Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report the first evidence of a ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA in a ΔybeY mutant at 45°C, making YbeY the first endoribonuclease to be implicated in the critically important processing of the 16S rRNA 3' terminus. These key roles in ribosome quality control and maturation indicate why YbeY is a member of the minimal bacterial gene set and suggest that it could be a potential target for antibacterial drugs.

SummaryThe UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5Ј-and 3Ј-ends of 16S rRNA as well as maturation of the 5Ј-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery.

The short-chain dehydrogenase/reductase (SDR) family is one of the largest and most ubiquitous protein families in bacterial genomes. Despite there being a few well-characterized examples, the substrate specificities or functions of most members of the family are unknown. In this study, we carried out a large-scale mutagenesis of the SDR gene family in the alfalfa root nodule symbiont Sinorhizobium meliloti. Subsequent phenotypic analysis revealed phenotypes for mutants of 21 of the SDR-encoding genes. This brings the total number of S. meliloti SDR-encoding genes with known function or associated phenotype to 25. Several of the mutants were deficient in the utilization of specific carbon sources, while others exhibited symbiotic deficiencies on alfalfa (Medicago sativa), ranging from partial ineffectiveness to complete inability to form root nodules. Five of the mutants had both symbiotic and carbon utilization phenotypes. These results clearly demonstrate the importance of the SDR family in both symbiosis and saprotrophy, and reinforce the complex nature of the interaction of S. meliloti with its plant hosts. Further analysis of the genes identified in this study will contribute to the overall understanding of the biology and metabolism of S. meliloti in relation to its interaction with alfalfa.

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.

We reported previously that murine L-929 cells expressing a human interferon (IFN)-gamma cDNA lacking a signal peptide sequence synthesize but fail to secrete human IFN-gamma and support viral replication at a reduced level. These cells also had elevated levels of IFN-inducible gene products. We show here that a similar response is seen in human cells expressing a mutated murine IFN-gamma cDNA. The ability of human IFN-gamma to induce gene expression in murine cells is shown to be due to the intracellular IFN-gamma rather than to clonal variation, induction of endogenous murine IFN, or alternative mediators of antiviral activity. We have used a murine cell line, Ltk-aprt-, which is resistant to both type I and II IFNs but responsive to combined treatment with both. Ltk-aprt- cells transfected with human IFN-gamma cDNA lacking a signal sequence support virus replication at the same level as control cells. However, unlike transfectants containing only the neoR selection gene, clones expressing the mutated human IFN-gamma gene show strong protection against viral infection and elevated levels of 2,5 A synthetase mRNA and MHC class I protein after treatment with IFN-beta alone. Reverse transcriptase-PCR rules out the induction of endogenous murine IFN expression as a mediator of these effects. Thus, expression of intracellular human IFN-gamma mimics treatment with extracellular murine IFN-gamma in permitting a synergistic response to IFN-beta. Given the inability of human IFN-gamma to bind to the murine cell-surface receptor our results show that intracellular IFN-gamma can activate certain responses independent of cell-surface binding.

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Abstract-Thymine is the one and only base transcribed into uracil during production of proteins. Thymine in DNA and uracil in mRNA plays a major role in producing proteins with appropriate carbon content for stability and activity. Thymine distribution is different frames of coding nucleic acids are investigated statistically. The results confirm that frame 1 supposed to have definite thymine content. Frame 3 prefers to have least thymine content. Frames 4 & 5 maintain some degree of thymine while 2 & 6 have a variable fraction of thymine.