by strongly elevating ytkD-MW1731 mRNA levels while reducing menE-menC expression.

Analysis of the Mechanisms Underlying the Regulation of Noncontiguous Operons Expression.

RNase III endoribonuclease is responsible for processing overlapping sense/antisense transcripts genome-wide in bacteria (21, 25, 26). Thus, we examined the importance of RNase III activity in the reduction of MW1733 transcript levels when an excess of menE-menC-ytkD-MW1731 is transcribed. Deletion of RNase III both in the wild-type strain and in the strain overproducing the tetracistronic operon (PblaZ-men) (SI Appendix, Fig. S5) caused a slight increase in the amount of MW1733 mRNA (Fig. 4A). Consequently, MW1733 protein levels only moderately increased in rnc mutants compared with those in the respective RNase III producing strains (Fig. 4A). However, we studied the involvement of RNase III in menE-menC-ytkD-MW1731 mRNA processing when MW1733 is overexpressed. A Northern blot, using probe A, with RNA from cells of the wild type, PblaZ-MW1733, and their corresponding rnc mutants showed no significant differences between strains, given the low detectability of menE-menC-ytkD-MW1731 mRNA (Fig. 4B). Second, we carried out a similar Northern blot, but with the use of probe B, specific to detect ytkD-MW1731 mRNA. Results revealed that processing of the tetracistronic mRNA when an excess of MW1733 is transcribed still occurred in the absence of RNase III. However, in this case, the processing pattern of the operon changed, leading to a significant decrease in the amount of the discrete 1.5-kb transcript and to the appearance of two additional larger mRNA fragments (Fig. 4C). Accordingly, MW1731 protein levels decreased in rnc mutants of the wild-type and PblaZ-MW1733 strains compared with those in their respective RNase III producing strains (Fig. 4C and SI Appendix, Fig. S5). Overall, these results indicated that RNase III explains, only to a certain extent, the MW1733-mediated cleavage of menE-menC-ytkD-MW1731 mRNA, suggesting that additional ribonuclease(s) might also be responsible for this processing.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

RNase III processing at the overlapping region and transcriptional interference are involved in reciprocal regulation of the overlapping transcripts generated from the noncontiguous operon. (A, Upper) Northern blot showing MW1733 mRNA levels in the WT, ΔPMW1733, Δrnc, PblaZ-men, and PblaZ-men Δrnc. A specific riboprobe (probe C) for MW1733 was used; Western blot showing MW1733 protein levels in the same strains producing a 3xFLAG-tagged MW1733 protein (A, Lower). (B, Upper) Northern blot showing menE-menC mRNA levels in the WT, Δrnc, PblaZ-MW1733, and PblaZ-MW1733 Δrnc. A specific riboprobe (probe A) for menE-menC was used; Western blot showing MenC protein levels in the same strains producing a 3xFLAG-tagged MenC protein (B, Lower). (C, Upper) Northern blot showing ytkD-MW1731 mRNA levels in the strains described in B. A specific riboprobe (probe B) for ytkD-MW1731 was used; (C, Lower) Western blot showing MW1731 protein levels in the same strains producing a 3xFLAG-tagged MW1731 protein. (D, Upper) Northern blot showing MW1733 mRNA levels in the WT, PblaZ-men, ΔPMW1733, ΔPMW1733 MW1733 trans and ΔPMW1733 PblaZ-men MW1733 trans. A specific riboprobe (probe C) for MW1733 was used; Western blot showing MW1733 protein levels in the same strains producing a 3xFLAG-tagged MW1733 protein (D, Lower). (E) Western blots showing MenC (Upper) and MW1733 (Lower) protein levels in the WT, WT pCN40, PblaZ-MW1733, and WT pCN40::MW1733-3xFLAG. Strains contained a chromosomal copy of either menC or MW1731 tagged with the 3xFLAG epitope. The positions of RNA standards are indicated, and 16S and 23S ribosome bands stained with ethidium bromide are shown as loading controls. The 3xFLAG-tagged proteins were detected with commercial anti-3xFLAG antibodies. Coomassie stained or stain-free gel portions are shown as a loading control. The strains used in this figure are depicted in SI Appendix, Fig. S5 for A–C and in SI Appendix, Fig. S6 for D and E.

