Biology subjects

Jetten, M. S. M.

Publications and source records attributed to Jetten, M. S. M..

33 records · Page 2Linked to original sources

bioRxiv2021

Unraveling nitrogen, sulfur and carbon metabolic pathways and microbial community transcriptional responses to substrate deprivation and toxicity stresses in a bioreactor mimicking anoxic brackish coastal sediment conditions

Microbial communities are key drivers of carbon, sulfur and nitrogen cycling in coastal ecosystems, where they are subjected to dynamic shifts in substrate availability and exposure to toxic compounds. However, how these shifts affect microbial interactions and function is poorly understood. Unraveling such microbial community responses is key to understand their environmental distribution and resilience under current and future disturbances. Here, we used metagenomics and metatranscriptomics to investigate microbial community structure and transcriptional responses to prolonged ammonium deprivation and sulfide and nitric oxide toxicity stresses in a controlled bioreactor system mimicking coastal sediment conditions. Candidatus Nitrobium versatile, identified in this study as a sulfide-oxidizing denitrifier, became a rare community member upon ammonium removal. The methanotroph Ca. Methanoperedens nitroreducens showed remarkable resilience to both experimental conditions, dominating transcriptional activity of dissimilatory nitrate reduction to ammonium (DNRA). After the ammonium removal experiment, a novel methanotroph species that we have named Ca. Methylomirabilis tolerans outcompeted Ca. Methylomirabilis lanthanidiphila and the anaerobic ammonium oxidizer (anammox) Ca. Kuenenia stuttgartiensis outcompeted Ca. Scalindua rubra. At the end of the sulfide and nitric oxide experiment, a gammaproteobacterium affiliated to the family Thiohalobacteraceae was enriched and dominated transcriptional activity of sulfide:quinone oxidoreductase. Our results indicate that some community members could be more resilient to stresses than others in coastal ecosystems, leading to dynamic microbial community shifts and novel functional states. Methane and sulfide oxidation could be ecosystem functions preserved across the investigated disturbances, while differing nitrogen cycling pathways might be favored in response to stresses. ImportanceCoastal ecosystems are primary zones of biogeochemical cycling, processing inputs of nutrients both generated in situ and derived from land runoff. Microbial communities that inhabit costal sediments perform these biogeochemical reactions, but microbial responses to dynamic, periodic substrate deprivation and exposure to toxic compounds remain elusive. In this study, we sought to address this knowledge gap in a controlled bioreactor system, unraveling microbial metabolic pathways and monitoring microbial responses to stresses that might occur in costal sediments. We identified key microbial players and shifts in their abundance and transcriptional activity. Our results indicated that methanotrophs were particularly resilient to stresses, sulfide oxidizers differed in resiliency but the community maintained sulfide oxidation function across stresses, and that anaerobic ammonium oxidizing (anammox) bacteria were sensitive to substrate deprivation but could restore activity once favorable conditions were reestablished. These insights will help to understand and predict coastal ecosystem responses to future disturbances.

microbiology↗

bioRxiv2021

Effect of concentration and hydraulic reaction time on the removal of pharmaceutical compounds in a membrane bioreactor inoculated with activated sludge

Pharmaceuticals are often not fully removed in wastewater treatment plants (WWTPs) and are thus being detected at trace levels in water bodies all over the world posing a risk to numerous organisms. These organic micropollutants (OMPs) reach WWTPs at concentrations sometimes too low to serve as growth substrate for microorganisms, thus co-metabolism is thought to be the main conversion mechanism. In this study, the microbial removal of six pharmaceuticals was investigated in a membrane bioreactor at increasing concentrations (4-800 nM) of the compounds and using three different hydraulic retention times (HRT; 1, 3.5, 5 days). The bioreactor was inoculated with activated sludge from a Dutch WWTP and fed with ammonium, acetate, and methanol as main growth substrates to stimulate and mimic co-metabolism in a WWTP. Each pharmaceutical compound had a different average removal efficiency: acetaminophen (100%) > fluoxetine (50%) > metoprolol (25%) > diclofenac (20%) > metformin (15%) > carbamazepine (10%). Higher pharmaceutical influent concentrations proportionally increased the removal rate of each compound, but surprisingly not the removal percentage. Furthermore, only metformin removal improved to 80-100% when HRT or biomass concentration was increased in the reactor. Microbial community changes were followed with 16S rRNA gene amplicon sequencing in response to the increment of supplied pharmaceutical concentration: it was found that Nitrospirae and Planctomycetes 16S rRNA relative gene abundance decreased, whereas Acidobacteria and Bacteroidetes increased. Remarkably, the Dokdonella genus, previously implicated in acetaminophen metabolism, showed a 30-fold increase in abundance at the highest (800 nM) concentration of pharmaceuticals applied. Taken together, these results suggest that the incomplete removal of most pharmaceutical compounds in WWTPs is neither dependent on concentration nor HRT. Accordingly, we propose a chemical equilibrium or a growth substrate limitation as the responsible mechanisms of the incomplete removal. Finally, Dokdonella could be the main acetaminophen degrader under activated sludge conditions, and non-antimicrobial pharmaceuticals might still be toxic to relevant WWTP bacteria.

