Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3′-phosphoadenosine 5′-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans

Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis.

International audienceEscherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn(-) strain. A triple mutant (nrnA(-), nrnB(-), yhaM(-)) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5'-to-3' exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis

3′-5′ Phosphoadenosine phosphate is an inhibitor of PARP-1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

pAp (3′-5′ phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies

An Efficient Vector System to Modify Cells Genetically

The transfer of foreign genes into mammalian cells has been essential for understanding the functions of genes and mechanisms of genetic diseases, for the production of coding proteins and for gene therapy applications. Currently, the identification and selection of cells that have received transferred genetic material can be accomplished by methods, including drug selection, reporter enzyme detection and GFP imaging. These methods may confer antibiotic resistance, or be disruptive, or require special equipment. In this study, we labeled genetically modified cells with a cell surface biotinylation tag by co-transfecting cells with BirA, a biotin ligase. The modified cells can be quickly isolated for downstream applications using a simple streptavidin bead method. This system can also be used to screen cells expressing two sets of genes from separate vectors

From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later

Comparative genomics is the cornerstone of identification of gene functions. The immense number of living organisms precludes experimental identification of functions except in a handful of model organisms. The bacterial domain is split into large branches, among which the Firmicutes occupy a considerable space. Bacillus subtilis has been the model of Firmicutes for decades and its genome has been a reference for more than 10 years. Sequencing the genome involved more than 30 laboratories, with different expertises, in a attempt to make the most of the experimental information that could be associated with the sequence. This had the expected drawback that the sequencing expertise was quite varied among the groups involved, especially at a time when sequencing genomes was extremely hard work. The recent development of very efficient, fast and accurate sequencing techniques, in parallel with the development of high-level annotation platforms, motivated the present resequencing work. The updated sequence has been reannotated in agreement with the UniProt protein knowledge base, keeping in perspective the split between the paleome (genes necessary for sustaining and perpetuating life) and the cenome (genes required for occupation of a niche, suggesting here that B. subtilis is an epiphyte). This should permit investigators to make reliable inferences to prepare validation experiments in a variety of domains of bacterial growth and development as well as build up accurate phylogenies

Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens