Transcriptome profiling of Nudix hydrolase gene deletions in the thermoacidophilic archaeon Sulfolobus acidocaldarius

Abstract

Nudix hydrolases comprise a large and ubiquitous protein superfamily that catalyzes the hydrolysis of a nucleoside diphosphate linked to another moiety X (Nudix). Sulfolobus acidocaldarius possesses four Nudix domain-containing proteins (SACI_RS00730/Saci_0153, SACI_RS02625/Saci_0550, SACI_RS00060/Saci_0013/Saci_NudT5, and SACI_RS00575/Saci_0121). Deletion strains were generated for the four individual Nudix genes and for both Nudix genes annotated to encode ADP-ribose pyrophosphatases (SACI_RS00730, SACI_RS00060) and did not reveal a distinct phenotype compared to the wild-type strain under standard growth conditions, nutrient stress or heat stress conditions. We employed RNA-seq to establish the transcriptome profiles of the Nudix deletion strains, revealing a large number of differentially regulated genes, most notably in the ΔSACI_RS00730/SACI_RS00060 double knock-out strain and the ΔSACI_RS00575 single deletion strain. The absence of Nudix hydrolases is suggested to impact transcription via differentially regulated transcriptional regulators. We observed downregulation of the lysine biosynthesis and the archaellum formation iModulons in stationary phase cells, as well as upregulation of two genes involved in the de novo NAD⁺ biosynthesis pathway. Furthermore, the deletion strains exhibited upregulation of two thermosome subunits (α, β) and the toxin-antitoxin system VapBC, which are implicated in the archaeal heat shock response. These results uncover a defined set of pathways that involve archaeal Nudix protein activities and assist in their functional characterization.

Full text

Frontiers in Microbiology 01 frontiersin.org
Transcriptome profiling of Nudix
hydrolase gene deletions in the
thermoacidophilic archaeon
Sulfolobus acidocaldarius
RuthBreuer
1, JoséVicenteGomes-Filho
1, JingYuan
2,3 and
LennartRandau
1,3*
1 Prokaryotic RNA Biology, Department of Biology, Philipps-Universität Marburg, Marburg, Germany,
2 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany, 3 SYNMIKRO, Center for Synthetic
Microbiology, Marburg, Germany
Nudix hydrolases comprise a large and ubiquitous protein superfamily that
catalyzes the hydrolysis of a nucleoside diphosphate linked to another moiety
X (Nudix). Sulfolobus acidocaldarius possesses four Nudix domain-containing
proteins (SACI_RS00730/Saci_0153, SACI_RS02625/Saci_0550, SACI_RS00060/
Saci_0013/Saci_NudT5, and SACI_RS00575/Saci_0121). Deletion strains
were generated for the four individual Nudix genes and for both Nudix genes
annotated to encode ADP-ribose pyrophosphatases (SACI_RS00730, SACI_
RS00060) and did not reveal a distinct phenotype compared to the wild-type
strain under standard growth conditions, nutrient stress or heat stress conditions.
We employed RNA-seq to establish the transcriptome profiles of the Nudix
deletion strains, revealing a large number of dierentially regulated genes, most
notably in the ΔSACI_RS00730/SACI_RS00060 double knock-out strain and
the ΔSACI_RS00575 single deletion strain. The absence of Nudix hydrolases
is suggested to impact transcription via dierentially regulated transcriptional
regulators. We observed downregulation of the lysine biosynthesis and the
archaellum formation iModulons in stationary phase cells, as well as upregulation
of two genes involved in the de novo NAD+ biosynthesis pathway. Furthermore,
the deletion strains exhibited upregulation of two thermosome subunits (α, β)
and the toxin-antitoxin system VapBC, which are implicated in the archaeal heat
shock response. These results uncover a defined set of pathways that involve
archaeal Nudix protein activities and assist in their functional characterization.
KEYWORDS
Nudix hydrolase, transcriptomics, thermophile, RNA processing, gene regulation
1. Introduction
Nudix hydrolases constitute an evolutionary conserved protein superfamily of functionally
versatile proteins present in all three domains of life. ey catalyze the hydrolysis of a wide range
of small nucleotide substrates composed of a nucleoside diphosphate linked to another moiety
X (Nudix) (Figure1) identiable by the conserved Nudix motif with the consensus sequence
GX
5
EX
5
U/AXREX
2
EEXGU (U for a hydrophobic residue, X for any residue) (Bessman etal.,
1996; McLennan, 2006). Initially characterized as “housecleaning enzymes” which cleanse the
cell of potentially toxic metabolites (Bessman etal., 1996), it has since been revealed that their
biological roles are more diverse than previously thought. In Escherichia coli, the Nudix
OPEN ACCESS
EDITED BY
Solenne Ithurbide,
University of Freiburg, Germany
REVIEWED BY
Alejandra Recalde,
University of Freiburg, Germany
Elżbieta Kraszewska,
Polish Academy of Sciences, Poland
*CORRESPONDENCE
Lennart Randau
lennart.randau@sta.uni-marburg.de
RECEIVED 31 March 2023
ACCEPTED 01 June 2023
PUBLISHED 15 June 2023
CITATION
Breuer R, Gomes-Filho JV, Yuan J and
Randau L (2023) Transcriptome profiling of
Nudix hydrolase gene deletions in the
thermoacidophilic archaeon Sulfolobus
acidocaldarius.
Front. Microbiol. 14:1197877.
doi: 10.3389/fmicb.2023.1197877
COPYRIGHT
© 2023 Breuer, Gomes-Filho, Yuan and
Randau. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
TYPE Original Research
PUBLISHED 15 June 2023
DOI 10.3389/fmicb.2023.1197877

