Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae.

Bacillus thuringiensis-based products are key in the biopesticides market. Bacillus thuringiensis kurstaki strains Lip and BLB1 were isolated from Lebanese and Tunisian soils, respectively. These strains are highly toxic against lepidopteran larvae, Ephestia kuehniella. Here, we report Lip and BLB1 complete genomes, including their plasmid and toxin contents.

Multiomics Study of Bacterial Growth Arrest in a Synthetic Biology Application.

We investigated the scalability of a previously developed growth switch based on external control of RNA polymerase expression. Our results indicate that, in liter-scale bioreactors operating in fed-batch mode, growth-arrested Escherichia coli cells are able to convert glucose to glycerol at an increased yield. A multiomics quantification of the physiology of the cells shows that, apart from acetate production, few metabolic side effects occur. However, a number of specific responses to growth slow-down and growth arrest are launched at the transcriptional level. These notably include the downregulation of genes involved in growth-associated processes, such as amino acid and nucleotide metabolism and translation. Interestingly, the transcriptional responses are buffered at the proteome level, probably due to the strong decrease of the total mRNA concentration after the diminution of transcriptional activity and the absence of growth dilution of proteins. Growth arrest thus reduces the opportunities for dynamically adjusting the proteome composition, which poses constraints on the design of biotechnological production processes but may also avoid the initiation of deleterious stress responses.

The dual role of amyloid-ß-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae.

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein.

Polyunsaturated fatty acid metabolites: biosynthesis in Leishmania and role in parasite/host interaction.

Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.

One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.

RETRACTED:A new function for the yeast trehalose-6P synthase (Tps1) protein, as key pro-survival factor during growth, chronological ageing, and apoptotic stress.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of Marie-Ange Teste, Isabelle Léger-Silvestre, Jean M François and Jean-Luc Parrou. Marjorie Petitjean could not be reached. The corresponding author identified major issues and brought them to the attention of the Journal. These issues span from significant errors in the Material and Methods section of the article and major flaws in cytometry data analysis to data fabrication on the part of one of the authors. Given these errors, the retracting authors state that the only responsible course of action would be to retract the article, to respect scientific integrity and maintain the standards and rigor of literature from the retracting authors' group as well as the Journal. The retracting authors sincerely apologize to the readers and editors.

Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress.

UNLABELLED: A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells. IMPORTANCE: Ethanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties.

Yeast Tolerance to Various Stresses Relies on the Trehalose-6P Synthase (Tps1) Protein, Not on Trehalose.

Trehalose is a stable disaccharide commonly found in nature, from bacteria to fungi and plants. For the model yeast Saccharomyces cerevisiae, claims that trehalose is a stress protectant were based indirectly either on correlation between accumulation of trehalose and high resistance to various stresses or on stress hypersensitivity of mutants deleted for TPS1, which encodes the first enzyme in trehalose biosynthetic pathway. Our goal was to investigate more directly which one, between trehalose and/or the Tps1 protein, may serve yeast cells to withstand exposure to stress. By employing an original strategy that combined the use of mutant strains expressing catalytically inactive variants of Tps1, with MAL(+) yeast strains able to accumulate trehalose from an exogenous supply, we bring for the first time unbiased proof that trehalose does not protect yeast cells from dying and that the stress-protecting role of trehalose in this eukaryotic model was largely overestimated. Conversely, we identified the Tps1 protein as a key player for yeast survival in response to temperature, oxidative, and desiccation stress. We also showed by robust RT-quantitative PCR and genetic interaction analysis that the role of Tps1 in thermotolerance is not dependent upon Hsf1-dependent transcription activity. Finally, our results revealed that the Tps1 protein is essential to maintain ATP levels during heat shock. Altogether, these findings supported the idea that Tps1 is endowed with a regulatory function in energy homeostasis, which is essential to withstand adverse conditions and maintain cellular integrity.

Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6) disaccharides isomaltose and palatinose, with Michaëlis-Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6) linkage, but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties.

Characterization of a new multigene family encoding isomaltases in the yeast Saccharomyces cerevisiae, the IMA family.

It has been known for a long time that the yeast Saccharomyces cerevisiae can assimilate alpha-methylglucopyranoside and isomaltose. We here report the identification of 5 genes (YGR287c, YIL172c, YJL216c, YJL221c and YOL157c), which, similar to the SUCx, MALx, or HXTx multigene families, are located in the subtelomeric regions of different chromosomes. They share high nucleotide sequence identities between themselves (66-100%) and with the MALx2 genes (63-74%). Comparison of their amino acid sequences underlined a substitution of threonine by valine in region II, one of the four highly conserved regions of the alpha-glucosidase family. This change was previously shown to be sufficient to discriminate alpha-1,4- to alpha-1,6-glucosidase activity in YGR287c (Yamamoto, K., Nakayama, A., Yamamoto, Y., and Tabata, S. (2004) Eur. J. Biochem. 271, 3414-3420). We showed that each of these five genes encodes a protein with alpha-glucosidase activity on isomaltose, and we therefore renamed these genes IMA1 to IMA5 for IsoMAltase. Our results also illustrated that sequence polymorphisms among this family led to interesting variability of gene expression patterns and of catalytic efficiencies on different substrates, which altogether should account for the absence of functional redundancy for growth on isomaltose. Indeed, deletion studies revealed that IMA1/YGR287c encodes the major isomaltase and that growth on isomaltose required the presence of AGT1, which encodes an alpha-glucoside transporter. Expressions of IMA1 and IMA5/YJL216c were strongly induced by maltose, isomaltose, and alpha-methylglucopyranoside, in accordance with their regulation by the Malx3p-transcription system. The physiological relevance of this IMAx multigene family in S. cerevisiae is discussed.

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.

BACKGROUND: Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. RESULTS: From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. CONCLUSION: In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.

Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose.

It is well known that glycogen and trehalose accumulate in yeast under nutrient starvation or entering into the stationary phase of growth, and that high levels of trehalose are found in heat-shocked cells. However, effects of various types of stress on trehalose, and especially on glycogen, are poorly documented. Taking into account that almost all genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stress-responsive element (STRE), an investigation was made of the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. Using transcriptional fusions, it was found that all these genes were induced in a similar fashion, although to various extents, by temperature, osmotic and oxidative stresses. Experiments performed with an msn2/msn4 double mutant proved that the transcriptional induction of the genes encoding glycogen synthase (GSY2) and trehalose-6-phosphate synthase (TPS1) was needed for the small increase in glycogen and trehalose upon exposure to a mild heat stress and salt shock. However, the extent of transcriptional activation of these genes upon stresses in wild-type strains was not correlated with a proportional rise in either glycogen or trehalose. The major explanation for this lack of correlation comes from the fact that genes encoding the enzymes of the biosynthetic and of the biodegradative pathways were almost equally induced. Hence, trehalose and glycogen accumulated to much higher levels in cells lacking neutral trehalose or glycogen phosphorylase exposed to stress conditions, which suggested that one of the major effects of stress in yeast is to induce a wasteful expenditure of energy by increasing the recycling of these molecules. We also found that transcriptional induction of STRE-controlled genes was abolished at temperatures above 40 degree C, while induction was still observed for a heat-shock-element regulated gene. Remarkably, trehalose accumulated to very high levels under this condition. This can be explained by a stimulation of trehalose synthase and inhibition of trehalose by high temperature.