Assessment of the Accuracy of High-Throughput Sequencing of the ITS1 Region of Neocallimastigomycota for Community Composition Analysis

Anaerobic fungi (Neocallimastigomycota) are common inhabitants of the digestive tract of large mammalian herbivores, where they make an important contribution to plant biomass degradation. The internal transcribed spacer 1 (ITS1) region is currently the molecular marker of choice for anaerobic fungal community analysis, despite its known size polymorphism and heterogeneity. The aim of this study was to assess the accuracy of high-throughput sequencing of the ITS1 region of anaerobic fungi for community composition analysis. To this end, full-length ITS1 clone libraries from five pure cultures, representing the ITS1 region size range, were Sanger sequenced to generate a reference dataset. Barcoded amplicons of the same five pure cultures, and four different mock communities derived from them, were then sequenced using Illumina HiSeq. The resulting sequences were then assessed in relation to either the reference dataset (for the pure cultures) or the corresponding theoretical mock communities. Annotation of sequences obtained from individual pure cultures was not always consistent at the clade or genus level, irrespective of whether data from clone libraries or high-throughput sequencing were analyzed. The detection limit of the high-throughput sequencing method appeared to be influenced by factors other than the parameters used during data processing, as some taxa with theoretical values >0.6% were not detected in the mock communities. The high number of PCR cycles used was considered to be a potential explanation for this observation. Accuracy of two of the four mock communities was limited, and this was speculated to be due to preferential amplification of smaller sized ITS1 regions. If this is true, then this is predicted to be an issue with only six of the 32 named anaerobic fungal clades. Whilst high-throughput sequencing of the ITS1 region from anaerobic fungi can be used for environmental sample analysis, we conclude that the accuracy of the method is influenced by sample community composition. Furthermore, ambiguity in the annotation of sequences within pure cultures due to ITS1 heterogeneity reinforces the limitations of the ITS1 region for the taxonomic assignment of anaerobic fungi. In order to overcome these issues, there is a need to develop an alternative taxonomic marker for anaerobic fungi.</p

Perchlorate and chlorate reduction by the Crenarchaeon Aeropyrum pernix and two thermophilic Firmicutes

This study reports the ability of one hyperthermophile and two thermophilic microorganisms to grow anaerobically by the reduction of chlorate and perchlorate. Physiological, genomic and proteome analyses suggest that the Crenarchaeon Aeropyrum pernix reduces perchlorate with a periplasmic enzyme related to nitrate reductases, but that it lacks a functional chlorite-disproportionating enzyme (Cld) to complete the pathway. A. pernix, previously described as a strictly aerobic microorganism, seems to rely on the chemical reactivity of reduced sulfur compounds with chlorite, a mechanism previously reported for perchlorate-reducing Archaeoglobus fulgidus. The chemical oxidation of thiosulfate (in excessive amounts present in the medium) and the reduction of chlorite result in the release of sulfate and chloride, which are the products of a biotic-abiotic perchlorate reduction pathway in A. pernix. The apparent absence of Cld in two other perchlorate-reducing microorganisms, Carboxydothermus hydrogenoformans and Moorella glycerini strain NMP, and their dependence on sulfide for perchlorate reduction is consistent with observations made on A. fulgidus. Our findings suggest that microbial perchlorate reduction at high temperature differs notably from the physiology of perchlorate- and chlorate-reducing mesophiles and that it is characterized by the lack of a chlorite dismutase and is enabled by a combination of biotic and abiotic reactions.This research was financially supported by Shell Global Solutions International BV. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO). Sequencing data for strain NMP have been submitted to the European Nucleotide Archive (ENA) under accession number PRJEB8377. Mass spectrometry proteomics data and database search results have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014) via the PRIDE partner repository with the dataset identifier PXD001683 and DOI 0.6019/PXD001683

Forward genetics by genome sequencing uncovers the central role of the aspergillus niger goxB locus in hydrogen peroxide induced glucose oxidase expression