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 02 frontiersin.org
hydrolase NudB hydrolyses 8-oxo-dADP, 8-oxo-dGDP and 2-oxo-
dADP and was thus proposed to possess antimutator activity (Hori
et al., 2005). Subsequent studies revealed dihydroneopterin
triphosphate (DHNTP), which is structurally similar to GTP, to bethe
preferred substrate and deletion of nudB led to impaired folate
synthesis in vivo, where DHNTP plays an integral intermediary role
(Gabelli etal., 2007). Recently, Nudix hydrolases have gained attention
due to their ability to remove non-canonical metabolite caps from
RNA molecules. Two prominent examples are the bacterial Nudix
hydrolases NudC and RppH. Initially described as a NAD/H
pyrophosphohydrolase, NAD-RNA decapping by NudC results in
5′-monosphosphorylated RNA and nicotinamide mononucleotide
(NMN) (Frick and Bessman, 1995; Cahová etal., 2015). e RNA
pyrophosphohydrolase RppH converts 5′-triphosphorylated RNA into
5′-monophosphorylated RNA triggering RNA degradation by RNase
E or RNase J (Deana etal., 2008; Richards etal., 2011) and can also act
as NAD-decapping enzyme (Frindert etal., 2018; Grudzien-Nogalska
etal., 2019). Furthermore, E. coli RppH removes non-methylated
5′-Np
n
N-caps from RNA (Hudeček etal., 2020). In eukaryotes, various
Nudix hydrolases (termed “NudT”) capable of removing dierent caps
from RNA in vitro have been identied (Abdelraheim etal., 2003;
Song etal., 2010, 2013; Grudzien-Nogalska etal., 2019). NAD-capped
RNA has been identied in bacteria and eukaryotes and recently also
in the archaeal domain in Sulfolobus acidocaldarius and Haloferax
volcanii (Chen etal., 2009; Cahová etal., 2015; Jiao etal., 2017; Walters
etal., 2017; Ruiz-Larrabeiti etal., 2021; Gomes-Filho etal., 2022).
Furthermore, methylated and non-methylated dinucleoside
polyphosphates (NpnNs) were identied at the 5′ ends of E. coli RNA
(Hudeček etal., 2020) and most recently, ADPR-capped RNA was
identied in human cells (Weixler etal., 2022).
Following the discovery of NAD-capped RNA in S. acidocaldarius,
weaimed to identify the respective decapping enzyme(s) among the
Nudix hydrolases present in this organism and identied four Nudix
domain-containing proteins encoded in the genome. S. acidocaldarius
is a thermophilic crenarchaeon with an optimal growth temperature
between 75 to 80°C and the need for acidic growth medium with an
optimal pH of 2–3. It might likely possess more than one decapping
enzyme, e.g., mammalian cells possess more than 20 decapping
enzymes, each regulating specic subsets of capped RNA (McLennan,
2006; Song et al., 2010; Li etal., 2011). Additionally, it has been
reported that the number of Nudix family representatives in bacteria,
eukaryotic microorganisms and fungi is linearly correlated with
genome size (McLennan, 2006). Currently, E. coli presents 13 genes
encoding Nudix family hydrolases, Saccharomyces cerevisiae 4,
Caenorhabditis elegans 14, Drosophila melanogaster 20 and Arabidopsis
thaliana 25–28 members (McLennan, 2006; Bessman, 2019). In the
archaeal domain, Haloferax volcanii exhibits 12 genes encoding Nudix
domain-containing proteins, while Methanococcous jannaschii,
M. maripaludis and ermococcus kodakarensis each possess a single
Nudix domain protein. Among the other Sulfolobales/Saccharolobales,
Sa. solfataricus possesses 3, S. tokodaii 4 and S. islandicus 4 genes
encoding Nudix domain-containing proteins. In the organisms
presenting only one Nudix protein, it is annotated as a putative ADPR
hydrolase (Alm etal., 2005). e Nudix family is known for displaying
a large substrate range, especially in vitro, encompassing canonical and
oxidized nucleotides, nucleotide sugars, dinucleotide coenzymes,
diadenosine polyphosphates and capped RNAs, as well as
non-nucleotide substrates such as inositol pyrophosphates
(McLennan, 2013; Srouji etal., 2017). While the Nudix protein Saci_
NudT5 demonstrated ADPR-RNA decapping ability in vitro, the other
proteins’ activities remain elusive (Gomes-Filho etal., 2022). To this
end, this study presents the impact of Nudix hydrolase absence on the
transcriptome of S. acidocaldarius. None of the gene deletions elicited
an obvious phenotype, but all strains exhibit a considerable number
of dierentially regulated transcriptional regulators. Overall, the
transcriptome of the Nudix deletion strains resembles that of samples
FIGURE1
Schematic representation of the substrate backbone for Nudix hydrolases. In S. acidocaldarius, four Nudix hydrolases were identified, and their
tridimensional structure was predicted using AlphaFold (AlphaFoldDB accession codes: Q4JCN6, Q4JCD4, Q4JCA5, Q4JB83, Jumper etal., 2021). The
Nudix motif (orange, consensus sequence: GX5EX7REUXEEXGU) and the corresponding functional amino acids (yellow) are shown. The amino acid at
position 16 (green) after the Nudix motif can beused as an initial guide for substrate identification of newly discovered Nudix hydrolases.

