In Bacillus subtilis W23, the duet sigmaXsigmaM, two sigma factors of the extracytoplasmic function subfamily, are required for septum and wall synthesis under batch culture conditions.

The synthesis of poly(RboP), the main Bacillus subtilis W23 teichoic acid, is encoded by tarDF-tarABIJKL operons, the latter being controlled by two promoters designated PtarA-int and PtarA-ext. Analysis by lacZ fusions reveals that PtarA-int activity exhibits sharp increases at the beginning and end of the transition between exponential and stationary growth phase. As confirmed by mRNA quantification, these increases are mediated by ECF sigma factors sigmaX and sigmaM respectively. In liquid media, strain W23 sigX sigM double mutants experience serious difficulties in the transition and stationary growth phases. Inactivation of sigmaX- and sigmaM-controlled regulons, which precludes transcription from PtarA-int, leads to (i) delays in chromosome segregation and septation and (ii) a transient loss of up to 30% of the culture OD or lysis. However, specific inactivation of PtarA-int, leading mainly to a shortage of poly(RboP), does not affect growth while, nevertheless, interfering with normal septation, as revealed by electron microscopy. The different sigM transcription in strains W23 and 168 is discussed. In W23, expression of tarA and sigM, which is shown to control divIC, is inversely correlated with growth rate, suggesting that the sigM regulon is involved in the control of cell division.

SCAP is required for timely and proper myelin membrane synthesis.

Myelination requires a massive increase in glial cell membrane synthesis. Here, we demonstrate that the acute phase of myelin lipid synthesis is regulated by sterol regulatory element-binding protein (SREBP) cleavage activation protein (SCAP), an activator of SREBPs. Deletion of SCAP in Schwann cells led to a loss of SREBP-mediated gene expression involving cholesterol and fatty acid synthesis. Schwann cell SCAP mutant mice show congenital hypomyelination and abnormal gait. Interestingly, aging SCAP mutant mice showed partial regain of function; they exhibited improved gait and produced small amounts of myelin indicating a slow SCAP-independent uptake of external lipids. Accordingly, extracellular lipoproteins partially rescued myelination by SCAP mutant Schwann cells. However, SCAP mutant myelin never reached normal thickness and had biophysical abnormalities concordant with abnormal lipid composition. These data demonstrate that SCAP-mediated regulation of glial lipogenesis is key to the proper synthesis of myelin membrane, and provide insight into abnormal Schwann cell function under conditions affecting lipid metabolism.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization.

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor.

The HER-2/ErbB-2 oncoprotein is overexpressed in human breast and ovarian adenocarcinomas and is clearly associated with the malignant phenotype. Although no specific ligand for this receptor has been positively identified, ErbB-2 was shown to play a central role in a network of interactions with the related ErbB-1, ErbB-3 and ErbB-4 receptors. We have selected new peptides binding to ErbB-2 extracellular domain protein (ECD) by screening 2 newly developed constrained and unconstrained random hexapeptide phage libraries. Out of 37 phage clones, which bound specifically to ErbB-2 ECD, we found 6 constrained and 10 linear different hexapeptide sequences. Among the latter, 5 consensus motifs, all with a common methionine and a positively charged residue at positions 1 and 3, respectively, were identified. Furthermore, 3 representative hexapeptides were fused to a coiled-coil pentameric recombinant protein to form the so-called peptabodies recently developed in our laboratory. The 3 peptabodies bound specifically to the ErbB-2 ECD, as determined by enzyme-linked immunosorbent assay and BIAcore analysis and to tumor cells overexpressing ErbB-2, as shown by flow cytometry. Interestingly, one of the free selected linear peptides and all 3 peptabodies inhibited the proliferation of tumor cells overexpressing ErbB-2. In conclusion, a novel type of ErbB-2-specific ligand is described that might complement presently available monoclonal antibodies.

