Listeria monocytogenes is an intracellular, foodborne gastrointestinal pathogen that is primarily responsible for causing listeriosis or food poisoning in otherwise healthy individuals. Infections ...that arise during pregnancy or within immune compromised individuals are much more serious resulting in the risk of fetal termination or fetal fatality postpartum in the former and septicemia or meningitis with a 20% fatality rate in the latter. While the roles of internalin proteins and listeriolysin-O in the infection process are well characterized, the specific roles of lysine-modified phospholipids in the membrane of L. monocytogenes are not. Investigation into the lipid bilayer composition of L. monocytogenes indicated that the overall proportions of lipids, including lysylcardiolipin and lysylphosphatidylglycerol (LysPG), vary with growth temperature and growth phase. In addition, we demonstrate that LysPG formation is essential for L. monocytogenes survival in the presence of increased osmolytic stress but has no effect on bacterial adherence, invasion or survival in the presence of physiologically relevant concentrations of human neutrophil peptide (HNP-1). In the absence of LysPG synthesis, L. monocytogenes unexpectedly retained flagellum-mediated motility at 37 °C. Taken together, these findings show that LysPG formation in L. monocytogenes has broader functions in virulence and survival beyond its known role in the modification of membrane potential previously observed in other bacteria.
Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a ...pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3' and 5'-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5' leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.
Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA ...synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNAPhe. Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNAPhe, an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.
Aminoacylphosphatidylglycerol synthases (aaPGSs) are enzymes that transfer amino acids from aminoacyl-tRNAs (aa-tRNAs) to phosphatidylglycerol (PG) to form aa-PG in the cytoplasmic membrane of ...bacteria. aa-PGs provide bacteria with resistance to a range of antimicrobial compounds and stress conditions. Enterococcus faecium encodes a triple-specific aaPGS (RakPGS) that utilizes arginine, alanine, and lysine as substrates. Here we identify a novel hydrolase (AhyD), encoded immediately adjacent to rakPGS in E. faecium, which is responsible for the hydrolysis of aa-PG. The genetic synteny of aaPGS and ahyD is conserved in >60 different bacterial species. Deletion of ahyD in E. faecium resulted in increased formation of Ala-PG and Lys-PG and increased sensitivity to bacitracin. Our results suggest that AhyD and RakPGS act together to maintain optimal levels of aa-PG in the bacterial membrane to confer resistance to certain antimicrobial compounds and stress conditions.
Background: Aminoacylation of phosphatidylglycerol (PG) alters the charge of the bacterial membrane and confers resistance to antimicrobial peptides.
Results: AhyD catalyzes the hydrolysis of aminoacylated PG in Enterococcus faecium and increases tolerance to bacitracin.
Conclusion: AhyD participates in maintaining aminoacyl-PG homeostasis in the membrane.
Significance: Understanding how bacteria modulate their membranes is crucial to understanding how they adapt to environmental challenges.
Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to ...metabolism and the activation of stress response systems. Mutations that abolish the ability of the pathogen to respond to external cues are typically attenuating. Here we discuss our recent discovery of a novel post-transcriptional regulatory pathway critical for Salmonella virulence and stress resistance. The enzymes PoxA and YjeK coordinately attach a unique beta-amino acid onto a highly conserved lysine residue in the translation factor elongation factor P (EF-P). Strains in which EF-P is unmodified due to the absence of PoxA or YjeK are attenuated for virulence and display highly pleiotropic phenotypes, including hypersusceptibility to a wide range of unrelated antimicrobial compounds. Work from our laboratory and others now suggests that EF-P, previously thought to be essential, instead plays an ancillary role in translation by regulating the synthesis of a relatively limited subset of proteins. Other observations suggest that the eukaryotic homolog of EF-P, eIF5A, may illicit similar changes in the translation machinery during stress adaptation, indicating that the role of these factors in physiology may be broadly conserved.
In some bacteria Lys-tRNA(Lys) is used both in translation and for the specific addition of Lys to phosphatidylglycerol in the cytoplasmic membrane. This reaction is catalyzed by the membrane protein ...MprF, and the lysyl-phosphatidylglycerol formed contributes to the resistance of these bacteria to various cationic antibacterial molecules. Obtaining proteins and reconstituting an in vitro system mimicking membrane conditions is a major challenge to studying the function of membrane proteins, especially when labile substrates such as Lys-tRNA(Lys) are required. Here we report methods to obtain a stable enriched membrane fraction containing MprF, and the techniques necessary to quantitatively monitor its activity in vitro and in vivo.
Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Because the current understanding ...of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we use a variety of known mutations in hmt-tRNAPhe to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-tRNAPhe mutations on aminoacylation, structure, and translation elongation-factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure, and elongation-factor binding. One notable exception was the G34A anticodon mutation of hmt-tRNAPhe (mitochondrial DNA mutation G611A), which is associated with MERRF (myoclonic epilepsy with ragged red fibers). In vitro, the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-tRNAPhe does not undergo adenosine-to-inosine (A-to-I) editing, ruling out miscoding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-tRNAPhe with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene.
Medical personnel represent the largest group of workers occupationally exposed to ionizing radiation. Although the health risks associated with occupational exposure to low doses of ionizing ...radiation in the medical field have been investigated in several national cohorts, no study has been conducted in France to date. The ORICAMs (Occupational Radiation Induced Cancer in Medical staff) cohort is a nationwide French longitudinal cohort of medical workers exposed to ionizing radiation aiming to investigate the risk of radiation-associated cancer and non-cancer mortality. The ORICAMs cohort was set up in 2011 and includes all medical personnel monitored for ionizing radiation exposure with at least one dosimetric record in the SISERI database (the national registry for monitoring ionizing radiation exposure in workers) over the period 2002-2012. Causes of death were abstracted from death certificates and coded according to ICD-10. The follow-up ended on 31/12/2013. Standardized mortality ratios (SMRs) were calculated by cause of death to compare the mortality in the cohort to that in the French population, by gender, age group and calendar period. Among the 164,015 workers included in the cohort (60% women) a total of 1,358 deaths (892 in male and 466 in female) were reported. The observed number of all-cause deaths was significantly lower than expected based on national rates in both male (SMR = 0.35; 95% CI: 0.33, 0.38; ndeaths = 892) and female (SMR = 0.41; 95% CI: 0.38, 0.45; ndeaths = 466). This analysis leads to the conclusion that mortality in French workers exposed to medical radiation is significantly lower than the national reference rates. However, these results based on a comparative analysis with national rates may be impacted by the healthy worker effect towards low SMRs, and do not enable to establish a potential relationship between occupational exposure and mortality risk, even if we may suspect an impact of high SES of these professionals on the observed decreased mortality. Thus, further dose-response analyses based on individual ionizing radiation exposure and job's type will be conducted to characterize correlation between risk of cancer mortality and occupational exposure.
Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all ...archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsnsynthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.
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