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

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 , which modulates the ammonium transporter AmtB1 according to nitrogen availability.

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).

Newly Established Genetic System for Functional Analysis of MetSV.

The linear chromosome of the 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.

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

spherical virus (MetSV), infecting 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 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.

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).

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany.

Small Proteins in Archaea, a Mainly Unexplored World.

In recent years, increasing numbers of small proteins have moved into the focus of science. Small proteins have been identified and characterized in all three domains of life, but the majority remains functionally uncharacterized, lack secondary structure, and exhibit limited evolutionary conservation. While quite a few have already been described for bacteria and eukaryotic organisms, the amount of known and functionally analyzed archaeal small proteins is still very limited.

High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity.

Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein.

Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame-encoded peptides in top-down proteomics.

Short open reading frame-encoded peptides (SEP) represent a widely undiscovered part of the proteome. The detailed analysis of SEP has, despite inherent limitations such as incomplete sequence coverage, challenges encountered with protein inference, the identification of posttranslational modifications and the assignment of potential N- and C-terminal truncations, predominantly been assessed using bottom-up proteomic workflows. The use of top-down based proteomic workflows is capable of providing an unparalleled level of characterization information, which is of increased importance in the case of alternatively encoded protein products.

The CARF Protein MM_0565 Affects Transcription of the Casposon-Encoded Gene in Gö1.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 associated to the type I-B CRISPR-locus of Gö1.

Surgery triage during the COVID-19 pandemic.

Background: The novel coronavirus, SARS-CoV-2, caused the COVID-19 global pandemic. In response, the Australian and New Zealand governments activated their respective emergency plans and hospital frameworks to deal with the potential increased demand on scarce resources. Surgical triage formed an important part of this response to protect the healthcare system's capacity to respond to COVID-19.

Complementarity of Different SDS-PAGE Gel Staining Methods for the Identification of Short Open Reading Frame-Encoded Peptides.

Short open reading frame-encoded peptides (SEP) have been identified across all domains of life and are predicted to be involved in many biochemical processes, however, for the vast majority of SEP their biological function is still unknown. Optimized methodologies have to be used for the mass spectrometric analysis of SEP, because traditional methods of bottom-up proteomics show a bias against small proteins. Here, different staining methods for SDS-PAGE gels prior in-gel digestion following LC-MS/MS analysis for the identification of SEP in the archaeon Methanosarcina mazei are investigated.

Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization.

Archaeosine (G) is a structurally complex modified nucleoside found quasi-universally in the tRNA of and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the G is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G at position 15, coupled with its novel molecular structure, led to a hypothesis that G stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G contributes to tRNA stability and define the biological role of G, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G into the genome of the hyperthermophilic archaeon and the mesophilic archaeon , resulting in modification of the tRNA with the G precursor 7-cyano-7-deazaguansine (preQ) (deletion of ) or no modification at position 15 (deletion of ).

Homologs of aquifex aeolicus protein-only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei.

The mature 5'-ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein-only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa).

Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1.

The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs).

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains.

A novel archaeal lytic virus targeting species of the genus was isolated using strain Gö1 as the host. Due to its spherical morphology, the virus was designated hanosarcina pherical irus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction.

The transcriptional activator NrpA is crucial for inducing nitrogen fixation in Methanosarcina mazei Gö1 under nitrogen-limited conditions.

With the aim of unraveling their potential involvement in the regulation of nitrogen metabolism in Methanosarcina mazei strain Gö1, we characterized five genes that are differentially transcribed in response to changing nitrogen availability and encoding putative transcriptional regulators. Study of the respective mutant strains under nitrogen-limited conditions revealed a growth delay for M. mazei MM0444::pac and MM1708::pac, and strongly reduced diazotrophic growth for MM0872::pac, whereas the absence of MM2441 or MM2525 did not affect growth behaviour.

The intestinal archaea Methanosphaera stadtmanae and Methanobrevibacter smithii activate human dendritic cells.

The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in human's health and disease was rarely examined. Consequently, we studied and report here on the effects of M.

National estimates of insulin-related hypoglycemia and errors leading to emergency department visits and hospitalizations.

Importance: Detailed, nationally representative data describing high-risk populations and circumstances involved in insulin-related hypoglycemia and errors (IHEs) can inform approaches to individualizing glycemic targets.

Objective: To describe the US burden, rates, and characteristics of emergency department (ED) visits and emergency hospitalizations for IHEs.

Design, Setting, And Participants: Nationally representative public health surveillance of adverse drug events among insulin-treated patients seeking ED care (National Electronic Injury Surveillance System-Cooperative Adverse Drug Event Surveillance project) and a national household survey of insulin use (the National Health Interview Survey) were used to obtain data from January 1, 2007, through December 31, 2011.

Two CRISPR-Cas systems in Methanosarcina mazei strain Gö1 display common processing features despite belonging to different types I and III.

The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical.

Effects of antimicrobial peptides on methanogenic archaea.

As members of the indigenous human microbiota found on several mucosal tissues, Methanobrevibacter smithii and Methanosphaera stadtmanae are exposed to the effects of antimicrobial peptides (AMPs) secreted by these epithelia. Although antimicrobial and molecular effects of AMPs on bacteria are well described, data for archaea are not available yet. Besides, it is not clear whether AMPs affect them as the archaeal cell envelope differs profoundly in terms of chemical composition and structure from that of bacteria.

Connection between multimetal(loid) methylation in methanoarchaea and central intermediates of methanogenesis.

In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH₃Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis.

NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon Methanosarcina mazei Gö1.

We report here on the formation of a complex between the two NrpR homologs present in Methanosarcina mazei Gö1 and their binding properties to the nifH and glnK(1) promoters. Reciprocal co-chromatography demonstrated that NrpRI forms stable complexes with NrpRII (at an NrpRI : NrpRII molar ratio of ∼ 1 : 3), which are not affected by 2-oxoglutarate. Promoter-binding, analyses using DNA-affinity chromatography and electrophoretic gel mobility shift assays, verified that NrpRII is not able to bind to either the nifH promoter or the glnK(1) promoter except when in complex with NrpRI.