The archaeome in metaorganism research, with a focus on marine models and their bacteria-archaea interactions.

Metaorganism research contributes substantially to our understanding of the interaction between microbes and their hosts, as well as their co-evolution. Most research is currently focused on the bacterial community, while archaea often remain at the sidelines of metaorganism-related research. Here, we describe the archaeome of a total of eleven classical and emerging multicellular model organisms across the phylogenetic tree of life. To determine the microbial community composition of each host, we utilized a combination of archaea and bacteria-specific 16S rRNA gene amplicons. Members of the two prokaryotic domains were described regarding their community composition, diversity, and richness in each multicellular host. Moreover, association with specific hosts and possible interaction partners between the bacterial and archaeal communities were determined for the marine models. Our data show that the archaeome in marine hosts predominantly consists of Nitrosopumilaceae and Nanoarchaeota, which represent keystone taxa among the porifera. The presence of an archaeome in the terrestrial hosts varies substantially. With respect to abundant archaeal taxa, they harbor a higher proportion of methanoarchaea over the aquatic environment. We find that the archaeal community is much less diverse than its bacterial counterpart. Archaeal amplicon sequence variants are usually host-specific, suggesting adaptation through co-evolution with the host. While bacterial richness was higher in the aquatic than the terrestrial hosts, a significant difference in diversity and richness between these groups could not be observed in the archaeal dataset. Our data show a large proportion of unclassifiable archaeal taxa, highlighting the need for improved cultivation efforts and expanded databases.

In the footsteps of Heinz Schlesner and Peter Hirsch: Exploring the untapped diversity of the phylum Planctomycetota in isolates from the 1980s to the early 2000s.

Recent sampling and strain isolation campaigns have accelerated research on the bacterial phylum Planctomycetota. The contribution of more than 100 novel isolates to the open collection of currently 123 described planctomycetal species in the last decade benefited greatly from pioneering work conducted in the second half of the last century. One of those pioneers was Heinz Schlesner, who investigated budding and prosthecate bacteria from habitats world-wide during his time at Christian-Albrechts-University Kiel. An outcome of his research was a strain collection with more than 500 isolates belonging to different bacterial phyla, many of which are uncharacterised members of the phylum Planctomycetota. Due to the lack of affordable genome sequencing techniques at the time of their isolation, most of them were characterised based on phenotypic features and DNA-DNA hybridisation experiments. After the retirement of Heinz Schlesner in 2002, the collection was stored for several years and transferred to Jena in 2019. To get a glimpse on the diversity of members from the phylum Planctomycetota in Schlesner's collection, we here summarised from his records and publications all available information about the collection regarding sampling habitat and phylogeny. Furthermore, we conducted an updated phylogenetic analysis for a representative excerpt of the collection based on the 16S rRNA gene sequence of 59 strains Schlesner deposited in the NCBI database during strain characterisation studies published in the 1980s until the early 2000s. The results support that strains from his collection are still a valuable contribution to expand the cultivated diversity of the understudied phylum Planctomycetota.

Small protein mediates inhibition of ammonium transport in Methanosarcina mazei-an ancient mechanism?

IMPORTANCE: Small proteins containing fewer than 70 amino acids, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role of the small protein sP36 in the nitrogen metabolism of M. mazei, which modulates the ammonium transporter AmtB1 according to nitrogen availability. This modulation might represent an ancient archaeal mechanism of AmtB1 inhibition, in contrast to the well-studied uridylylation-dependent regulation in bacteria.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate.

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.

The power of the small: the underestimated role of small proteins in bacterial and archaeal physiology.

Small proteins encoded by small open-reading frames (sORFs) (≤70 aa) were overlooked for decades due to methodological reasons and are thus often missing in genome annotations. Novel detection methods such as ribosome profiling (Ribo-Seq) and mass spectrometry optimized for small proteins (peptidomics) have opened up a new field of interest and several catalogs of small proteins in bacteria and archaea have been recently reported. Many translated sORFs have been discovered in genomic locations previously thought to be noncoding, such as 5' or 3' untranslated regions or well-studied regulatory small RNAs (sRNAs). Even within longer ORFs, additional functional sORFs have been detected. Today, only a small proportion is characterized, but those small proteins indicate important and diverse functions in cellular physiology. Here, we summarize recently characterized small proteins involved in microbial metabolism.