Divinyl ether fatty acid synthesis in late blight-diseased potato leaves.

We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems.

Synthesis and transport of creatine in the CNS: importance for cerebral functions.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06935.x Abstract Apart of its well known function of 'energetic buffer' through the creatine/phosphocreatine/creatine kinase system allowing the regeneration of ATP, creatine has been recently suggested as a potential neuromodulator of even true neurotransmitter. Moreover, the recent discovery of primary creatine deficiency syndromes, due to deficiencies in l-arginine : glycine amidinotransferase or guanidinoacetate methyltransferase (the two enzymes allowing creatine synthesis) or in the creatine transporter, has shed new light on creatine synthesis, metabolism and transport, in particular in CNS which appears as the main tissue affected by these creatine deficiencies. Recent data suggest that creatine can cross blood-brain barrier but only with a poor efficiency, and that the brain must ensure parts of its needs in creatine by its own endogenous synthesis. Finally, the recent years have demonstrated the interest to use creatine as a neuroprotective agent in a growing number of neurodegenerative diseases, including Parkinson's and Huntington's diseases. This article aims at reviewing the latest data on creatine metabolism and transport in the brain, in relation to creatine deficiencies and to the potential use of creatine as neuroprotective molecule. Emphasis is also given to the importance of creatine for cerebral function.

Convenient synthesis of heterobifunctional poly(ethylene glycol) suitable for the functionalization of iron oxide nanoparticles for biomedical applications.

A straightforward route is proposed for the multi-gram scale synthesis of heterobifunctional poly(ethylene glycol) (PEG) oligomers containing combination of triethyloxysilane extremity for surface modification of metal oxides and amino or azido active end groups for further functionalization. The suitability of these PEG derivatives to be conjugated to nanomaterials was shown by pegylation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), followed by functionalization with small peptide ligands for biomedical applications.

Methicillin-resistant Staphylococcus aureus resensitization by protein synthesis inhibitors : proposal for a mechanism

