Characterization of key triacylglycerol biosynthesis processes in rhodococci.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39–3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18–0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon

Métabolisme énergétique et régulation de la biosynthèse d'antibiotiques chez streptomyces

Les Streptomyces sont des bactéries filamenteuses du sol. Ces bactéries présentent un cycle de différenciation morphologique complexe et produisent de très nombreux métabolites secondaires bioactifs, dont la plupart des antibiotiques connus. La biosynthèse de ces métabolites est contrôlée par un réseau de régulation complexe dont l'étude de mutants sur-producteurs ou non-producteurs permet une meilleure compréhension. Un mutant du gène ppk (polyphosphate kinase) de S. lividans présente une production accrue d'antibiotiques, et l'analyse de ce mutant a suggéré qu' une diminution de la concentration en ATP dans la cellule serait à l'origine de cette sur-production. Cette hypothèse a été testée en exprimant des gènes codant une ATPase chez S. lividans, afin de générer un déficit artificiel en ATP dans la cellule. Il a été démontré que l'expression de l'ATPase stimule la production d'un antibiotique en particulier et que les profils de transcription de certains gènes impliqués dans la biosynthèse d'autres métabolites sont aussi modifiés par la diminution de la concentration en ATP dans la cellule. Un gène dont la sur-expression entraine l'inhibition de la différenciation morphologique et de la biosynthèse d'antibiotiques chez S. coelicolor a également été identifié. Il a été démontré que ce gène code un régulateur transcriptionnel de type TetR dont la caractérisation a été initiée. Enfin, une banque de promoteurs synthétiques de forces variables a été construite pour permettre différents niveaux d'expression de gènes chez Streptomyces et a permis de révéler certaines caractéristiques du gène aphII, utilisé comme rapporteur pour l'analyse de ces promoteurs.Streptomyces are soil-dwelling bacteria characterized by both a complex life cycle and the ability to synthesize numerous bio-active compounds such as antibiotics. Secondary metabolism is controlled by a highly intricate regulatory network and analysis of non-producing or over-producing strains can lead to a better understanding of gene regulation among this network. Deletion of the ppk gene (polyphosphate kinase) in S. lividans leads to antibiotic overproduction. Analysis of the ppk mutant suggested that reduced ATP concentrations in the cell could trigger antibiotic biosynthesis. In order to test this hypothesis, genes encoding an ATPase were expressed in S. lividans. By lowering intracellular ATP concentration, ATPase expression stimulated the biosynthesis of a specific antibiotic compound. It was also shown that transcription profiles of some others biosynthetic genes was also modified by ATP concentration. A gene encoding a negative regulator of both antibiotic biosynthesis and morphological differentiation in S. coelicolor was also isolated. It was shown that this regulator belongs to the TetR transcriptional regulator family and its functional characterization was started. Finally, a library of synthetic promoters of various strengths was engineered in order to allow different levels of gene expression in Streptomyces. This work revealed interesting features of aphII gene used as a reporter in this study.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Novel insights regarding the sigmoidal pattern of resistance to neomycin conferred by the aphII gene, in Streptomyces lividans.

International audienceA library of synthetic promoters of various strengths, specifically constructed for Streptomyces species, was cloned in the promoter-probe plasmid pIJ487, upstream of the promoter-less aphII gene that confers resistance to neomycin. The survival rates conferred by promoters were assessed in the presence of 100 mug.ml-1 neomycin. The correlation between the transcriptional activity of the aphII gene (estimated by RT-PCR) and the resistance to neomycin (expressed as survival rate) indicated a sigmoid rather than a linear correlation. In this issue, we propose a tentative explanation for this sigmoidal pattern of resistance in relation with the level of aphII gene expression. Beyond this specific example, our model might constitute a sound explanation for the generally observed but never explained sigmoidal shape of classical inhibition curves obtained in the presence of linearly increasing antibiotic concentrations

Repression of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator ▿ †

The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches

Characterization of the Sporulation Control Protein SsgA by Use of an Efficient Method To Create and Screen Random Mutant Libraries in Streptomycetes

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function

The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters

International audienceStreptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the -35 and -10 consensus promoter sequences were completely randomized and some variability was introduced in the -35 (position 6) and -10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 μgml(-1)). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the -10 box, the -10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters

Characterization of key triacylglycerol biosynthesis processes in rhodococci.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans

International audienceIn most Streptomyces species, antibiotic production is triggered in a condition of phosphate limitation, a condition that is known to be correlated with a low intracellular ATP content compared to growth in a condition of phosphate proficiency. This observation suggests that a low ATP content might be a direct trigger of antibiotic biosynthesis. In order to test this hypothesis, we introduced into the model strain Streptomyces lividans, a functional and a non-functional ATPase cloned into the replicative vector pOSV206 and expressed under the control of the strong ErmE* promoter. The functional ATPase was constituted by the α (AtpA), β (AtpB) and γ (AtpD) sub-units of the native F1 part of the ATP synthase of S. lividans that, when separated from the membrane-bound F0 part, bears an ATPase activity. The non-functional ATPase was a mutated version of the latter, bearing a 12 amino acids deletion encompassing the active site of the AtpD sub-unit. S. lividans was chosen to test our hypothesis since this strain hardly produces any antibiotics. However, it possesses the same biosynthetic pathways of various specialized metabolites as S. coelicolor, a phylogenetically closely related strain that produces these metabolites in abundance. Our results demonstrated that the over-expression of the functional ATPase, but not that of its mutated version, indeed correlated with the production of the bioactive metabolites of the CDA, RED and ACT clusters. These results confirmed the long known and mysterious link existing between a phosphate limitation leading to an ATP deficit and the triggering of antibiotic biosynthesis. Based on this work and the previous published results of our group, we propose an entirely novel conception of the nature of this lin