Genome Analysis of a Variant of Streptomyces coelicolor M145 with High Lipid Content and Poor Ability to Synthetize Antibiotics.

Streptomyces coelicolor M145 is a model strain extensively studied to elucidate the regulation of antibiotic biosynthesis in Streptomyces species. This strain abundantly produces the blue polyketide antibiotic, actinorhodin (ACT), and has a low lipid content. In a process designed to delete the gene encoding the isocitrate lyase (sco0982) of the glyoxylate cycle, an unexpected variant of S. coelicolor was obtained besides bona fide sco0982 deletion mutants. This variant produces 7- to 15-fold less ACT and has a 3-fold higher triacylglycerol and phosphatidylethanolamine content than the original strain. The genome of this variant was sequenced and revealed that 704 genes were deleted (9% of total number of genes) through deletions of various sizes accompanied by the massive loss of mobile genetic elements. Some deletions include genes whose absence could be related to the high total lipid content of this variant such as those encoding enzymes of the TCA and glyoxylate cycles, enzymes involved in nitrogen assimilation as well as enzymes belonging to some polyketide and possibly trehalose biosynthetic pathways. The characteristics of this deleted variant of S. coelicolor are consistent with the existence of the previously reported negative correlation existing between lipid content and antibiotic production in Streptomyces species.

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

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

In 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), ß (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 link.

Bullous Dermatoses and Depression: A Systematic Review.

IMPORTANCE: There is a lack of evidence synthesis on the association between bullous skin disease and depression. OBJECTIVE: To synthesize and interpret the current evidence on the association between bullous skin disease and depression. EVIDENCE REVIEW: This review was conducted according to PRISMA guidelines and reviewed literature related to bullous skin disease and depression in the PubMed, Embase, PsycInfo, and Cochrane databases published between 1945 and February 2021. The quality of each included article was assessed via the Newcastle-Ottawa Scale. This review was registered with PROSPERO (CRD42021230750). FINDINGS: A total of 17 articles were identified that analyzed a total of 83 910 patients (55.2% female; specifically, 6951 patients with bullous pemphigoid, 1669 patients with pemphigus, and 79 patients with epidermolysis bullosa were analyzed). The prevalence of depressive symptoms among patients with bullous dermatoses ranged from 40% to 80%. The prevalence of depression diagnosis among patients with bullous dermatoses ranged from 11.4% to 28%. CONCLUSIONS AND RELEVANCE: In this systematic review, high rates of depression and depressive symptoms existed among patients with bullous skin disease. Adequate treatment of bullous dermatoses may be associated with a decrease in mental health burden on patients.

Impact of Phosphate Availability on Membrane Lipid Content of the Model Strains, Streptomyces lividans and Streptomyces coelicolor.

In this issue we demonstrated that the phospholipid content of Streptomyces lividans varies greatly with Pi availability being was much lower in Pi limitation than in Pi proficiency whereas that of Streptomyces coelicolor varied little with Pi availability. In contrast the content in phosphate free ornithine lipids was enhanced in both strains in condition of phosphate limitation. Ornithine lipids biosynthesis starts with the N-acylation of ornithine to form lyso-ornithine that is then O-acylated to yield ornithine lipid. The operon sco1222-23 was proposed to be involved in the conversion of specific amino acids into ornithine in condition of phosphate limitation whereas the sco0921-20 operon encoding N- and O-acyltransferase, respectively, was shown to be involved in the biosynthesis of these lipids. The expression of these two operons was shown to be under the positive control of the two components system PhoR/PhoP and thus induced in phosphate limitation. The expression of phoR/phoP being weak in S. coelicolor, the poor expression of these operons resulted into a fivefold lower ornithine lipids content in this strain compared to S. lividans. In the deletion mutant of the sco0921-20 operon of S. lividans, lyso-ornithine and ornithine lipids were barely detectable and TAG content was enhanced. The complementation of this mutant by the sco0921-20 operon or by sco0920 alone restored ornithine lipids and TAG content to wild type level and was correlated with a twofold increase in the cardiolipin content. This suggested that SCO0920 bears, besides its broad O-acyltransferase activity, an N-acyltransferase activity and this was confirmed by the detection of lyso-ornithine in this strain. In contrast, the complementation of the mutant by sco0921 alone had no impact on ornithine lipids, TAG nor cardiolipin content but was correlated with a high lyso-ornithine content. This confirmed that SCO0921 is a strict N-acyltransferase. However, interestingly, the over-expression of the sco0921-20 operon or of sco0921 alone in S. coelicolor, led to an almost total disappearance of phosphatidylinositol that was correlated with an enhanced DAG and TAG content. This suggested that SCO0921 also acts as a phospholipase C, degrading phosphatidylinositol to indirectly supply of phosphate in condition of phosphate limitation.