RESUME Staphylococcus aureus est un important pathogène à gram-positif, à la fois responsable d'infections nosocomiales et communautaires. Le S. aureus résistant à la méthicilline est intrinsèquement résistant aux bêta-lactamines, inhibiteurs de la synthèse de la paroi bactérienne, grâce à une enzyme nouvellement acquise, la protéine liant la pénicilline 2A, caractérisée par une faible affinité pour ces agents et pouvant poursuivre la synthèse de la paroi, alors que les autres enzymes sont bloquées. Ce micro-organisme a également développé des résistances contre quasiment tous les antibiotiques couramment utilisés en clinique. Parallèlement au développement de molécules entièrement nouvelles, il peut être utile d'explorer d'éventuelles caractéristiques inattendues de médicaments déjà existants, par exemple en les combinant, dans l'espoir d'un potentiel effet synergique. Comprendre les mécanismes de tels effets synergiques pourrait contribuer à la justification de leur utilisation clinique potentielle. Récemment, un effet synergique contre le S. aureus résistant à la méthicilline a été décrit entre la streptogramine quinupristine-datfopristine et les bêta-lactamines, aussi bien in vitro qu'in vivo. Le présent travail a pour but de proposer un modèle pour le mécanisme de cette interaction positive et de l'étendre à d'autres classes d'antibiotiques. Premièrement, un certain nombre de méthodes microbiologiques ont permis de mieux cerner la nature de cette interaction, en montrant qu'elle agissait spécifiquement sur le S. aureus résistant à la méthicilline et qu'elle était restreinte à l'association entre inhibiteurs de la synthèse des protéines et bêta-lactamines. Deuxièmement, L'observation de l'influence des inhibiteurs de la synthèse des protéines sur la machinerie de la paroi bactérienne, c'est-à-dire sur l'expression des protéines liant la pénicilline, responsables de la synthèse du peptidoglycan, a montré une diminution de la quantité de ta protéine liant la pénicilline 2, connue pour posséder une activité de transglycosylation, indispensable au bon fonctionnement de la protéine liant la pénicilline 2A, responsable de la résistance à la méthicilline. Troisièmement, l'analyse fine de la composition du peptidoglycan extrait de bactéries, avant ou après traitement par des inhibiteurs de la synthèse des protéines, a montré des altérations corrélant avec leur capacité à agir en synergie avec les bêta-lactamines contre S. aureus résistant à ta méthicilline. Ces altérations dans les muropeptides pourraient représenter une signature de la diminution de la quantité de la protéine liant la pénicilline 2. Le modèle mécanistique retenu considère que les inhibiteurs de la synthèse des protéines pourraient diminuer l'expression de la protéine Liant la pénicilline 2, indispensable à la résistance à la méthiciltine, et que ce déséquilibre dans les enzymes synthétisant la paroi bactérienne pourrait générer une signature dans les muropeptides. SUMMARY Staphylococcus aureus is a major gram-positive pathogen causing both hospital-acquired and community-acquired infections. Methicillin- resistant Staphylococcus aureus is intrinsically resistant to the cell wall inhibitors beta-lactams by virtue of a newly acquired cell-wall-building enzyme, tow-affinity penicillin-binding protein 2A, which can build the wall when other penicillin-binding proteins are blocked. Moreover, the microorganism has developed resistance to virtually all non-experimental antibiotics. In addition of producing entirely new molecules, it is useful to explore unexpected features of existing drugs, for example by using them in combination, expecting drug synergisms. Understanding the mechanisms of such synergisms would help justify their putative clinical utilization. Recently, a synergism between the streptogramin quinupristin-dalfopristin and beta-lactams was reported against methicillin-resistant S. aureus, both in vitro and in vivo. The present work intends to propose a model for the mechanism of this positive interaction and to extend it to other drug classes. First, microbiological experimentation helped better defining the nature of this interaction, restricting it to methicillin-resistant S. aureus, and to the association of protein synthesis inhibitors with beta-lactams. Second, the observation of inhibitors of protein synthesis influence on the cell-wall-building machinery, i.e. on the expression of penicillin-binding proteins responsible for peptidoglycan synthesis, showed a decrease in the amount of penicillin-binding protein 2, known to provide a transglycosylase activity for glycan chain elongation, indispensable for the functionality of the low-affinity penicillin-binding protein 2A responsible for methicillin resistance. Third, the fine analysis of the peptidoglycan composition purified from bacteria before or after treatment with inhibitors of protein synthesis showed alterations that correlated with their ability to synergize with beta-lactams against methicillin-resistant S. aureus. These muropeptide alterations could be the signature of decrease in the amount of penicillin-binding protein 2. The retained mechanistic model is that inhibitors of protein synthesis could decrease the expression of penicillin-binding protein 2, wich is indispensable for methicillin-resistance, and that this imbalance in cell-wall-building enzymes could generate a muropeptide signature.

Saccharomyces cerevisiae, a tool to study the β-oxidation through the synthesis of polyhydroxyalkanoates