Besides processing by RNase III, another possible explanation for the reciprocal regulation of overlapping transcripts described above might be transcriptional interference (27), defined as the suppressive influence that the convergent RNA synthesis machinery from one DNA strand causes in cis on the transcription of the neighboring gene. Thus, we next sought to determine whether the observed antisense regulation of menE-menC-ytkD-MW1731 over MW1733 occurred when the MW1733 gene was expressed in another location of the chromosome. To do so, we inserted a 3xFLAG-tagged MW1733 gene under its own promoter next to the innocuous attB site of the lipase gene in both ΔPMW1733 and ΔPMW1733 PblaZ-men genetic backgrounds (SI Appendix, Fig. S6). Importantly, and contrary to what happens when MW1733 is located in its natural location, Northern blot analysis of MW1733 transcript levels showed that these were only slightly reduced in the presence of an excess of menE-menC-ytkD-MW1731 mRNA when the MW1733 gene was placed and expressed in trans (Fig. 4D). Note that there is a marked difference in the size and abundance of MW1733 mRNA when it is ectopically expressed from the attB chromosomal location. Consistent with Northern blot results, Western blot analysis showed that MW1733 protein levels were unaffected in the ΔPMW1733 PblaZ-men MW1733 trans strain compared with those in the wild-type strain (Fig. 4D). These results indicated that menE-menC-ytkD-MW1731–mediated suppressive influence on MW1733 expression requires cis localization of both transcripts. Lastly, to reinforce these results, we overexpressed a 3xFLAG-tagged MW1733 gene ectopically from a plasmid in the wild-type strain harboring a chromosomal copy of either menC or MW1731 tagged with the 3xFLAG epitope (SI Appendix, Fig. S6) and analyzed MenC and MW1731 levels by Western blot. Overexpression of MW1733 in trans did not have any impact on MenC or MW1731 production, showing that MW1733 effect in discoordinating menE-menC-YtkD-MW1731 operon expression also requires cis localization of both transcripts (Fig. 4E). Overall, the above results indicate the existence of a transcriptional interference mechanism of gene regulation between the machinery that synthesizes the noncontiguous operon mRNA and the one synthesizing the mRNA of the interspersed gene.

High Transcriptional Levels of the MW1733 Gene Can Lead to the Appearance of Small Colony Variants.

The experiments shown above demonstrated that overexpression of MW1733 mRNA leads to reduced MenC protein levels (Fig. 3B). In S. aureus, the inhibition of the synthesis of menaquinone has been associated with a slowed growth phenotype, known as small colony variants (SCVs) (28). SCVs are frequently isolated from clinical samples obtained from patients experiencing chronic infections by S. aureus. We observed that the ΔPmen strain constructed in this work, which still shows some residual production of the MenC protein (Fig. 2A), produces colonies whose size are smaller than the ones corresponding to the wild-type strain although they are not as small as the SCVs generated by deletion of menE-menC genes (ΔmenEC) (SI Appendix, Fig. S7A). Therefore, we wondered whether constitutive expression of MW1733 might be followed by the appearance of SCVs phenotypic hallmarks. To test this hypothesis, the promoter of MW1733 was replaced by the constitutive blaZ promoter in ΔPmen strain (SI Appendix, Fig. S8). The resulting strain produced colonies significantly smaller than the ΔPmen strain and exhibited several characteristics associated to S. aureus SCVs such as decreased pigmentation and increased resistance to aminoglycosides (tobramycin, streptomycin, gentamycin, and amikacin) than the wild-type strain (29) (SI Appendix, Fig. S7 B and C). These results suggest that overexpression of MW1733 suppresses the expression of its convergent menE-menC genes, which, in turn, leads to suppressed menaquinone synthesis and the appearance of a SCV phenotype.

To confirm that appearance of SCVs by MW1733 overexpression in ΔPmen strain exclusively happened when MW1733 and menE-menC-ytkD-MW1731 mRNAs were expressed in cis, we overexpressed the MW1733 gene ectopically from a plasmid in ΔPmen strain and analyzed colony size on TSA plates. The resulting strain, ΔPmen pCN40::MW1733 (SI Appendix, Figs. S7A and S8), showed the same phenotype as the ΔPmen strain. Thus, we conclude that this noncontiguous operon transcriptional organization constitutes an effective mechanism for regulating gene expression and ultimately for controlling cell growth.