microbiology↗

bioRxiv2021

An activity-based labelling method for the detection of ammonia and methane-oxidizing bacteria.

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia- and methane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. However, the in situ detection and phylogenetic identification of novel ammonia- and methane-oxidizing bacteria remains a challenge. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and methane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labelling of cells harboring active ammonia and methane monooxygenases. The biotinylation of these enzymes in combination with immunogold labelling reveals the subcellular localization of the tagged proteins, while the fluorescent labelling of cells harboring active ammonia or methane monooxygenases provides a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labelling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and methanotrophs from complex environmental samples, facilitating the retrieval of their high quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and methane-oxidizing bacteria present in complex microbial communities.

microbiology↗

bioRxiv2020

Enrichment of novel Verrucomicrobia, Bacteroidetes and Krumholzibacteria in an oxygen-limited, methane- and iron-fed bioreactor inoculated with Bothnian Sea sediments

Microbial methane oxidation is a major biofilter preventing larger emissions of this powerful greenhouse gas from marine coastal areas into the atmosphere. In these zones, various electron acceptors such as sulfate, metal oxides, nitrate or oxygen can be utilized. However, the key microbial players and mechanisms of methane oxidation are poorly understood. In this study, we inoculated a bioreactor with methane- and iron-rich sediments from the Bothnian Sea in order to investigate microbial methane and iron cycling under low oxygen concentrations. Using metagenomics, we observed shifts in the microbial community over approximately 2.5 years of bioreactor operation. Marker genes for methane and iron cycling, as well as respiratory and fermentative metabolism, were investigated. Metagenome-assembled genomes representing novel Verrucomicrobia, Bacteroidetes and Krumholzibacteria were recovered and revealed potential for methane oxidation, organic matter degradation, and iron cycling, respectively. This work brings new insights into the identity and metabolic versatility of microorganisms that may be members of such functional guilds in coastal marine sediments and highlights that the methane biofilter in these sediments may be more diverse than previously appreciated. ImportanceDespite the essential role of microorganisms in preventing most methane in the ocean floor to reach the atmosphere, comprehensive knowledge on the identity and the mechanisms employed by these microorganisms is still lacking. This is problematic because such information is needed to understand how the ecosystem functions in the present and how microorganisms may respond to climate change in the future. Here, we enriched and identified novel taxa potentially involved in methane and iron cycling in an oxygen-limited bioreactor inoculated with methane- and iron-rich coastal sediments. Metagenomic analyses provided hypotheses about the mechanisms they may employ, such as the use of oxygen at very low concentrations. The implication of our results is that in more shallow sediments, where oxygen-limited conditions are present, the methane biofilter is potentially composed of novel, metabolically versatile Verrucomicrobia that could contribute to mitigating methane emissions from coastal marine zones.

microbiology↗

bioRxiv2020

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Furthermore, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named "Candidatus Nitrospira kreftii", and performed a detailed genomic and physiological characterization. The complete genome of "Ca. N. kreftii" allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (Km(app)_NH3 {approx} 0.036 {micro}M), but a higher nitrite affinity (Km(app)_NO2- {approx} 13.8 {micro}M), indicating an adaptation to highly oligotrophic environments. Counterintuitively for a nitrifying microorganism, we also observed an inhibition of ammonia oxidation at ammonium concentrations as low as 25 {micro}M. This substrate inhibition of "Ca. N. kreftii" indicate that differences in ammonium tolerance rather than affinity can be a niche determining factor for different comammox Nitrospira.

microbiology↗

bioRxiv2020

Metagenomic profiling of ammonia- and methane-oxidizing microorganisms in a Dutch drinking water treatment plant