The Inhibition of Antibiotic Production in Streptomyces coelicolor Over-Expressing the TetR Regulator SCO3201 IS Correlated With Changes in the Lipidome of the Strain.

In condition of over-expression, SCO3201, a regulator of the TetR family was previously shown to strongly inhibit antibiotic production and morphological differentiation in Streptomyces coelicolor M145. In order to elucidate the molecular processes underlying this interesting, but poorly understood phenomenon, a comparative analysis of the lipidomes and transcriptomes of the strain over-expressing sco3201 and of the control strain containing the empty plasmid, was carried out. This study revealed that the strain over-expressing sco3201 had a higher triacylglycerol content and a lower phospholipids content than the control strain. This was correlated with up- and down- regulation of some genes involved in fatty acids biosynthesis (fab) and degradation (fad) respectively, indicating a direct or indirect control of the expression of these genes by SCO3201. In some instances, indirect control might involve TetR regulators, whose encoding genes present in close vicinity of genes involved in lipid metabolism, were shown to be differentially expressed in the two strains. Direct interaction of purified His6-SCO3201 with the promoter regions of four of such TetR regulators encoding genes (sco0116, sco0430, sco4167, and sco6792) was demonstrated. Furthermore, fasR (sco2386), encoding the activator of the main fatty acid biosynthetic operon, sco2386-sco2390, has been shown to be an illegitimate positive regulatory target of SCO3201. Altogether our data demonstrated that the sco3201 over-expressing strain accumulates TAG and suggested that degradation of fatty acids was reduced in this strain. This is expected to result into a reduced acetyl-CoA availability that would impair antibiotic biosynthesis either directly or indirectly.

Expression of genes of the Pho regulon is altered in Streptomyces coelicolor.

Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.

Negative Correlation between Lipid Content and Antibiotic Activity in Streptomyces: General Rule and Exceptions.

Streptomycetes are well known antibiotic producers and are among the rare prokaryotes able to store carbon as lipids. Previous comparative studies of the weak antibiotic producer Streptomyces lividans with its ppk mutant and with Streptomyces coelicolor, which both produce antibiotics, suggested the existence of a negative correlation between total lipid content and the ability to produce antibiotics. To determine whether such a negative correlation can be generalized to other Streptomyces species, fifty-four strains were picked randomly and grown on modified R2YE medium, limited in phosphate, with glucose or glycerol as the main carbon source. The total lipid content and antibiotic activity against Micrococcus luteus were assessed for each strain. This study revealed that the ability to accumulate lipids was not evenly distributed among strains and that glycerol was more lipogenic than glucose and had a negative impact on antibiotic biosynthesis. Furthermore, a statistically significant negative Pearson correlation between lipid content and antibiotic activity could be established for most strains, but a few strains escape this general law. These exceptions are likely due to limits and biases linked to the type of test used to determine antibiotic activity, which relies exclusively on Micrococcus luteus sensitivity. They are characterized either by high lipid content and high antibiotic activity or by low lipid content and undetectable antibiotic activity against Micrococcus luteus. Lastly, the comparative genomic analysis of two strains with contrasting lipid content, and both named Streptomyces antibioticus (DSM 41,481 and DSM 40,868, which we found to be phylogenetically related to Streptomyces lavenduligriseus), indicated that some genetic differences in various pathways related to the generation/consumption of acetylCoA could be responsible for such a difference.

Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) for Rapid Determination of Microbial Cell Lipid Content: Correlation with Gas Chromatography-Mass Spectrometry (GC-MS).

There is a growing interest worldwide for the production of renewable oil without mobilizing agriculture lands; fast and reliable methods are needed to identify highly oleaginous microorganisms of potential industrial interest. The aim of this study was to demonstrate the relevance of attenuated total reflection (ATR) spectroscopy to achieve this goal. To do so, the total lipid content of lyophilized samples of five Streptomyces strains with varying lipid content was assessed with two classical quantitative but time-consuming methods, gas chromatography-mass spectrometry (GC-MS) and ATR Fourier transform infrared (ATR FT-IR) spectroscopy in transmission mode with KBr pellets and the fast ATR method, often questioned for its lack of reliability. A linear correlation between these three methods was demonstrated allowing the establishment of equations to convert ATR values expressed as CO/amide I ratio, into micrograms of lipid per milligram of biomass. The ATR method proved to be as reliable and quantitative as the classical GC-MS and FT-IR in transmission mode methods but faster and more reproducible than the latter since it involves far less manipulation for sample preparation than the two others. Attenuated total reflection could be regarded as an efficient fast screening method to identify natural or genetically modified oleaginous microorganisms by the scientific community working in the field of bio-lipids.

Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145.

The Streptomyces genus is well known for its ability to produce bio-active secondary metabolites of great medical interest. However, the metabolic features accompanying these bio-productions remain to be defined. In this study, the comparison of related model strains producing differing levels of actinorhoddin (ACT), showed that S. lividans, a weak producer, had high TriAcylGlycerol (TAG) content indicative of a glycolytic metabolism. In contrast, the strong producer, S. coelicolor, was characterized by low TAG content, active consumption of its polyphosphate (PolyP) stores and extremely high ATP/ADP ratios. This indicated highly active oxidative metabolism that was correlated with induction of ACT biosynthesis. Interestingly, in conditions of phosphate limitation, the ppk mutant had TAG content and ACT production levels intermediary between those of S. lividans and S. coelicolor. This strain was characterized by high ADP levels indicating that Ppk was acting as an Adenosine Di Phosphate Kinase. Its absence resulted in energetic stress that is proposed to trigger an activation of oxidative metabolism to restore its energetic balance. This process, which is correlated with ACT biosynthesis, requires acetylCoA to fuel the Krebs cycle and phosphate for ATP generation by the ATP synthase coupled to the respiratory chain, resulting in low TAG and polyP content of the ACT producing strains.

Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator.

Recently, we introduced a new electrochemical imaging technique called fluorescence-enabled electrochemical microscopy (FFEM). The central idea of FEEM is that a closed bipolar electrode is utilized to electrically couple a redox reaction of interest to a complementary fluorogenic reaction converting an electrochemical signal into a fluorescent signal. This simple strategy enables one to use fluorescence microscopy to observe conventional electrochemical processes on very large electrochemical arrays. The initial demonstration of FEEM focused on the use of a specific fluorogenic indicator, resazurin, which is reduced to generate highly fluorescent resorufin. The use of resazurin has enabled the study of analyte oxidation reactions, such as the oxidation of dopamine and H2O2. In this report, we extend the capability of FEEM to the study of cathodic reactions using a new fluorogenic indicator, dihydroresorufin. Dihydroresorufin is a nonfluorescent molecule, which can be electrochemically oxidized to generate resorufin. The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+). Here we demonstrate the correlation between the simultaneously recorded fluorescence intensity of resorufin and electrochemical oxidation current during potential sweep experiments. FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 µM on a 25 µm ultramicroelectrode. We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

Recombination proteins mediate meiotic spatial chromosome organization and pairing.

Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4, and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure "entanglement avoidance." Entanglements that remain at zygotene, i.e., "interlockings," require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4, and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes.

The potential efficacy of omega-3 fatty acids as anti-angiogenic agents in benign vascular tumors of infancy.

Hemangiomas of infancy are benign vascular tumors frequently encountered in pediatrics. Medical treatment (corticosteroids, interferon, chemotherapy, embolization and radiation) in high-risk hemangioma cases could greatly benefit from the addition of new and safer therapies. The rapid growth of hemangiomas during the proliferative phase occurs secondary to a process of local uncontrolled angiogenesis, involving potent mediators such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We hypothesize that omega-3 fatty acids, naturally occurring nutrients of proven health benefit to infants, could become an alternative or an adjuvant treatment for hemangiomas, by slowing down their rapid proliferation phase through anti-angiogenic and anti-tumoral effects. Suggested mechanisms of action of omega-3 fatty acids include the downregulation of VEGF and bFGF, and the suppression of pro-angiogenic eicosanoids such as cylooxygenase-2. In this article, we review recent animal and human studies using dietary omega-3 fatty acids supplements, alone or in conjunction with chemotherapy, for the treatment of a variety of tumors dependent on angiogenesis for growth. Available murine hemangioma models offer the opportunity to determine optimal omega-3 fatty acid dose, while taking in account related immunohistochemical markers, clinical outcome and secondary effects, before planning clinical trials. Lessons learned in hemangiomas of infancy may have a broad impact in understanding the phenomenon of dysregulated angiogenesis in cancer research.