Aspergillus niger is an industrially important source for gluconic acid and glucose oxidase (GOx), a secreted commercially important flavoprotein which catalyses the oxidation of b-D-glucose by molecular oxygen to D-glucolactone and hydrogen peroxide. Expression of goxC, the GOx encoding gene and the concomitant two step conversion of glucose to gluconic acid requires oxygen and the presence of significant amounts of glucose in the medium and is optimally induced at pH 5.5. The molecular mechanisms underlying regulation of goxC expression are, however, still enigmatic. Genetic studies aimed at understanding GOx induction have indicated the involvement of at least seven complementation groups, for none of which the molecular basis has been resolved. In this study, a mapping-by-sequencing forward genetics approach was used to uncover the molecular role of the goxB locus in goxC expression. Using the Illumina and PacBio sequencing platforms a hybrid high quality draft genome assembly of laboratory strain N402 was obtained and used as a reference for mapping of genomic reads obtained from the derivative NW103:goxB mutant strain. The goxB locus encodes a thioredoxin reductase. A deletion of the encoding gene in the N402 parent strain led to a high constitutive expression level of the GOx and the lactonase encoding genes required for the two-step conversion of glucose in gluconic acid and of the catR gene encoding catalase R. This high constitutive level of expression was observed to be irrespective of the carbon source and oxidative stress applied. A model clarifying the role of GoxB in the regulation of the expression of goxC involving hydrogen peroxide as second messenger is presented

Intestinimonas-like bacteria are important butyrate producers that utilize Nε-fructosyllysine and lysine in formula-fed infants and adults

Our study aim is to investigate the role of Intestinimonas, Nε-fructosyllysine (FL)-degrading bacterium, in infants and adults. We used lysine and subsequently FL in anaerobic serial dilutions of stools of infants and adults to enrich lysine and FL-degrading species. The fecal microbiota of adults were able to ferment lysine and FL to butyrate. Different groups of Intestinimonas spp. were detected from all lysine enrichments whereas the FL enrichments consisted of broader taxonomic groups with a reduced abundance of Intestinimonas-related species. Remarkably, the capability to degrade FL was only observed in formula-fed but not in breast-fed infants, which may relate to high contents of FL in formulas after thermal treatment. This possibility was supported by analyzing metagenomic datasets of 3-month and 4-month infants. Our data indicate the key role of Intestinimonas-like bacteria in FL degradation in formula-fed infants and adults as a profound example of adaptation of intestinal bacteria to dietary components.Peer reviewe

Global transcriptional response of aspergillus niger to blocked active citrate export through deletion of the exporter gene

Aspergillus niger is the major industrial citrate producer worldwide. Export as well as uptake of citric acid are believed to occur by active, proton-dependent, symport systems. Both are major bottlenecks for industrial citrate production. Therefore, we assessed the consequences of deleting the citT gene encoding the A. niger citrate exporter, effectively blocking active citrate export. We followed the consumption of glucose and citrate as carbon sources, monitored the secretion of organic acids and carried out a thorough transcriptome pathway enrichment analysis. Under controlled cultivation conditions that normally promote citrate secretion, the knock-out strain secreted negligible amounts of citrate. Blocking active citrate export in this way led to a reduced glucose uptake and a reduced expression of high-affinity glucose transporter genes, mstG and mstH. The glyoxylate shunt was strongly activated and an increased expression of the OAH gene was observed, resulting in a more than two-fold higher concentration of oxalate in the medium. Deletion of citT did not affect citrate uptake suggesting that citrate export and citrate uptake are uncoupled from the system

Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes associates with host health

Here, the authors report an anaerobic metabolic pathway from the dominant gut butyrogen Anaerostipes, showing several strains of this genus to be capable of producing propionate from dietary myo-inositol that associates with reduced fasting-glucose levels in mice. We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [C-13]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [C-13]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.Peer reviewe

A Continuous Battle for Host-Derived Glycans Between a Mucus Specialist and a Glycan Generalist in vitro and in vivo

The human gastrointestinal tract is colonized by a diverse microbial community, which plays a crucial role in human health. In the gut, a protective mucus layer that consists of glycan structures separates the bacteria from the host epithelial cells. These host-derived glycans are utilized by bacteria that have adapted to this specific compound in the gastrointestinal tract. Our study investigated the close interaction between two distinct gut microbiota members known to use mucus glycans, the generalist Bacteroides thetaiotaomicron and the specialist Akkermansia muciniphila in vitro and in vivo. The in vitro study, in which mucin was the only nutrient source, indicated that B. thetaiotaomicron significantly upregulated genes coding for Glycoside Hydrolases (GHs) and mucin degradation activity when cultured in the presence of A. muciniphila. Furthermore, B. thetaiotaomicron significantly upregulated the expression of a gene encoding for membrane attack complex/perforin (MACPF) domain in co-culture. The transcriptome analysis also indicated that A. muciniphila was less affected by the environmental changes and was able to sustain its abundance in the presence of B. thetaiotaomicron while increasing the expression of LPS core biosynthesis activity encoding genes (O-antigen ligase, Lipid A and Glycosyl transferases) as well as ABC transporters. Using germ-free mice colonized with B. thetaiotaomicron and/or A. muciniphila, we observed a more general glycan degrading profile in B. thetaiotaomicron while the expression profile of A. muciniphila was not significantly affected when colonizing together, indicating that two different nutritional niches were established in mice gut. Thus, our results indicate that a mucin degrading generalist adapts to its changing environment, depending on available carbohydrates while a mucin degrading specialist adapts by coping with competing microorganism through upregulation of defense related genes.Peer reviewe