Antimicrobial Peptides Originating from Expression Libraries of Aurelia aurita and Mnemiopsis leidyi Prevent Biofilm Formation of Opportunistic Pathogens.

The demand for novel antimicrobial compounds is rapidly growing due to the rising appearance of antibiotic resistance in bacteria; accordingly, alternative approaches are urgently needed. Antimicrobial peptides (AMPs) are promising, since they are a naturally occurring part of the innate immune system and display remarkable broad-spectrum activity and high selectivity against various microbes. Marine invertebrates are a primary resource of natural AMPs. Consequently, cDNA expression (EST) libraries from the Cnidarian moon jellyfish Aurelia aurita and the Ctenophore comb jelly Mnemiopsis leidyi were constructed in Escherichia coli. Cell-free size-fractionated cell extracts (<3 kDa) of the two libraries (each with 29,952 clones) were consecutively screened for peptides preventing the biofilm formation of opportunistic pathogens using the crystal violet assay. The 3 kDa fraction of ten individual clones demonstrated promising biofilm-preventing activities against Klebsiella oxytoca and Staphylococcus epidermidis. Sequencing the respective activity-conferring inserts allowed for the identification of small ORFs encoding peptides (10-22 aa), which were subsequently chemically synthesized to validate their inhibitory potential. Although the peptides are likely artificial products from a random translation of EST inserts, the biofilm-preventing effects against K. oxytoca, Pseudomonas aeruginosa, S. epidermidis, and S. aureus were verified for five synthetic peptides in a concentration-dependent manner, with peptide BiP_Aa_5 showing the strongest effects. The impact of BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 on the dynamic biofilm formation of K. oxytoca was further validated in microfluidic flow cells, demonstrating a significant reduction in biofilm thickness and volume by BiP_Aa_2 and BiP_Aa_5. Overall, the structural characteristics of the marine invertebrate-derived AMPs, their physicochemical properties, and their promising antibiofilm effects highlight them as attractive candidates for discovering new antimicrobials.

First insights into the Aurelia aurita transcriptome response upon manipulation of its microbiome.

Introduction: The associated diverse microbiome contributes to the overall fitness of Aurelia aurita, particularly to asexual reproduction. However, how A. aurita maintains this specific microbiome or reacts to manipulations is unknown. Methods: In this report, the response of A. aurita to manipulations of its native microbiome was studied by a transcriptomics approach. Microbiome-manipulated polyps were generated by antibiotic treatment and challenging polyps with a non-native, native, and potentially pathogenic bacterium. Total RNA extraction followed by RNAseq resulted in over 155 million reads used for a de novo assembly. Results: The transcriptome analysis showed that the antibiotic-induced change and resulting reduction of the microbiome significantly affected the host transcriptome, e.g., genes involved in processes related to immune response and defense mechanisms were highly upregulated. Similarly, manipulating the microbiome by challenging the polyp with a high load of bacteria (2 × 107 cells/polyp) resulted in induced transcription of apoptosis-, defense-, and immune response genes. A second focus was on host-derived quorum sensing interference as a potential defense strategy. Quorum Quenching (QQ) activities and the respective encoding QQ-ORFs of A. aurita were identified by functional screening a cDNA-based expression library generated in Escherichia coli. Corresponding sequences were identified in the transcriptome assembly. Moreover, gene expression analysis revealed differential expression of QQ genes depending on the treatment, strongly suggesting QQ as an additional defense strategy. Discussion: Overall, this study allows first insights into A. aurita's response to manipulating its microbiome, thus paving the way for an in-depth analysis of the basal immune system and additional fundamental defense strategies.

Newly Established Genetic System for Functional Analysis of MetSV.