Summary Polyhydroxyalkanoates (PHAs) represent a family of polyesters naturally synthesized by a wide variety of bacteria. Through their thermoplastic and elastomeric qualities, together with their biodegradable and renewable properties, they are predicted to be a good alternative to the petroleum- derived plastics. Nevertheless, as PHA production costs using bacteria fermentation are still too high, PHA synthesis within eukaryotic systems, such as plants, has been elaborated. Although the costs were then efficiently lowered, the yield of PHAs produced remained low. In this study, Saccharomyces cerevisae has been used as another eukaryotic model in order to reveal the steps which limit PHA production. These cells express the PHA synthase of Pseudomonas aeruginosa and the PHAs obtained were analyzed to understand the flux of fatty acids towards and through the peroxisomal β-oxidation core cycle, generating the main substrate of the PHA synthase. When S. cerevisiae wild-type cells are grown in a media containing glucose as carbon source as well as fatty acids, the PHA monomer composition is largely influenced by the nature of the external fatty acid used. Thus, even-chain PHA monomers are generated from oleic acid (18:1Δ9cis) and odd- chain PHA monomers are generated from heptadecenoic acid (17:1Δ. 10 cis). Moreover, PHA synthesis is dependent on the first two enzymes of the 0-oxidation core cycle, the acyl-CoA oxidase and the multifunctional enzyme enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA dehydrogenase. S. cerevisiae mutant cells growing on oleic or heptadecenoic acid and deficient in either the R-3- hydroxyacyl-CoA dehydrogenase or in the 3-ketothiolase activity, the last β-oxidation cycle steps, surprisingly contained PHAs of predominantly even-chain monomers. This is also noticed in wild- type and mutants grown on glucose or raffinose, indicating that the substrate used for PHA synthesis is generated from the degradation of intracellular short- and medium-chain fatty acids by the 3- oxidation cycle. Inhibition of fatty acid biosynthesis by cerulenin blocks the synthesis of PHAs from intracellular fatty acids but still enables the use of extracellular fatty acids for polymer production. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed towards the peroxisomal β-oxidation pathway. In this thesis, no increase of the yield of PHA produced could be obtained. But the PHA synthesis confirmed the carbon flux into and through the β-oxidation core cycle and unveiled the existence of novel mechanisms. It is thus a good tool to study in vivo the flux of carbons in S. cerevisiae cells. Résumé Les polyhydroxyalkanoates (PHAs) sont une famille de polyesters naturellement synthétisés par un grand nombre de bactéries. Ayant des propriétés de thermoplastiques, d'élastomères et étant des ressources biodégradables et renouvelables, les PHAs représentent une bonne alternative aux plastiques dérivés du pétrole. Pour pallier aux coûts considérables de la production de PHAs par fermentation bactérienne, la synthèse de PHAs par des systèmes eucaryotes telles les plantes a été élaborée. Les coûts ont ainsi efficacement été diminués, mais le rendement de PHAs produits reste faible. Dans cette étude, Saccharomyces cerevisiae a été utilisé comme autre modèle eucaryote pour révéler les étapes limitantes de la production de PHAs. Les PHAs obtenus dans les cellules exprimant la F'HA synthase de Pseudomonas aeruginosa ont été analysés afin de comprendre le flux d'acides gras vers et à travers le cycle péroxisomal de la β-oxidation, principal producteur du substrat de la PHA synthase. Lorsque la souche S. cerevisiae de type sauvage se développe dans un milieu contenant du glucose et des acides gras, la composition des monomères de PHAs est influencée par la nature des acides gras extracellulaires. Ainsi, les monomères pairs sont générés par l'acide oléique (18:1Δ9cis), tandis que les impairs le sont par l'acide heptadécénoïque (17:1Δ10cis). La synthèse de PHAs est dépendante des deux premières enzymes de la β-oxidation; l'acyl-CoA oxidase et l'enzyme multifonctionnelle enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA déshydrogénase. Les souches mutantes ne possédant pas les activités de la R-3-hydroxyacyl-CoA déshydrogénase ou de la 3- ketothiolase contiennent, en présence d'acide oléique ou heptadécénoïque, des PHAs composés essentiellement de monomères pairs. Cela a également été observé en présence de glucose ou de raffinose uniquement. Le substrat utilisé pour la synthèse de PHAs a ainsi été généré par la dégradation d'acides gras intracellulaires à chaîne courte et moyenne via le cycle de la β-oxidation. L'inhibition de la synthèse d'acides gras par la cérulénine a bloqué la synthèse de PHAs par les acides gras internes. Ces résultats ont révélés l'existence d'un cycle futile par lequel des intermédiaires à chaîne courte et moyenne de la synthèse cytoplasmique d'acides gras sont dirigés vers le cycle péroxisomal de la β-oxidation. Dans cette étude, le rendement de PHAs produits reste inchangé, mais l'analyse des PHAs permet de confirmer le flux de carbones vers et à travers le cycle péroxisomal de la β-oxidation et l'existence de nouveaux méchanismes a été dévoilée. Cette synthèse s'avère être un bon outil pour étudier in vivo le flux de carbones dans les cellules de S. cerevisiae.