Elevated concentrations of ammonium and methane in groundwater can cause severe problems during drinking water production. To avoid their accumulation, raw water in the Netherlands, and many other countries, is purified by sand filtration. These drinking water filtration systems select for microbial communities that mediate the biodegradation of organic and inorganic compounds. In this study, the active layers and wall biofilm of a Dutch drinking water treatment plant (DWTP) were sampled at different locations along the filtration units of the plant over three years. We used high-throughput sequencing in combination with differential coverage and sequence composition-based binning to recover 56 near-complete metagenome-assembled genomes (MAGs) with an estimated completion of [≥]70% and with [≤]10% redundancy. These MAGs were used to characterize the microbial communities involved in the conversion of ammonia and methane. The methanotrophic microbial communities colonizing the wall biofilm (WB) and the granular material of the primary rapid sand filter (P-RSF) were dominated by members of the Methylococcaceae and Methylophilaceae. The abundance of these bacteria drastically decreased in the secondary rapid sand filter (S-RSF) samples. In all samples, complete ammonia-oxidizing (comammox) Nitrospira were the most abundant nitrifying guild. Clade A comammox Nitrospira dominated the P-RSF, while clade B was most abundant in WB and S-RSF, where ammonium concentrations were much lower. In conclusion, the knowledge obtained in this study contributes to understanding the role of microorganisms in the removal of carbon and nitrogen compounds during drinking water production. We furthermore found that drinking water treatment plants represent valuable model systems to study microbial community function and interaction. HighlightsO_LIMicrobial distribution was mainly influenced by sampling location within the DWTP C_LIO_LIClade A comammox Nitrospira were the dominant nitrifiers in the primary sand filter C_LIO_LIClade B was most abundant in samples from wall biofilm and the secondary filter C_LIO_LIA novel Methylophilaceae-affiliated methanotroph dominated the primary sand filter C_LI

microbiology↗

bioRxiv2019

Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria

Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle, and plays a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using nitrite (NO2-) or nitric oxide (NO) as the electron acceptor. In the absence of NO2- or NO, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as Fe(III) or Mn(IV). Their genomes contain homologs of Geobacter and Shewanella cytochromes involved in extracellular electron transfer (EET). However, it is still unknown whether anammox bacteria have EET capability and can couple the oxidation of NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors. Here we show using complementary approaches that in the absence of NO2-, freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (GO) or electrodes poised at a certain potential in microbial electrolysis cells (MECs). Metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with MEC performance confirmed that anammox electrode biofilms were responsible for current generation through EET-dependent oxidation of NH4+. 15N-labelling experiments revealed the molecular mechanism of the EET-dependent anammox process. NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate when electrode was the terminal electron acceptor. Comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for NH4+ oxidation coupled to EET when electrode is used as electron acceptor compared to NO2-as electron acceptor. To our knowledge, our results provide the first experimental evidence that marine and freshwater anammox bacteria can couple NH4+ oxidation with EET, which is a significant finding, and challenges our perception of a key player of anaerobic oxidation of NH4+ in natural environments and engineered systems.

microbiology↗

bioRxiv2019

Autotrophic and mixotrophic metabolic network fluxes suggest versatile lifestyle for the anammox bacterium Candidatus Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus Kuenenia stuttgartiensis using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of an oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggests that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactors side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

microbiology↗

bioRxiv2019

The novel shapeshifting bacterial phylum Saltatorellota

Our current understanding of a free-living bacterium - capable of withstanding a variety of environmental stresses-is represented by the image of a peptidoglycan-armored rigid casket. The making and breaking of peptidoglycan greatly determines cell shape. The cytoplasmic membrane follows this shape, pressed towards the cell wall by turgor pressure. Consequently, bacteria are morphologically static organisms, in contrast to eukaryotic cells that can facilitate shape changes. Here we report the discovery of the novel bacterial phylum Saltatorellota, that challenges this concept of a bacterial cell. Members of this phylum can change their shape, are capable of amoeba-like locomotion and trunk-formation through the creation of extensive pseudopodia-like structures. Two independent Saltatorellota cells can fuse, and they employ various forms of cell division from budding to canonical binary fission. Despite their polymorphisms, members of the Saltatorellota do possess a peptidoglycan cell wall. Their genomes encode flagella and type IV pili as well as a bacterial actin homolog, the saltatorellin. This protein is most similar to MamK, a dynamic filament-forming protein, that aligns and segregates magnetosome organelles via treadmilling. We found saltatorellin to form filaments in both, E. coli and Magnetospirillum gryphiswaldense, leading to the hypothesis that shapeshifting and pseudopodia formation might be driven by treadmilling of saltatorellin.

