Moy, J. K. | bioRxiv Science

bioRxiv2024

Diverse intrinsic properties shape transcript stability and stabilization in Mycolicibacterium smegmatis

In mycobacteria, regulation of transcript degradation is known to occur in response to environmental stress and to facilitate adaptation. However, the mechanisms underlying this regulation are unknown. Here we sought to gain an understanding of the mechanisms controlling mRNA stability by investigating the transcript properties associated with variance in transcript stability and stress-induced transcript stabilization. We performed transcriptome-wide mRNA degradation profiling of Mycolicibacterium smegmatis in both log phase growth and hypoxia-induced growth arrest. The transcriptome was globally stabilized in response to hypoxia, with all transcripts having longer half-lives, but some having greater degrees of stabilization than others. The transcripts of essential genes were generally stabilized more than those of non-essential genes. We then developed machine learning models that utilized a compendium of transcript properties and enabled us to identify the non-linear collective effect of diverse properties on transcript stability and stabilization. The comparisons of these properties confirmed the association of 5 UTRs with transcript stability, along with other differences between leadered and leaderless transcripts. Our analysis highlighted the protective effect of translation in log phase but not in hypoxia-induced growth arrest. Steady-state transcript abundance had a weak negative association with transcript half-life that was stronger in hypoxia, while coding sequence length showed an unexpected correlation with half-life in hypoxia only. In summary, we found that transcript properties are differentially associated with transcript stability depending on both the transcript type and the growth condition. Our results reveal the complex interplay between transcript features and microenvironment that shapes transcript stability in mycobacteria.

molecular biology↗

bioRxiv2022

The small non-coding RNA B11 regulates multiple facets of Mycobacterium abscessus virulence

Mycobacterium abscessus causes severe, virtually incurable disease in young patients with cystic fibrosis. Little is known in M. abscessus about the roles of small regulatory RNAs (sRNA) in gene expression regulation. Here, we show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough colony morphology, increased pro-inflammatory signaling and virulence in in-vivo infection models, and increased resistance to clinically relevant antibiotics. Examination of clinical isolate cohorts revealed some isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of two mutations found in clinical isolates. Approximate 230 genes were differentially expressed in the B11 deletion mutant. Strains with the clinical B11 mutations showed similar expression trends to the deletion mutant but of a lesser magnitude, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with a model of translational repression via base-pairing of B11 to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences in their RBS, consistent with B11 functioning as a negative regulator through direct binding of target mRNAs. Intriguingly, the genes upregulated in the absence of B11 included components of the ESX-4 secretion system, known to be critical for M. abscessus virulence. One of these genes had a B11-complementary sequence at its RBS, and fusing the UTR of this gene to a reporter was sufficient to make the reporter suppressible by B11. Taken together, our data show that B11 may act as either a negative or positive regulator with pleiotropic effects on gene expression and clinically important phenotypes in M. abscessus. The presence of hypomorphic B11 mutations in clinical strains supports the idea that lower B11 activity may be advantageous for M. abscessus in some clinical contexts. To our knowledge, this is the first report of the role of an sRNA in M. abscessus.

microbiology↗