Genetic dysregulation of glutathione synthesis predicts alteration of plasma thiol redox status in schizophrenia.

Abstract Genetic studies have shown an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL), the key enzyme for glutathione (GSH) synthesis. The present study was aimed at analyzing the influence of a GSH dysregulation of genetic origin on plasma thiols (total cysteine, homocysteine, and cysteine-glycine) and other free amino acid levels as well as fibroblast cultures GSH levels. Plasma thiols levels were also compared between patients and controls. As compared with patients with a low-risk GCLC GAG TNR genotype, patients with a high-risk genotype, having an impaired GSH synthesis, displayed a decrease of fibroblast GSH and plasma total cysteine levels, and an increase of the oxidized form of cysteine (cystine) content. Increased levels of plasma free serine, glutamine, citrulline, and arginine were also observed in the high-risk genotype. Taken together, the high-risk genotypes were associated with a subgroup of schizophrenia characterized by altered plasma thiols and free amino acid levels that reflect a dysregulation of redox control and an increased susceptibility to oxidative stress. This altered pattern potentially contributes to the development of a biomarker profile useful for early diagnosis and monitoring the effectiveness of novel drugs targeting redox dysregulation in schizophrenia. Antioxid. Redox Signal. 15, 2003-2010.

Reactivation of transcription from a vaccinia virus early promoter late in infection.

We have studied the kinetics of RNA synthesis from the vaccinia virus 7,500-molecular-weight gene (7.5K gene) which is regulated by early and late promoters arranged in tandem. Unexpectedly, after a first burst of RNA synthesis early in infection, transcription was reactivated late in infection. Reactivation was not dependent on the location of the promoter in the genome or on the presence of the upstream late regulatory sequences. The mRNA synthesized from the reactivated promoter in the late phase had the same 5' and 3' ends as the molecules transcribed in the early phase. Interestingly, these molecules were efficiently translated despite the absence of the poly(A) leader characteristic of late mRNAs. Reactivation appears to be dependent on virus assembly since it is prevented by rifampin, a specific inhibitor of morphogenesis. Finally, analysis of various other early genes showed that reactivation is not unique to the 7.5K early promoter.

Use of an isothermal microcalorimetry assay to characterize microbial oxalotrophic activity

Isothermal microcalorimetry (IMC) has been used in the past to monitor metabolic activities in living systems. A few studies have used it on ecological research. In this study, IMC was used to monitor oxalotrophic activity, a widespread bacterial metabolism found in the environment, and particularly in soils. Six model strains were inoculated in solid angle media with K-oxalate as the sole carbon source. Cupriavidus oxalaticus, Cupriavidus necator, and Streptomyces violaceoruber presented the highest activity (91, 40, and 55 μW, respectively) and a maximum growth rate (μmax h(-1) ) of 0.264, 0.185, and 0.199, respectively, among the strains tested. These three strains were selected to test the incidence of different oxalate sources (Ca, Cu, and Fe-oxalate salts) in the metabolic activity. The highest activity was obtained in Ca-oxalate for C. oxalaticus. Similar experiments were carried out with a model soil to test whether this approach can be used to measure oxalotrophic activity in field samples. Although measuring oxalotrophic activity in a soil was challenging, there was a clear effect of the amendment with oxalate on the metabolic activity measured in soil. The correlation between heat flow and growth suggests that IMC analysis is a powerful method to monitor bacterial oxalotrophic activity