Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1 M NaCl), hydrogen peroxide (40 mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fmet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entry channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

OBJECTIVE: Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

Despite having a highly reduced genome, Chlamydia trachomatis undergoes a complex developmental cycle in which the bacteria differentiate between the following two functionally and morphologically distinct forms: the infectious, nonreplicative elementary body (EB) and the noninfectious, replicative reticulate body (RB). The transitions between EBs and RBs are not mediated by division events that redistribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require bulk protein turnover. One system for targeted protein degradation is the trans-translation system for ribosomal rescue, where polypeptides stalled during translation are marked with an SsrA tag encoded by a hybrid tRNA-mRNA, tmRNA. ClpX recognizes the SsrA tag, leading to ClpXP-mediated degradation. We hypothesize that ClpX functions in chlamydial differentiation through targeted protein degradation. We found that mutation of a key residue (R230A) within the specific motif in ClpX associated with the recognition of SsrA-tagged substrates resulted in abrogated secondary differentiation while not reducing chlamydial replication or developmental cycle progression as measured by transcripts. Furthermore, inhibition of trans-translation through chemical and targeted genetic approaches also impeded chlamydial development. Knockdown of tmRNA and subsequent complementation with an allele mutated in the SsrA tag closely phenocopied the overexpression of ClpXR230A, thus suggesting that ClpX recognition of SsrA-tagged substrates plays a critical function in secondary differentiation. Taken together, these data provide mechanistic insight into the requirements for transitions between chlamydial developmental forms. IMPORTANCE Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and preventable infectious blindness. This unique organism undergoes developmental transitions between infectious, nondividing forms and noninfectious, dividing forms. Therefore, the chlamydial developmental cycle is an attractive target for Chlamydia-specific antibiotics, which would minimize effects of broad-spectrum antibiotics on the spread of antibiotic resistance in other organisms. However, the lack of knowledge about chlamydial development on a molecular level impedes the identification of specific, druggable targets. This work describes a mechanism through which both the fundamental processes of trans-translation and proteomic turnover by ClpXP contribute to chlamydial differentiation, a critical facet of chlamydial growth and survival. Given the almost universal presence of trans-translation and ClpX in eubacteria, this mechanism may be conserved in developmental cycles of other bacterial species. Additionally, this study expands the fields of trans-translation and Clp proteases by emphasizing the functional diversity of these systems throughout bacterial evolution.

Streptomyces coelicolor is a model organism for studying streptomycetes. This genus possesses relevant medical and economical roles, because it produces many biologically active metabolites of pharmaceutical interest, including the majority of commercialized antibiotics. In this bioinformatic study, the transcriptome of S. coelicolor has been analyzed to identify novel RNA species and quantify the expression of both annotated and novel transcripts in solid and liquid growth medium cultures at different times. The major characteristics disclosed in this study are: (i) the diffuse antisense transcription; (ii) the great abundance of transfer-messenger RNAs (tmRNA); (iii) the abundance of rnpB transcripts, paramount for the RNase-P complex; and (iv) the presence of abundant fragments derived from pre-ribosomal RNA leader sequences of unknown biological function. Overall, this study extends the catalogue of ncRNAs in S. coelicolor and suggests an important role of non-coding transcription in the regulation of biologically active molecule production.

Bacillus strains are widely distributed in terrestrial and marine environments, and some of them are used as biocontrol organisms for their biofilm-formation ability. In Bacillus subtilis, biofilm formation is fine-tuned by a complex network, a clear understanding of which still requires study. In bacteria, tmRNA, encoded by the ssrA gene, catalyzes trans-translation that can rescue ribosomes stalled on mRNA transcripts lacking a functional stop codon. tmRNA also affects physiological bioprocesses in some bacteria. In this study, we constructed a ssrA mutant in B. subtilis and found that the biofilm formation in the ssrA mutant was largely impaired. Moreover, we isolated a biofilm-formation suppressor of ssrA, in which the biofilm formation was restored to a level even stronger than that in the wild type. We further performed RNAseq assays with the wild type, ssrA mutant, and suppressor of ssrA for comparisons of their transcriptomes. By analyzing the transcriptomic data, we predicted the possible functions of some differentially expressed genes (DEGs) in the tmRNA regulation of biofilm formation in B. subtilis. Finally, we found that the overexpression of two DEGs, acoA and yhjR, could restore the biofilm formation in the ssrA mutant, indicating that AcoA and YhjR were immediate regulators involved in the tmRNA regulatory web controlling biofilm formation in B. subtilis. Our data can improve the knowledge about the molecular network involved in Bacillus biofilm formation and provide new targets for manipulation of Bacillus biofilms for future investigation.

