Streptomyces Venezuelae Research Articles

The Novelty of Amino Acid Production From Feathers Degradation Em-ploying Keratinase from Streptomyces Venezuelae AZ15

Keratinases are enzymes belonging to the serine hydrolases group which are capable of degradation of keratin, an insoluble and fibrous structural protein widely cross-linked with hydrogen, disulfide, and hydrophobic bonds. Attempts to find new sources of enzymes and amino acids for fundamental knowledge of enzyme evolution, structure‐function relationships, catalysis mechanisms of enzymes, and even for the identification of novel protein folds. In this study, seventy-nine samples were collected from different places in the University of Baghdad, and the best isolate for amino acid production by feathers degradation was by using Streptomyces venezuelae AZ15. The best fermentation system and the optimum culture conditions for amino acid production from feathers as substrate utilizing S. venezuelae AZ15 were elucidated. Using feathers as a substrate, the results showed that the submerged fermentation (SmF) system was the best to give the highest yield of amino acid. Furthermore, the highest yield of total amino acid was gained in feather broth supplemented with 2% glucose and 0.5 % ammonium nitrate, with a pH of 6.0 at 37 °C for 8 days.

Global Effects of the Developmental Regulator BldB in Streptomyces venezuelae.

In Streptomyces, the Bld (Bald) regulators control formation of the reproductive aerial hyphae. The functions of some of these regulators have been well characterized, but BldB has remained enigmatic. In addition to the bldB gene itself, Streptomyces venezuelae has 10 paralogs of bldB that sit next to paralogs of whiJ and abaA. Transcriptome sequencing (RNA-seq) revealed that loss of BldB function causes the dramatic transcriptional upregulation of the abaA paralogs and a novel inhibitor of sporulation, iosA, and that cooverexpression of just two of these genes, iosA and abaA6, was sufficient to recapitulate the bldB mutant phenotype. Further RNA-seq analysis showed that the transcription factor WhiJ9 is required for the activation of iosA seen in the bldB mutant, and biochemical studies showed that WhiJ9 mediates the activation of iosA expression by binding to direct repeats in the iosA-whiJ9 intergenic region. BldB and BldB9 hetero-oligomerize, providing a potential link between BldB and the iosA-whiJ9-bldB9 locus. This work greatly expands our overall understanding of the global effects of the BldB developmental regulator. IMPORTANCE To reproduce and disperse, the filamentous bacterium Streptomyces develops specialized reproductive structures called aerial hyphae. The formation of these structures is controlled by the bld (bald) genes, many of which encode transcription factors whose functions have been characterized. An exception is BldB, a protein whose biochemical function is unknown. In this study, we gain insight into the global effects of BldB function by examining the genome-wide transcriptional effects of deleting bldB. We identify a small set of genes that are dramatically upregulated in the absence of BldB. We show that their overexpression causes the bldB phenotype and characterize a transcription factor that mediates the upregulation of one of these target genes. Our results provide new insight into how BldB influences Streptomyces development.

Xanthine Dehydrogenase Is a Modulator of Dopaminergic Neurodegeneration in Response to Bacterial Metabolite Exposure in C. elegans.

Oxidative stress is a contributing factor to Parkinson's disease (PD). Considering the prevalence of sporadic PD, environmental exposures are postulated to increase reactive oxygen species and either incite or exacerbate neurodegeneration. We previously determined that exposure to the common soil bacterium, Streptomyces venezuelae (S. ven), enhanced oxidative stress and mitochondrial dysfunction in Caenorhabditis elegans, leading to dopaminergic (DA) neurodegeneration. Here, S. ven metabolite exposure in C. elegans was followed by RNA-Seq analysis. Half of the differentially identified genes (DEGs) were associated with the transcription factor DAF-16 (FOXO), which is a key node in regulating stress response. Our DEGs were enriched for Phase I (CYP) and Phase II (UGT) detoxification genes and non-CYP Phase I enzymes associated with oxidative metabolism, including the downregulated xanthine dehydrogenase gene, xdh-1. The XDH-1 enzyme exhibits reversible interconversion to xanthine oxidase (XO) in response to calcium. S. ven metabolite exposure enhanced XO activity in C. elegans. The chelation of calcium diminishes the conversion of XDH-1 to XO and results in neuroprotection from S. ven exposure, whereas CaCl2 supplementation enhanced neurodegeneration. These results suggest a defense mechanism that delimits the pool of XDH-1 available for interconversion to XO, and associated ROS production, in response to metabolite exposure.

Targeted Metabolomics and High-Throughput RNA Sequencing-Based Transcriptomics Reveal Massive Changes in the Streptomyces venezuelae NRRL B-65442 Metabolism Caused by Ethanol Shock.

The species Streptomyces venezuelae is represented by several distinct strains with variable abilities to biosynthesize structurally diverse secondary metabolites. In this work, we examined the effect of ethanol shock on the transcriptome and metabolome of Streptomyces venezuelae NRRL B-65442 using high-throughput RNA sequencing (RNA-seq) and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ethanol shock caused massive changes in the gene expression profile, differentially affecting genes for secondary metabolite biosynthesis and central metabolic pathways. Most of the data from the transcriptome analysis correlated well with the metabolome changes, including the overproduction of jadomycin congeners and a downshift in the production of desferrioxamines, legonoxamine, foroxymithin, and a small cryptic ribosomally synthesized peptide. Some of the metabolome changes, such as the overproduction of chloramphenicol, could not be explained by overexpression of the cognate biosynthetic genes but correlated with the expression profiles of genes for precursor biosynthesis. Changes in the transcriptome were also observed for several genes known to play a role in stress response in other bacteria and included at least 10 extracytoplasmic function σ factors. This study provides important new insights into the stress response in antibiotic-producing bacteria and will help to understand the complex mechanisms behind the environmental factor-induced regulation of secondary metabolite biosynthesis. IMPORTANCE Streptomyces spp. are filamentous Gram-positive bacteria known as versatile producers of secondary metabolites, of which some have been developed into human medicines against infections and cancer. The genomes of these bacteria harbor dozens of gene clusters governing the biosynthesis of secondary metabolites (BGCs), of which most are not expressed under laboratory conditions. Detailed knowledge of the complex regulation of BGC expression is still lacking, although certain growth conditions are known to trigger the production of previously undetected secondary metabolites. In this work, we investigated the effect of ethanol shock on the production of secondary metabolites by Streptomyces venezuelae and correlated these findings with the expression of cognate BGCs and primary metabolic pathways involved in the generation of cofactors and precursors. The findings of this study set the stage for the rational manipulation of bacterial genomes aimed at enhanced production of industrially important bioactive natural products.

Discovery of venediols by activation of a silent type I polyketide biosynthetic gene cluster in Streptomyces venezuelae ATCC 15439

The genus of Streptomyces plays an indispensable role in discovery of bioactive secondary metabolites. As a model strain, Streptomyces venezuelae ATCC 15439 possesses more than 30 biosynthetic gene clusters (BGCs) in its genome; however, many of which are transcriptionally silent and hence uncharacterized. Here, we apply CRISPR/Cas9 genome editing technology to activate a cryptic type I polyketide biosynthetic gene cluster by site-specific insertion of the constitutive promoter kasOp∗, leading to the discovery of two new polyketide metabolites including venediols A (1) and B (2). The elucidated structures and heterologous expression of this BGC suggest an unusual loss of colinearity of the modular polyketide synthases during their biosynthesis.

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems.

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

An Environmental Contributor to Parkinson’s Disease Causes a Hormetic Lifespan Effect in C. elegans

Only 5-10% of Parkinson’s Disease (PD) cases have a direct genetic origin; however, exposure to herbicides, pesticides, and interactions with soil are potential risk factors. PD is characterized by the loss of dopaminergic (DA) neurons and the formation of protein inclusions that contain α-synuclein (α-syn). Conversely, a soil bacterium, Streptomyces venezuelae (S. ven), produces a secondary metabolite that causes age- and dose- dependent DA neurodegeneration in C. elegans; it also exacerbates α-syn-induced DA neurodegeneration. Previous studies from our lab determined that exposure to the S. ven metabolite caused oxidative stress, mitochondrial fragmentation and enhanced reactive oxygen species (ROS). Here we report that exposure to S. ven metabolite causes a hormetic effect on C. elegans lifespan, where low concentrations (5X) extend lifespan in N2 animals, but at higher concentrations (20X) lifespan is decreased. To further examine this hormetic response, we examined daf-16 mutants in this assay. daf-16 mutants displayed no significant differences between solvent and metabolite at both high and low concentrations, suggesting the hormetic response is daf-16 dependent. We also studied S. ven metabolite on C. elegans aging mutants. We investigated mutants in the AMPK signaling pathway and found when exposed to the 20X concentration of S. ven metabolite, aak-2 mutants displayed no significant difference between solvent and metabolite over lifespan. However, when aak-2 mutants were exposed to solvent control and the 5X concentration, mutants displayed a decreased lifespan. This suggests that functional aak-2 might be important for increased lifespan when combating toxicants following chronic exposure.

Caracterización de bacterias rizosféricas aisladas de suelo cultivado con caña de azúcar en el estado de Tamaulipas, México

The state of Tamaulipas, Mexico, has two important sugar mills, with plantations of sugarcane (Saccharum officinarum L.) of more than 50 years, the objective of the study was the identification and bacterial characterization in the production of indole-3-acetic acid (IAA), the solubilization of phosphorus and plant growth of bacterial isolates from the rhizosphere of sugarcane. The isolation and morphological characterization were in 30 rhizosphere soil samples of the sugarcane variety CP 72-2086 using the Luria-Bertani media, Congo Red Agar and Potato Dextrose Agar. The molecular characterization was with the 16S rRNA gene and the bacterial inoculation consisted of seedlings of the variety CP 72-2086. 121 strains (38 species) were isolated, being Bacillus sp. most frequently, 14 species were positive for phosphorus solubilization: Bacillus sp. (6), Pseudomonas spp. (5), Paenibacillus (2) Streptomyces venezuelae (1) stand out. The greater phosphorus solubilization was Pseudomonas mediterranea (21.6 mm). Nine bacteria showed production close to 5 ppm IAA: Bacillus aryabhattai (6 ppm), Bacillus pumilus (5.8 ppm) and Ensifer adhaerens (5.6 ppm). Bacillus megaterium showed a higher percentage of chlorophyll and foliar nitrogen. In the present analysis, 38 bacterial species associated with the rhizosphere of the sugarcane variety CP 72-2086 were identified, so these results showed the potential to select native bacteria that have the ability to stimulate plant growth of the variety CP 72-2086.

Selection of the Optimal L-asparaginase II Against Acute Lymphoblastic Leukemia: An In Silico Approach.

L-asparaginase II (asnB), a periplasmic protein commercially extracted from E coli and Erwinia, is often used to treat acute lymphoblastic leukemia. L-asparaginase is an enzyme that converts L-asparagine to aspartic acid and ammonia. Cancer cells are dependent on asparagine from other sources for growth, and when these cells are deprived of asparagine by the action of the enzyme, the cancer cells selectively die. Questions remain as to whether asnB from E coli and Erwinia is the best asparaginase as they have many side effects. asnBs with the lowest Michaelis constant (Km; most potent) and lowest immunogenicity are considered the most optimal enzymes. In this paper, we have attempted the development of a method to screen for optimal enzymes that are better than commercially available enzymes. In this paper, the asnB sequence of E coli was used to search for homologous proteins in different bacterial and archaeal phyla, and a maximum likelihood phylogenetic tree was constructed. The sequences that are most distant from E coli and Erwinia were considered the best candidates in terms of immunogenicity and were chosen for further processing. The structures of these proteins were built by homology modeling, and asparagine was docked with these proteins to calculate the binding energy. asnBs from Streptomyces griseus, Streptomyces venezuelae, and Streptomyces collinus were found to have the highest binding energy (-5.3 kcal/mol, -5.2 kcal/mol, and -5.3 kcal/mol, respectively; higher than the E coli and Erwinia asnBs) and were predicted to have the lowest Kms, as we found that there is an inverse relationship between binding energy and Km. Besides predicting the most optimal asparaginase, this technique can also be used to predict the most optimal enzymes where the substrate is known and the structure of one of the homologs is solved. We have devised an in silico method to predict the enzyme kinetics from a sequence of an enzyme along with being able to screen for optimal alternative asnBs against acute lymphoblastic leukemia.

Interplay between Nucleoid-Associated Proteins and Transcription Factors in Controlling Specialized Metabolism in Streptomyces.

ABSTRACTLsr2 is a small nucleoid-associated protein found throughout the actinobacteria. Lsr2 functions similarly to the well-studied H-NS, in that it preferentially binds AT-rich sequences and represses gene expression. In Streptomyces venezuelae, Lsr2 represses the expression of many specialized metabolic clusters, including the chloramphenicol antibiotic biosynthetic gene cluster, and deleting lsr2 leads to significant upregulation of chloramphenicol cluster expression. We show here that Lsr2 likely exerts its repressive effects on the chloramphenicol cluster by polymerizing along the chromosome and by bridging sites within and adjacent to the chloramphenicol cluster. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not upregulated in an lsr2 mutant strain. We demonstrate that CmlR is essential for chloramphenicol production, and further reveal that CmlR functions to “countersilence” Lsr2’s repressive effects by recruiting RNA polymerase and enhancing transcription, with RNA polymerase effectively clearing bound Lsr2 from the chloramphenicol cluster DNA. Our results provide insight into the interplay between opposing regulatory proteins that govern antibiotic production in S. venezuelae, which could be exploited to maximize the production of bioactive natural products in other systems.

Evolution of a σ–(c-di-GMP)–anti-σ switch

Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG-(c-di-GMP)2-WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3S(X)2R(X)3Q(X)3D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2-(c-di-GMP)2-WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans, the (RsiG)2-(c-di-GMP)2-WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.

Streptomyces venezuelae NRRL B-65442: genome sequence of a model strain used to study morphological differentiation in filamentous actinobacteria.

For over a decade, Streptomyces venezuelae has been used to study the molecular mechanisms that control morphological development in streptomycetes and is now a well-established model strain. Its rapid growth and ability to sporulate in a near-synchronised manner in liquid culture, unusual among streptomycetes, greatly facilitates the application of modern molecular techniques such as ChIP-seq and RNA-seq, as well as time-lapse fluorescence imaging of the complete Streptomyces life cycle. Here we describe a high-quality genome sequence of our isolate of the strain (Northern Regional Research Laboratory [NRRL] B-65442) consisting of an 8.2 Mb chromosome and a 158 kb plasmid, pSVJI1, which had not been reported previously. Surprisingly, while NRRL B-65442 yields green spores on MYM agar, the American Type Culture Collection (ATCC) type strain 10712 (from which NRRL B-65442 was derived) produces grey spores. While comparison of the genome sequences of the two isolates revealed almost total identity, it did reveal a single nucleotide substitution in a gene, vnz_33525, involved in spore pigment biosynthesis. Replacement of the vnz_33525 allele of ATCC 10712 with that of NRRL B-65442 resulted in green spores, explaining the discrepancy in spore pigmentation. We also applied CRISPR-Cas9 to delete the essential parB of pSVJI1 to cure the plasmid from the strain without obvious phenotypic consequences.

Lysine acetylation of the housekeeping sigma factor enhances the activity of the RNA polymerase holoenzyme

Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC–MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity.

Construction of a new integratingvector from actinophage ϕOZJ and its use in multiplex Streptomycestransformation.

Streptomyces and other closely-related actinobacteria are important sources of bioactive molecules. Streptomyces synthetic biology and genetics empower therapeutic and agrichemical development through strain improvement and biosynthetic understanding. Such efforts rely on the availability of developed molecular toolsets. Among these tools, vectors that enable combinatorial chromosomal manipulations are particularly desirable. Towards developing tools for facile multiplex engineering, we herein describe the development of new integrating vectors derived from BD1 subgroup actinophage OzzyJ (ϕOZJ). By demonstrating the transformation of several Streptomyces spp. using ϕOZJ-derived vectors, we reveal their potential for strain engineering. We further report the development of new ϕC31 and ϕBT1-based vectors having orthogonal resistance, replication and integration features for concomitant transformation with our ϕOZJ-derived vectors. Importantly, the resulting compatible vector panel enabled us to demonstrate the transfer of up to three plasmids each into Streptomyces venezuelae, Streptomyces roseosporus and Streptomyces pristinaespiralis during a single conjugation experiment. To our knowledge this is the first documentation of conjugation-mediated multiplex plasmid transformation, a useful approach for rapid combinatorial strain development.

Harnessing the intracellular triacylglycerols for titer improvement of polyketides in Streptomyces.

Pharmaceutically important polyketides such as avermectin are mainly produced as secondary metabolites during the stationary phase of growth of Streptomyces species in fermenters. The source of intracellular metabolites that are funneled into polyketide biosynthesis has proven elusive. We applied multi-omics to reveal that intracellular triacylglycerols (TAGs), which accumulates in primary metabolism, are degraded during stationary phase. This process could channel carbon flux from both intracellular TAGs and extracellular substrates into polyketide biosynthesis. We devised a strategy named 'dynamic degradation of TAG' (ddTAG) to mobilize the TAG pool and increase polyketide biosynthesis. Using ddTAG we increased the titers of actinorhodin, jadomycin B, oxytetracycline and avermectin B1a in Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces rimosus and Streptomyces avermitilis. Application of ddTAG increased the titer of avermectin B1a by 50% to 9.31 g l-1 in a 180-m3 industrial-scale fermentation, which is the highest titer ever reported. Our strategy could improve polyketide titers for pharmaceutical production.

Stinky molecules undermine competitors

Soil Microbes Soil bacteria produce several earthy and unpleasant-smelling compounds, but for what reason? The filamentous bacterium Streptomyces venezuelae is known to both produce and respond to fishy trimethylamine as a volatile signaling molecule. Jones et al. found that airborne trimethylamine from S. venezuelae reduced growth and survival of other microbes growing nearby. This effect was reversed by iron supplementation, suggesting a role for trimethylamine in making iron less available to competing bacteria, likely by increasing the local pH. S. venezuelae may compensate for the unhospitable environment it has created by secreting iron-chelating compounds to recover recalcitrant iron. mBio 10 , e00171-19 (2019).

Regulation of exploratory growth and antibiotic production in Streptomyces venezuelae by the two-component system CutRS

Antibiotic production and cellular development in bacteria are intimately linked to the extracellular environment. One key mechanism by which bacteria recognise and respond to these external cues is through two-component regulatory systems (2CS). Consisting of a membrane-bound sensor kinase and a cognate DNA-binding response regulator, these 2CSs are essential in the response to a myriad of signals including antibiotic attack, microbial interaction and nutrient availability. The soil-associated filamentous actinobacteria Streptomyces spp. are prolific antibiotic producers with a large number of 2CS. In addition to their complex life cycle a new developmental stage has recently been described termed exploration. When exploring, the streptomycetes rapidly expand via non-branching vegetative hyphae when contacted by fungi, a juxtaposition to the canonical lifecycle which ends in sporulation. Fifteen of the fifty-six 2CS in the model organism Streptomyces venezuelae are highly conserved throughout Streptomycetaceae. Having developed and screened a 2CS operon deletion library within S. venezuelae we determined that one of these, cutRS, is involved in co-ordinating exploration and antibiotic production. The cutRS deletion mutant displays unrepressed exploration and overproduction of chloramphenicol. With a greater understanding of how Streptomyces spp. identify and respond to external signals we can imitate and subvert these systems. Using this we aim to activate cryptic biosynthetic gene clusters enabling the discovery of novel antimicrobial products which may prove beneficial in the clinical setting.

No Country for Old Worms: A Systematic Review of the Application of C. elegans to Investigate a Bacterial Source of Environmental Neurotoxicity in Parkinson's Disease.

While progress has been made in discerning genetic associations with Parkinson’s disease (PD), identifying elusive environmental contributors necessitates the application of unconventional hypotheses and experimental strategies. Here, we provide an overview of studies that we conducted on a neurotoxic metabolite produced by a species of common soil bacteria, Streptomyces venezuelae (S. ven), indicating that the toxicity displayed by this bacterium causes stress in diverse cellular mechanisms, such as the ubiquitin proteasome system and mitochondrial homeostasis. This dysfunction eventually leads to age and dose-dependent neurodegeneration in the nematode Caenorhabditis elegans. Notably, dopaminergic neurons have heightened susceptibility, but all of the neuronal classes eventually degenerate following exposure. Toxicity further extends to human SH-SY5Y cells, which also degenerate following exposure. Additionally, the neurons of nematodes expressing heterologous aggregation-prone proteins display enhanced metabolite vulnerability. These mechanistic analyses collectively reveal a unique metabolomic fingerprint for this bacterially-derived neurotoxin. In considering that epidemiological distinctions in locales influence the incidence of PD, we surveyed soils from diverse regions of Alabama, and found that exposure to ~30% of isolated Streptomyces species caused worm dopaminergic neurons to die. In addition to aging, one of the few established contributors to PD appears to be a rural lifestyle, where exposure to soil on a regular basis might increase the risk of interaction with bacteria producing such toxins. Taken together, these data suggest that a novel toxicant within the Streptomyces genus might represent an environmental contributor to the progressive neurodegeneration that is associated with PD.

Gene-by-environment interactions that disrupt mitochondrial homeostasis cause neurodegeneration in C. elegans Parkinson\u2019s models

Parkinson’s disease (PD) is a complex multifactorial disorder where environmental factors interact with genetic susceptibility. Accumulating evidence suggests that mitochondria have a central role in the progression of neurodegeneration in sporadic and/or genetic forms of PD. We previously reported that exposure to a secondary metabolite from the soil bacterium, Streptomyces venezuelae, results in age- and dose-dependent dopaminergic (DA) neurodegeneration in Caenorhabditis elegans and human SH-SY5Y neurons. Initial characterization of this environmental factor indicated that neurodegeneration occurs through a combination of oxidative stress, mitochondrial complex I impairment, and proteostatic disruption. Here we present extended evidence to elucidate the interaction between this bacterial metabolite and mitochondrial dysfunction in the development of DA neurodegeneration. We demonstrate that it causes a time-dependent increase in mitochondrial fragmentation through concomitant changes in the gene expression of mitochondrial fission and fusion components. In particular, the outer mitochondrial membrane fission and fusion genes, drp-1 (a dynamin-related GTPase) and fzo-1 (a mitofusin homolog), are up- and down-regulated, respectively. Additionally, eat-3, an inner mitochondrial membrane fusion component, an OPA1 homolog, is also down regulated. These changes are associated with a metabolite-induced decline in mitochondrial membrane potential and enhanced DA neurodegeneration that is dependent on PINK-1 function. Genetic analysis also indicates an association between the cell death pathway and drp-1 following S. ven exposure. Metabolite-induced neurotoxicity can be suppressed by DA-neuron-specific RNAi knockdown of eat-3. AMPK activation by 5-amino-4-imidazole carboxamide riboside (AICAR) ameliorated metabolite- or PINK-1-induced neurotoxicity; however, it enhanced neurotoxicity under normal conditions. These studies underscore the critical role of mitochondrial dynamics in DA neurodegeneration. Moreover, given the largely undefined environmental components of PD etiology, these results highlight a response to an environmental factor that defines distinct mechanisms underlying a potential contributor to the progressive DA neurodegeneration observed in PD.

A mechanism for FtsZ-independent proliferation in Streptomyces

The central player in bacterial cell division, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces. Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ-dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.