Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence.

International audienceBACKGROUND: Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. RESULTS: C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. CONCLUSIONS: Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes

Truncating mutations in SPAST patients are associated with a high rate of psychiatric comorbidities in hereditary spastic paraplegia.

BACKGROUND: The hereditary spastic paraplegias (HSPs) are a rare and heterogeneous group of neurodegenerative disorders that are clinically characterised by progressive lower limb spasticity. They are classified as either 'pure' or 'complex' where spastic paraplegia is complicated with additional neurological features. Mutations in the spastin gene (SPAST) are the most common cause of HSP and typically present with a pure form. METHODS: We assessed in detail the phenotypic and genetic spectrum of SPAST-related HSP focused on 118 patients carrying SPAST mutations. RESULTS: This study, one of the largest cohorts of genetically confirmed spastin patients to date, contributes with the discovery of a significant number of novel SPAST mutations. Our data reveal a high rate of complex cases (25%), with psychiatric disorders among the most common comorbidity (10% of all SPASTpatients). Further, we identify a genotype-phenotype correlation between patients carrying loss-of-function mutations in SPAST and the presence of psychiatric disorders

Tubulin polyglutamylation stimulates spastin-mediated microtubule severing

Microtubules with long polyglutamylated C-terminal tails are more prone to severing by spastin, establishing the importance of tubulin posttranslational modifications

Aged-engineered nanoparticles effect on sludge anaerobic digestion performance and associated microbial communities

To investigate the potential effect of aged engineered nanoparticles (a-ENPs) on sludge digestion performance, 150 L pilot anaerobic digesters (AD) were fed with a blend of primary and waste activated sludge spiked either with a mixture of silver oxide, titanium dioxide and zinc oxide or a mixture of their equivalent bulk metal salts to achieve a target concentration of 250, 2000, and 2800 mg kg− 1 dry weight, respectively. Volatile fatty acids (VFA) were 1.2 times higher in the spiked digesters and significantly different (p = 0.05) from the control conditions. Specifically, isovaleric acid concentration was 2 times lower in the control digester compared to the spiked digesters, whereas hydrogen sulfide was 2 times lower in the ENPs spiked digester indicating inhibitory effect on sulfate reducing microorganisms. Based on the ether-linked isoprenoids concentration, the total abundance of methanogens was 1.4 times lower in the ENPs spiked digester than in the control and metal salt spiked digesters. Pyrosequencing indicated 80% decrease in abundance and diversity of methanogens in ENPs spiked digester compared to the control digester. Methanosarcina acetivorans and Methanosarcina barkeri were identified as nano-tolerant as their relative abundance increased by a factor of 6 and 11, respectively, compared to the other digesters. The results further provide compelling evidence on the resilience of Fusobacteria, Actinobacteria and the Trojan horse-like effect of ENPs which offered a competitive advantage to some organisms while reducing microbial abundance and diversity

Congenital bovine spinal dysmyelination is caused by a missense mutation in the SPAST gene

Bovine spinal dysmyelination (BSD) is a recessive congenital neurodegenerative disease in cattle (Bos taurus) characterized by pathological changes of the myelin sheaths in the spinal cord. The occurrence of BSD is a longstanding problem in the American Brown Swiss (ABS) breed and in several European cattle breeds upgraded with ABS. Here, we show that the disease locus on bovine chromosome 11 harbors the SPAST gene that, when mutated, is responsible for the human disorder hereditary spastic paraplegia (HSP). Initially, SPAST encoding Spastin was considered a less likely candidate gene for BSD since the modes of inheritance as well as the time of onset and severity of symptoms differ widely between HSP and BSD. However, sequence analysis of the bovine SPAST gene in affected animals identified a R560Q substitution at a position in the ATPase domain of the Spastin protein that is invariant from insects to mammals. Interestingly, three different mutations in human SPAST gene at the equivalent position are known to cause HSP. To explore this observation further, we genotyped more than 3,100 animals of various cattle breeds and found that the glutamine allele exclusively occurred in breeds upgraded with ABS. Furthermore, all confirmed BSD carriers were heterozygous, while all affected calves were homozygous for the glutamine allele consistent with recessive transmission of the underlying mutation and complete penetrance in the homozygous state. Subsequent analysis of recombinant Spastin in vitro showed that the R560Q substitution severely impaired the ATPase activity, demonstrating a causal relationship between the SPAST mutation and BSD

The CanOE Strategy: Integrating Genomic and Metabolic Contexts across Multiple Prokaryote Genomes to Find Candidate Genes for Orphan Enzymes