microbiology↗

bioRxiv2019

Metabolic overlap in environmentally diverse microbial communities

The majority of microbial communities consist of hundreds to thousands of species, creating a massive network of organisms competing for available resources within an ecosystem. In natural microbial communities it appears that many microbial species have highly redundant metabolisms and seemingly are capable of utilizing the same substrates. This is paradoxical, as theory indicates that species requiring a common resource should outcompete one another. To better understand why microbial species can co-exist, we developed Metabolic Overlap (MO) as a new metric to survey the functional redundancy of microbial communities at the genome scale across a wide variety of ecosystems. Using metagenome-assembled genomes, we surveyed over 1200 studies across ten ecosystem types. We found the highest MO in extreme (i.e., low pH/high temperature) and aquatic environments, while the lowest MO was observed in communities associated with animal hosts, or the built/engineered environment. In addition, different metabolism subcategories were explored for their degree of metabolic overlap. For instance, overlap in nitrogen metabolism was among the lowest in Animal and Engineered ecosystems, while the most was in species from the Built environment. Together, we present a metric that utilizes whole genome information to explore overlapping niches of microbes. This provides a detailed picture of potential metabolic competition and cooperation between species present in an ecosystem, indicates the main substrate types sustaining the community and serves as a valuable tool to generate hypotheses for future research.

microbiology↗

bioRxiv2019

Metagenomic analysis reveals large potential for carbon, nitrogen and sulfur cycling in coastal methanic sediments of the Bothnian Sea

The Bothnian Sea is an oligotrophic brackish basin characterized by low salinity and high concentrations of reactive iron, methane and ammonium in the sediments potentially enabling an intricate microbial network. Therefore, we analyzed and compared biogeochemical and microbial profiles at one offshore and two near coastal sites in the Bothnian Sea. 16S rRNA amplicon sequence analysis revealed stratification of both bacterial and archaeal taxa in accordance with the geochemical gradients of iron, sulfate and methane. The communities at the two near coastal sites were more similar to each other than that at the offshore site located at a greater water depth. To obtain insights into the metabolic networks within the iron-rich methanic sediment layer located below the sulfate-methane transition zone (SMTZ), we performed metagenomic sequencing of sediment-derived DNA. Genome bins retrieved from the most abundant bacterial and archaeal community members revealed a broad potential for respiratory sulfur metabolism via partially reduced sulfur species. Nitrogen cycling was dominated by reductive processes via a truncated denitrification pathway encoded exclusively by bacterial lineages. Gene-centric fermentative metabolism analysis indicated the central role of acetate, formate, alcohols and hydrogen in the analyzed anaerobic sediment. Methanogenic/-trophic pathways were dominated by Methanosaetaceae, Methanosarcinaceae, Methanomassiliicoccaceae, Methanoregulaceae and ANME-2 archaea. Thorarchaeota and Bathyarchaeota encoded pathways for acetogenesis. Our results indicate flexible metabolic capabilities of core community bacterial and archaeal taxa, which can adapt to changing redox conditions, and with a spatial distribution in Bothnian Sea sediments that is likely governed by the quality of available organic substrates.

ecology↗

bioRxiv2019

A novel mesocosm set-up reveals strong methane emission reduction in submerged peat moss Sphagnum cuspidatum by tightly associated methanotrophs.

Wetlands present the largest natural sources of methane (CH4) and their potential CH4 emissions greatly vary due to the activity of CH4-oxidizing bacteria associated with wetland plant species. In this study, the association of CH4-oxidizing bacteria with submerged Sphagnum peat mosses was studied, followed by the development of a novel mesocosm set-up. This set-up enabled the precise control of CH4 input and allowed for monitoring the dissolved CH4 in a Sphagnum moss layer while mimicking natural conditions. Two mesocosm set-ups were used in parallel: one containing a Sphagnum moss layer in peat water, and a control only containing peat water. Moss-associated CH4 oxidizers in the field could reduce net CH4 emission up to 93%, and in the mesocosm set-up up to 31%. Furthermore, CH4 oxidation was only associated with Sphagnum, and did not occur in peat water. Especially methanotrophs containing a soluble methane monooxygenase enzyme were significantly enriched during the 32 day mesocosm incubations. Together these findings showed the new mesocosm setup is very suited to study CH4 cycling in submerged Sphagnum moss community under controlled conditions. Furthermore, the tight associated between Sphagnum peat mosses and methanotrophs can significantly reduce CH4 emissions in submerged peatlands.

ecology↗

bioRxiv2019

Comparison of the bacterial and methanotrophic diversities between an Italian paddy field and its neighboring meadow