Bacteria use trans-translation to rescue stalled ribosomes and target incomplete proteins for proteolysis. Despite similarities between tRNAs and transfer-messenger RNA (tmRNA), the key molecule for trans-translation, new structural and biochemical data show important differences between translation and trans-translation at most steps of the pathways. tmRNA and its binding partner, SmpB, bind in the A site of the ribosome but do not trigger the same movements of nucleotides in the rRNA that are required for codon recognition by tRNA. tmRNA-SmpB moves from the A site to the P site of the ribosome without subunit rotation to generate hybrid states, and moves from the P site to a site outside the ribosome instead of to the E site. During catalysis, transpeptidation to tmRNA appears to require the ribosomal protein bL27, which is dispensable for translation, suggesting that this protein may be conserved in bacteria due to trans-translation. These differences provide insights into the fundamental nature of trans-translation, and provide targets for new antibiotics that may have decrease cross-reactivity with eukaryotic ribosomes.

The trans-translation process is a ribosomal rescue system for stalled ribosomes processing truncated mRNA. The genes ssrA and smpB fulfil the key functions in most bacteria, but some species have either lost these genes or the function of the ribosomal rescue system is taken over by other genes. To date, the ribosomal rescue system has not been analysed in detail for the Acholeplasmataceae. This family, in the Mollicutes class, comprises the genus Acholeplasma and the provisional taxon \"Candidatus Phytoplasma\". Despite their monophyletic origin, the two clades can be separated by traits such as not representing primary pathogens for acholeplasmas versus being phytopathogenic for the majority of phytoplasmas. Both taxa share reduced genomes, but only phytoplasma genomes are characterised by a remarkable level of instability and reduction. Despite the general relevance of the ribosomal rescue system, information is lacking on coding, the genomic context and pseudogenisation of smpB and ssrA and their possible application as a phylogenetic marker. Herein, we provide a comprehensive analysis of the ribosomal rescue system in members of Acholeplasmataceae. The examined Acholeplasmataceae genomes encode a ribosomal rescue system, which depends on tmRNA encoded by ssrA acting in combination with its binding protein SmpB. Conserved gene synteny is evident for smpB, while ssrA shows a less conserved genomic context. Analysis of the tmRNA sequences highlights the variability of proteolysis tag sequences and short conserved sites at the 5\'- and 3\'-ends. Analyses of smpB provided no hints regarding the coding of pseudogenes, but they did suggest its application as a phylogenetic marker of Acholeplasmataceae - in accordance with 16S rDNA topology. Sequence variability of smpB provides sufficient information for species assignment and phylogenetic analysis.

Because of the ever-increasing multidrug resistance in microorganisms, it is crucial that we find and develop new antibiotics, especially molecules with different targets and mechanisms of action than those of the antibiotics in use today. Translation is a fundamental process that uses a large portion of the cell\'s energy, and the ribosome is already the target of more than half of the antibiotics in clinical use. However, this process is highly regulated, and its quality control machinery is actively studied as a possible target for new inhibitors. In bacteria, ribosomal stalling is a frequent event that jeopardizes bacterial wellness, and the most severe form occurs when ribosomes stall at the 3\'-end of mRNA molecules devoid of a stop codon. Trans-translation is the principal and most sophisticated quality control mechanism for solving this problem, which would otherwise result in inefficient or even toxic protein synthesis. It is based on the complex made by tmRNA and SmpB, and because trans-translation is absent in eukaryotes, but necessary for bacterial fitness or survival, it is an exciting and realistic target for new antibiotics. Here, we describe the current and future prospects for developing what we hope will be a novel generation of trans-translation inhibitors.