Comparative genomics highlights symbiotic capacities and high metabolic flexibility of the marine genus Pseudovibrio

Pseudovibrio is a marine bacterial genus members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesised that Pseudovibrio spp. form mutualistic relationships with their hosts. Here, we studied Pseudovibrio phylogeny and genetic adaptations that may play a role in host colonization by comparative genomics of 31 Pseudovibrio strains, including 25 sponge isolates. All genomes were highly similar in terms of encoded core metabolic pathways, albeit with substantial differences in overall gene content. Based on gene composition, Pseudovibrio spp. clustered by geographic region, indicating geographic speciation. Furthermore, the fact that isolates from the Mediterranean Sea clustered by sponge species suggested host-specific adaptation or colonization. Genome analyses suggest that Pseudovibrio hongkongensis UST20140214-015BT is only distantly related to other Pseudovibrio spp., thereby challenging its status as typical Pseudovibrio member. All Pseudovibrio genomes were found to encode numerous proteins with SEL1 and tetratricopeptide repeats, which have been suggested to play a role in host colonization. For evasion of the host immune system, Pseudovibrio spp. may depend on type III, IV and VI secretion systems that can inject effector molecules into eukaryotic cells. Furthermore, Pseudovibrio genomes carry on average seven secondary metabolite biosynthesis clusters, reinforcing the role of Pseudovibrio spp. as potential producers of novel bioactive compounds. Tropodithietic acid, bacteriocin and terpene biosynthesis clusters were highly conserved within the genus, suggesting an essential role in survival e.g. through growth inhibition of bacterial competitors. Taken together, these results support the hypothesis that Pseudovibrio spp. have mutualistic relations with sponges

Long-chain fatty acids degradation by desulfomonile species and proposal of candidatus Desulfomonile palmitatoxidans

The datasets generated for this study can be found in the European Nucleotide Archive (ENA)–LS453291 (https://www.ebi.ac.uk/ena/browser/view/LS453291), PRJEB26656 (https://www.ebi.ac.uk/ena/browser/view/PRJEB26656), PRJEB35900 (https://www.ebi.ac.uk/ena/browser/view/PRJEB35900).Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16:0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of the Desulfomonile genus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong to Desulfomonile genus [ca. 92% 16S rRNA gene sequences closely related to Desulfomonile spp.; and ca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between the Desulfomonile sp. in HP culture and the type strain Desulfomonile tiedjei strain DCB-1T, we propose a novel species designated as Candidatus Desulfomonile palmitatoxidans. This bacterium shares 94.3 and 93.6% 16S rRNA gene identity with Desulfomonile limimaris strain DCB-MT and D. tiedjei strain DCB-1T, respectively. Based on sequence abundance of Desulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species Ca. Desulfomonile palmitatoxidans most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture and D. tiedjei strain DCB-1T with short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, nor D. tiedjei strain DCB-1T degraded palmitate when incubated with Methanobacterium formicicum as a possible methanogenic syntrophic partner. Unlike D. tiedjei strain DCB-1T, Ca. Desulfomonile palmitatoxidans lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genus Desulfomonile is proposed. Our study reveals an unrecognized LCFA degradation feature of the Desulfomonile genus.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (PTDC/AAG-TEC/3500/2014; POCI-01-0145-FEDER016575), and of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Organohalide-respiring Desulfoluna species isolated from marine environments

The online version of this article (https://doi.org/10.1038/s41396-019-0573-y) contains supplementary material, which is available to authorized usersThe genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20?mM sulfate or 20?mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2\% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.We thank Johanna Gutleben and Maryam Chaib de Mares for sediment sampling, W. Irene C. Rijpstra for fatty acid analysis, and Andreas Marquardt (Proteomics Centre of the University of Konstanz) for proteomic analyses. We acknowledge the China Scholarship Council (CSC) for the support to PP and YL. The authors thank BE-BASIC funds (grants F07.001.05 and F08.004.01) from the Dutch Ministry of Economic Affairs, ERC grant (project 323009), the Gravitation grant (project 024.002.002) and the UNLOCK project (NRGWI.obrug.2018.005) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO), and National Natural Science Foundation of China (project No.51709100) for funding.info:eu-repo/semantics/publishedVersio