Of all biochemically characterized metabolic reactions formalized by the IUBMB, over one out of four have yet to be associated with a nucleic or protein sequence, i.e. are sequence-orphan enzymatic activities. Few bioinformatics annotation tools are able to propose candidate genes for such activities by exploiting context-dependent rather than sequence-dependent data, and none are readily accessible and propose result integration across multiple genomes. Here, we present CanOE (Candidate genes for Orphan Enzymes), a four-step bioinformatics strategy that proposes ranked candidate genes for sequence-orphan enzymatic activities (or orphan enzymes for short). The first step locates “genomic metabolons”, i.e. groups of co-localized genes coding proteins catalyzing reactions linked by shared metabolites, in one genome at a time. These metabolons can be particularly helpful for aiding bioanalysts to visualize relevant metabolic data. In the second step, they are used to generate candidate associations between un-annotated genes and gene-less reactions. The third step integrates these gene-reaction associations over several genomes using gene families, and summarizes the strength of family-reaction associations by several scores. In the final step, these scores are used to rank members of gene families which are proposed for metabolic reactions. These associations are of particular interest when the metabolic reaction is a sequence-orphan enzymatic activity. Our strategy found over 60,000 genomic metabolons in more than 1,000 prokaryote organisms from the MicroScope platform, generating candidate genes for many metabolic reactions, of which more than 70 distinct orphan reactions. A computational validation of the approach is discussed. Finally, we present a case study on the anaerobic allantoin degradation pathway in Escherichia coli K-12

Oligomerization of ZFYVE27 (Protrudin) Is Necessary to Promote Neurite Extension

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27ΔHR3 failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27ΔHR3 was co-expressed with wild-type ZFYVE27 (ZFYVE27WT), it exerted a dominant negative effect on ZFYVE27WT as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions

The reductive glycine pathway allows autotrophic growth of Desulfovibrio desulfuricans

Supplementary informationis available for this paper athttps://doi.org/10.1038/s41467-020-18906-7Six CO2 fixation pathways are known to operate in photoautotrophic and chemoautotrophic microorganisms. Here, we describe chemolithoautotrophic growth of the sulphate-reducing bacterium Desulfovibrio desulfuricans (strain G11) with hydrogen and sulphate as energy substrates. Genomic, transcriptomic, proteomic and metabolomic analyses reveal that D. desulfuricans assimilates CO2 via the reductive glycine pathway, a seventh CO2 fixation pathway. In this pathway, CO2 is first reduced to formate, which is reduced and condensed with a second CO2 to generate glycine. Glycine is further reduced in D. desulfuricans by glycine reductase to acetyl-P, and then to acetyl-CoA, which is condensed with another CO2 to form pyruvate. Ammonia is involved in the operation of the pathway, which is reflected in the dependence of the autotrophic growth rate on the ammonia concentration. Our study demonstrates microbial autotrophic growth fully supported by this highly ATP-efficient CO2 fixation pathway.We acknowledge Änne-Michaelis and William Newell for assistance with the LC-MS forthe metabolomics experiments and Daniel Amador-Noguez for access to the LC-MS usedfor13C intracellular metabolomic analysis. We thank Ines Cardoso Pereira and John vander Oost for critically reading the manuscript. This research was funded by the Neth-erlands Organisation for Scientific Research (NWO) through SIAM Gravitation Grant024.002.002 and the Innovation Program Microbiology (WUR), NJC acknowledgesfunding from NWO through a Rubicon Grant (019.163LW.035) and a Veni Grant(VI.Veni.192.156).info:eu-repo/semantics/publishedVersio

Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

Many microbes in complex competitive environments share genes for acquiring and utilising nutrients, questioning whether niche specialisation exists and if so, how it is maintained. We investigated the genomic signatures of niche specialisation in the rumen microbiome, a highly competitive, anaerobic environment, with limited nutrient availability determined by the biomass consumed by the host. We generated individual metagenomic libraries from 14 cows fed an ad libitum diet of grass silage and calculated functional isoform diversity for each microbial gene identified. The animal replicates were used to calculate confidence intervals to test for differences in diversity of functional isoforms between microbes that may drive niche specialisation. We identified 153 genes with significant differences in functional isoform diversity between the two most abundant bacterial genera in the rumen (Prevotella and Clostridium). We found Prevotella possesses a more diverse range of isoforms capable of degrading hemicellulose, whereas Clostridium for cellulose. Furthermore, significant differences were observed in key metabolic processes indicating that isoform diversity plays an important role in maintaining their niche specialisation. The methods presented represent a novel approach for untangling complex interactions between microorganisms in natural environments and have resulted in an expanded catalogue of gene targets central to rumen cellulosic biomass degradation

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia

<p>Abstract</p> <p>Background</p> <p>Hereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the <it>SPAST </it>(<it>SPG4</it>) and <it>ATL1 </it>(<it>SPG3A</it>) genes would account for about 50% of the ADHSP cases.</p> <p>Methods</p> <p>We defined the <it>SPAST </it>and <it>ATL1 </it>mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype).</p> <p>Results</p> <p>We found 50 <it>SPAST </it>mutations (including two large deletions) in 54 patients and 7 <it>ATL1 </it>mutations in 11 patients. A total of 33 of the <it>SPAST </it>and 3 of the <it>ATL1 </it>were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the <it>SPAST </it>mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF.</p> <p>Conclusions</p> <p>In a large cohort of Spanish patients with spastic paraplegia, <it>SPAST </it>and <it>ATL1 </it>mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.</p