The linear chromosome of the Methanosarcina spherical virus with 10,567 bp exhibits 22 ORFs with mostly unknown functions. Annotation using common tools and databases predicted functions for a few genes like the type B DNA polymerase (MetSVORF07) or the small (MetSVORF15) and major (MetSVORF16) capsid proteins. For verification of assigned functions of additional ORFs, biochemical or genetic approaches were found to be essential. Consequently, we established a genetic system for MetSV by cloning its genome into the E. coli plasmid pCR-XL-2. Comparisons of candidate plasmids with the MetSV reference based on Nanopore sequencing revealed several mutations of yet unknown provenance with an impact on protein-coding sequences. Linear MetSV inserts were generated by BamHI restriction, purified and transformed in Methanosarcina mazei by an optimized liposome-mediated transformation protocol. Analysis of resulting MetSV virions by TEM imaging and infection experiments demonstrated no significant differences between plasmid-born viruses and native MetSV particles regarding their morphology or lytic behavior. The functionality of the genetic system was tested by the generation of a ΔMetSVORF09 mutant that was still infectious. Our genetic system of MetSV, the first functional system for a virus of methanoarchaea, now allows us to obtain deeper insights into MetSV protein functions and virus-host interactions.

Four Novel Caudoviricetes Bacteriophages Isolated from Baltic Sea Water Infect Colonizers of Aurelia aurita.

The moon jellyfish Aurelia aurita is associated with a highly diverse microbiota changing with provenance, tissue, and life stage. While the crucial relevance of bacteria to host fitness is well known, bacteriophages have often been neglected. Here, we aimed to isolate virulent phages targeting bacteria that are part of the A. aurita-associated microbiota. Four phages (Pseudomonas phage BSwM KMM1, Citrobacter phages BSwM KMM2-BSwM KMM4) were isolated from the Baltic Sea water column and characterized. Phages KMM2/3/4 infected representatives of Citrobacter, Shigella, and Escherichia (Enterobacteriaceae), whereas KMM1 showed a remarkably broad host range, infecting Gram-negative Pseudomonas as well as Gram-positive Staphylococcus. All phages showed an up to 99% adsorption to host cells within 5 min, short latent periods (around 30 min), large burst sizes (mean of 128 pfu/cell), and high efficiency of plating (EOP > 0.5), demonstrating decent virulence, efficiency, and infectivity. Transmission electron microscopy and viral genome analysis revealed that all phages are novel species and belong to the class of Caudoviricetes harboring a tail and linear double-stranded DNA (formerly known as Siphovirus-like (KMM3) and Myovirus-like (KMM1/2/4) bacteriophages) with genome sizes between 50 and 138 kbp. In the future, these isolates will allow manipulation of the A. aurita-associated microbiota and provide new insights into phage impact on the multicellular host.

Active in vivo translocation of the Methanosarcina mazei Gö1 Casposon.

Casposons are transposable elements containing the CRISPR associated gene Cas1solo. Identified in many archaeal genomes, casposons are discussed as the origin of CRISPR-Cas systems due to their proposed Cas1solo-dependent translocation. However, apart from bioinformatic approaches and the demonstration of Cas1solo integrase and endonuclease activity in vitro, casposon transposition has not yet been shown in vivo. Here, we report on active casposon translocations in Methanosarcina mazei Gö1 using two independent experimental approaches. First, mini-casposons, consisting of a R6Kγ origin and two antibiotic resistance cassettes, flanked by target site duplications (TSDs) and terminal inverted repeats (TIRs), were generated, and shown to actively translocate from a suicide plasmid and integrate into the chromosomal MetMaz-C1 TSD IS1a. Second, casposon excision activity was confirmed in a long-term evolution experiment using a Cas1solo overexpression strain in comparison to an empty vector control under four different treatments (native, high temperature, high salt, mitomycin C) to study stress-induced translocation. Analysis of genomic DNA using a nested qPCR approach provided clear evidence of casposon activity in single cells and revealed significantly different casposon excision frequencies between treatments and strains. Our results, providing the first experimental evidence for in vivo casposon activity are summarized in a modified hypothetical translocation model.