Methane is a potent greenhouse gas that contributes to global warming. However, under certain conditions, its release into the atmosphere can be mitigated by methane-oxidizing microorganisms. Typically, cultivated wetlands (i.e., paddy fields) are a major source of methane (CH4) while forests and meadow uplands are considered to be CH4 sinks. As the global need for rice production increases each year, more uplands are converted to inundated paddy fields. To investigate soils that may be converted into productive land for rice production, we investigated a paddy field and adjacent meadow in Northern Italy. Using a combination of 16S rRNA gene amplicon sequencing to analyze the bacterial community, and gas flux measurements to quantify CH4 emissions, we looked for differences between classically defined CH4 sinks (meadow soils) and CH4 sources (paddy fields). Analysis of the total bacterial community revealed that the family Fimbriimonadaceae, which belongs to the phylum Armatimonadetes, was significantly higher in paddy field soils driving the difference between paddy and meadow soils. Whereas, we found that the methylotrophic families Methyloligellaceae and Methylomirabilaceae were also present in higher relative abundance in the paddy field. Despite these major differences, CH4 fluxes were highly variable between the two sites with no significant differences observed. Furthermore, we found the Methylomonaceae family to be more abundant at the center of a neighboring paddy field compared to the edge of the paddy field from the current study, hinting at methanotrophic variation based on location. Taking these results into account, we propose a conceptual model to explain possible scenarios that may result in paddy and meadow fields not exhibiting classical source/sink properties. These findings call for caution when including paddy and meadow areas separately into global CH4 flux calculations, and urge further research to discern drivers of CH4 cycling under a range of environmental conditions rather than relying on assumptions.

microbiology↗

bioRxiv2017

Linking nitrogen load to the structure and function of wetland soil and rhizosphere microbial communities

Wetland ecosystems are important reservoirs of biodiversity and significantly contribute to emissions of the greenhouse gases CO2, NO2 and CH4. High anthropogenic nitrogen (N) inputs from agriculture and fossil fuel combustion have been recognized as a severe threat to biodiversity and ecosystem functioning such as control of greenhouse gas emissions. Therefore it is important to understand how increased N input into pristine wetlands affects the composition and activity of micro-organisms, especially in interaction with dominant wetland plants. In a series of incubations analyzed over 90 days, we disentangle the effects of N fertilization on the microbial community in bulk soil and the rhizosphere of Juncus acutiflorus, a common and abundant graminoid wetland plant. We observed an increase in greenhouse gas emissions when N is increased in incubations with J. acutiflorus, changing the system from a greenhouse gas sink to a source. Using 16S rRNA amplicon sequencing and metagenomics, we determined that the bacterial orders Opitutales, Subgroup-6 Acidobacteria and Sphingobacteriales significantly responded to high N availability and we hypothesize that these groups are contributing to the increased greenhouse gas emissions. These results indicated that increased N input leads to shifts in microbial activity within the rhizosphere, severely altering N cycling dynamics. Our study provides a framework for connecting environmental conditions of wetland bulk and rhizosphere soil to the structure and metabolic output of microbial communities.

microbiology↗

bioRxiv2017

The importance of Chargaff’s second parity rule for genomic signatures in metagenomics

An important problem in metagenomic data analysis is to identify the source organism, or at least taxon, of each sequence. Most methods tackle this problem in two steps by using an alignment-free approach: first the DNA sequences are represented as points of a real n-dimensional space via a mapping function then either clustering or classification algorithms are applied. Those mapping functions require to be genomic signatures: the dissimilarity between the mapped points must reflect the degree of phylogenetic similarity of the source species. Designing good signatures for metagenomics can be challenging due to the special characteristics of metagenomic sequences; most of the existing signatures were not designed accordingly and they were tested only on error-free sequences sampled from a few dozens of species.\n\nIn this work we analyze comparatively the goodness of existing and novel signatures based on tetranu-cleotide frequencies via statistical models and computational experiments; we also study how they are affected by the generalized Chargaffs second parity rule (GCSPR), which states that in a given sequence longer than 50kbp, inverse oligonucleotides are approximately equally frequent. We analyze 38 million sequences of 150 bp-1,000 bp with 1% base-calling error, sampled from 1,284 microbes. Our models indicate that GCSPR reduces strand-dependence of signatures, that is, their values are less affected by the source strand; GCSPR is further exploited by some signatures to reduce the intra-species dispersion. Two novel signatures stand out both in the models and in the experiments: the combination signature and the operation signature. The former achieves strand-independence without grouping oligonucleotides; this could be valuable for alignment-free sequence comparison methods when distinguishing inverse oligonucleotides matters. Operation signature sums the frequencies of reverse, complement, and inverse tetranucleotides; having 72 features it reduces the computational intensity of the analysis.

bioinformatics↗