Recurrent emergence of carbapenem resistance in Klebsiella pneumoniae mediated by an inhibitory ompK36 mRNA secondary structure

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

The Sequence of the Ribosomal Binding Site Controls the Gene Expression in Brucella

BackgroundBrucella is an important pathogen causing Brucellosis. Vaccine strains obtained by a single knockout cannot combine low virulence and immunogenicity. Our study modified the SD sequence and spacer sequence of the RBS of Brucella to affect its protein expression. We altered the RBS of LPS-associated genes to reduce LPS-associated protein expression while retaining LPS integrity.ResultsWe first established an evaluation system based on the reporter gene red fluorescent protein mCherry. The mCherry expression could be changed by altering the Shine Dalgarno sequence and spacer sequence of RBS. After optimizing the Shine Dalgarno sequence, mCherry expression was increased 4-fold in E. coli and decreased by 1/4 in Brucella. The mCherry expression was increased 1.5-fold in E. coli and decreased to 1/2 in Brucella when the length of the spacer sequence was 0. When the spacer sequence was NA (N = 4, 8, 12nt) or NG (N = 4, 8, 12nt), mCherry expression was reduced in both E. coli and Brucella. Accordingly, two mutant strains were constructed in an attempt to decrease the expression of LptA and LpxO, Brucella LPS-related genes, by 1/4. Silver staining experiments of LPS SDS-PAGE revealed an alteration in the composition of LPS in the two mutant strains. Polymyxin B experiments revealed that both mutant strains were more sensitive to Polymyxin B resistance.Conclusion: In Brucella, the expression of the target gene could be affected by changing the length or the composition of the RBS sequence. The LPS gene remained unchanged while reducing the expression of its associated protein, achieving the original goal of reducing bacterial virulence while retaining immunogenicity. It is a promising strategy to improve the safety and efficacy of vaccines.

Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5′ leader region, which usually contains the Shine–Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin–antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine–Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine–Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine–Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine–Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine–Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

A combination of mRNA features influence the efficiency of leaderless mRNA translation initiation

Bacterial translation is thought to initiate by base pairing of the 16S rRNA and the Shine–Dalgarno sequence in the mRNA’s 5′ untranslated region (UTR). However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5′ UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine–Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using these data, a simple computational model was built based on the combinatorial relationship of these mRNA features that can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

Virulent but not temperate bacteriophages display hallmarks of rapid translation initiation

Bacteriophages rely almost exclusively on host-cell machinery to produce their proteins, and their mRNAs must therefore compete with host mRNAs for valuable translational resources. In many bacterial species, highly translated mRNAs are characterized by the presence of a Shine-Dalgarno sequence motif upstream of the start codon and weak secondary structure within the start codon region. However, the general constraints and principles underlying the translation of phage mRNAs are largely unknown. Here, we show that phage mRNAs are highly enriched in strong Shine-Dalgarno sequences and have comparatively weaker secondary structures in the start codon region than host-cell mRNAs. Phage mRNAs appear statistically similar to the most highly expressed host genes in E. coli according to both features, strongly suggesting that they initiate translation at particularly high rates. Interestingly, we find that these observations are driven largely by virulent phages and that temperate phages encode mRNAs with similar start codon features to their host genes. These findings apply broadly across a wide-diversity of host-species and phage genomes. Further study of phage translational regulation — with a particular emphasis on virulent phages — may provide new strategies for engineering phage genomes and recombinant expression systems more generally.

Translation of a leaderless reporter is robust during exponential growth and well sustained during stress conditions in Mycobacterium tuberculosis

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5’ leader region, which usually contains the Shine-Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins with secondary adaptive functions are predominantly leaderless and the overall ratio of leaderless to Shine-Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during stress conditions that induce growth arrest and during optimal growth conditions, is unclear. Here, using bioluminescent reporter strains, we detect robust leaderless translation during exponential in vitro growth and we show that leaderless translation is more stable than Shine-Dalgarno translation during adaptation to stress conditions. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine-Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are more stable than those of Shine-Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein—bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.

Simplification of Ribosomes in Bacteria with Tiny Genomes

The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (<1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine–Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.