Mediation of gaseous emissions and improving plant productivity by DCD and DMPP nitrification inhibitors: Meta-analysis of last three decades.

Nitrification inhibitors (NIs), especially dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP), have been extensively investigated to mitigate nitrogen (N) losses from the soil and thus improve crop productivity by enhancing N use efficiency. However, to provide crop and soil-specific guidelines about using these NIs, a quantitative assessment of their efficacy in mitigating gaseous emissions, worth for nitrate leaching, and improving crop productivity under different crops and soils is yet required. Therefore, based upon 146 peer-reviewed research studies, we conducted a meta-analysis to quantify the effect of DCD and DMPP on gaseous emissions, nitrate leaching, soil inorganic N, and crop productivity under different variates. The efficacy of the NIs in reducing the emissions of CO2, CH4, NO, and N2O highly depends on the crop, soil, and experiment types. The comparative efficacy of DCD in reducing N2O emission was higher than the DMPP under maize, grasses, and fallow soils in both organic and chemical fertilizer amended soils. The use of DCD was linked to increased NH3 emission in vegetables, rice, and grasses. Depending upon the crop, soil, and fertilizer type, both the NIs decreased nitrate leaching from soils; however, DMPP was more effective. Nevertheless, the effect of DCD on crop productivity indicators, including N uptake, N use efficiency, and biomass/yield was higher than DMPP due to certain factors. Moreover, among soils, crops, and fertilizer types, the response by plant productivity indicators to the application of NIs ranged between 35 and 43%. Overall, the finding of this meta-analysis strongly suggests the use of DCD and DMPP while considering the crop, fertilizer, and soil types.

Functional Metagenomics as a Tool to Tap into Natural Diversity of Valuable Biotechnological Compounds.

The marine ecosystem covers more than 70% of the world's surface, and oceans represent a source of varied types of organisms due to the diversified environment. Consequently, the marine environment is an exceptional depot of novel bioactive natural products, with structural and chemical features generally not found in terrestrial habitats. Here, in particular, microbes represent a vast source of unknown and probably new physiological characteristics. They have evolved during extended evolutionary processes of physiological adaptations under various environmental conditions and selection pressures. However, to date, the biodiversity of marine microbes and the versatility of their bioactive compounds and metabolites have not been fully explored. Thus, metagenomic tools are required to exploit the untapped marine microbial diversity and their bioactive compounds. This chapter focuses on function-based marine metagenomics to screen for bioactive molecules of value for biotechnology. Functional metagenomic strategies are described, including sampling in the marine environment, constructing marine metagenomic large-insert libraries, and examples on function-based screens for quorum quenching and anti-biofilm activities.

Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei.

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study's results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis.

Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon.

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and-most likely due to its reduced size-shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified.

Endophytic bacteria perform better than endophytic fungi in improving plant growth under drought stress: A meta-comparison spanning 12 years (2010-2021).

Drought stress is a serious issue that affects agricultural productivity all around the world. Several researchers have reported using plant growth-promoting endophytic bacteria to enhance the drought resistance of crops. However, how endophytic bacteria and endophytic fungi are effectively stimulating plant growth under drought stress is still largely unknown. In this article, a global meta-analysis was undertaken to compare the plant growth-promoting effects of bacterial and fungal endophytes and to identify the processes by which both types of endophytes stimulate plant growth under drought stress. Moreover, this meta-analysis enlightens how plant growth promotion varies across crop types (C3 vs. C4 and monocot vs. dicot), experiment types (in vitro vs. pots vs. field), and the inoculation methods (seed vs. seedling). Specifically, this research included 75 peer-reviewed publications, 170 experiments, 20 distinct bacterial genera, and eight fungal classes. On average, both endophytic bacterial and fungal inoculation increased plant dry and fresh biomass under drought stress. The effect of endophytic bacterial inoculation on plant dry biomass, shoot dry biomass, root length, photosynthetic rate, leaf area, and gibberellins productions were at least two times greater than that of fungal inoculation. In addition, under drought stress, bacterial inoculation increased the proline content of C4 plants. Overall, the findings of this meta-analysis indicate that both endophytic bacterial and fungal inoculation of plants is beneficial under drought conditions, but the extent of benefit is higher with endophytic bacteria inoculation but it varies across crop type, experiment type, and inoculation method.

Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea.

Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.

Microscale Thermophoresis to Study RNA-RNA Binding Affinity.

Evaluation of RNA-RNA binding is crucial for in vitro studying of molecular mechanisms, for example, the interaction of noncoding RNAs (ncRNAs) with their respective targets. In recent years, the method of microscale thermophoresis (MST) has been developed, which is based on the physical phenomenon of thermophoresis (Ludwig-Soret Effect), defined as the migration of a molecule in a solution in response to a macroscopic temperature gradient. The method enables the fast detection and characterization of biophysical interaction between molecules, with the fundamental advantage that only small amounts of target and ligand are required. Here, we describe the characterization of RNA-RNA binding affinity using the example of the sRNA41 from Methanosarcina mazei and its native target, the 5' UTR of mRNA-MM2089, the first gene of the operon encoding the acetyl-CoA decarboxylase/synthase complex.

Bacteroides muris sp. nov. isolated from the cecum of wild-derived house mice.

Two bacterial strains, KH365_2T and KH569_7, were isolated from the cecum contents of wild-derived house mice. The strains were characterized as Gram-negative, rod-shaped, strictly anaerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains were most closely related to Bacteroides uniformis ATCC 8492T. Whole genome sequences of KH365_2T and KH569_7 strains have a DNA G + C content of 46.02% and 46.03% mol, respectively. Most morphological and biochemical characteristics did not differ between the newly isolated strains and classified Bacteroides strains. However, the average nucleotide identity (ANI) and dDNA-DNA hybridization (dDDH) values clearly distinguished the two strains from described members of the genus Bacteroides. Here, we present the phylogeny, morphology, and physiology of a novel species of the genus Bacteroides and propose the name Bacteroides muris sp. nov., with KH365_2T (DSM 114231T = CCUG 76277T) as type strain.

Label-Free Raman Microspectroscopy for Identifying Prokaryotic Virocells.

Raman microspectroscopy has been used to thoroughly assess growth dynamics and heterogeneity of prokaryotic cells, yet little is known about how the chemistry of individual cells changes during infection with virulent viruses, resulting in so-called virocells. Here, we investigate biochemical changes of bacterial and archaeal cells of three different species in laboratory cultures before and after addition of their respective viruses using single-cell Raman microspectroscopy. By applying multivariate statistics, we identified significant differences in the spectra of single cells with/without addition of virulent dsRNA phage (phi6) for Pseudomonas syringae. A general ratio of wavenumbers that contributed the greatest differences in the recorded spectra was defined as an indicator for virocells. Based on reference spectra, this difference is likely attributable to an increase in nucleic acid versus protein ratio of virocells. This method also proved successful for identification of Bacillus subtilis cells infected with the double-stranded DNA (dsDNA) phage phi29, displaying a decrease in respective ratio, but failed for archaeal virocells (Methanosarcina mazei with the dsDNA methanosarcina spherical virus) due to autofluorescence. Multivariate and univariate analyses suggest that Raman spectral data of infected cells can also be used to explore the complex biology behind viral infections of bacteria. Using this method, we confirmed the previously described two-stage infection of P. syringae's phi6 and that infection of B. subtilis with phi29 results in a stress response within single cells. We conclude that Raman microspectroscopy is a promising tool for chemical identification of Gram-positive and Gram-negative virocells undergoing infection with virulent DNA or RNA viruses. IMPORTANCE Viruses are highly diverse biological entities shaping many ecosystems across Earth. However, understanding the infection of individual microbial cells and the related biochemical changes remains limited. Using Raman microspectroscopy in conjunction with univariate and multivariate statistics, we established a marker for identification of infected Gram-positive and Gram-negative bacteria. This nondestructive, label-free analytical method at single-cell resolution paves the way for future studies geared towards analyzing virus-host systems of prokaryotes to further understand the complex chemistry and function of virocells.