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 03 frontiersin.org
taken under heat stress and nutrient limitation conditions (Bischof
etal., 2019), hence aiding our understanding of the gene network
regulating stress response in S. acidocaldarius.
2. Results and discussion
2.1. The Sulfolobus acidocaldarius genome
encodes four Nudix family hydrolases
BLAST analyses and multiple sequence alignments revealed four
genes encoding Nudix domain-containing proteins in the genome of
Sulfolobus acidocaldarius: SACI_RS00730, SACI_RS00060, SACI_
RS02625 and SACI_RS00575 (Figure 1). All proteins possess the
conserved glutamic acid residues in the Nudix motif crucial to Nudix
activity (Cahová etal., 2015; Höfer etal., 2016; Frindert etal., 2018;
Grudzien-Nogalska etal., 2019). e residue at position 16 following
the G of the Nudix motif correlates with possible substrates for the
respective Nudix protein and can serve to identify and distinguish
dierent subsets of Nudix hydrolases (Dunn etal., 1999). In SACI_
RS00060, a proline at this position suggests ADP-ribose (ADPR)
hydrolysis activity. Indeed, this protein was recently shown to decap
ADPR-RNA in vitro and hence renamed Saci_NudT5 to match the
nomenclature of its human homolog (Gomes-Filho etal., 2022). In
SACI_RS00575, a tyrosine at position 16 hints at specicity for
dinucleoside polyphosphate substrates, however its substrate
specicity remains unclear. Due to high sequence similarity to Saci_
NudT5, SACI_RS00730 might represent another ADPR hydrolase.
SACI_RS02625 is conserved across many archaeal species, but no
specic activity has been predicted thus far. In bacteria, a family of
Nudix-related transcriptional regulators (NrtR) regulates NAD
+
metabolism and interacts with ADPR as their eector molecule. ese
proteins are characterized by an N-terminal Nudix-like domain
homologous to ADPR pyrophosphatases and a C-terminal helix-turn-
helix (HTH)-like DNA-binding domain (Rodionov etal., 2008). On
this note, an HTH-domain was not identied in any of the Nudix
family hydrolases from S. acidocaldarius.
Protein structures of the four Nudix hydrolases from
S. acidocaldarius were modeled by AlphaFold sourced from the
UniProt database (Jumper etal., 2021; Varadi etal., 2022; Figure1).
All protein structure predictions reveal similar folding in which the
Nudix motif is present as an α-helix located on the outer part of the
structure, close to the substrate pocket. In the active site, three
conserved glutamic acid residues act as ligands to magnesium ions
and are directed toward the inside of the pocket. e indicator residue
at position 16 aer the G of the Nudix motif is located on the opposite
end of the pocket. Saci_NudT5, the largest of the four proteins,
additionally exhibits an extended structure which is not present in the
other three proteins (Figure1).
2.2. The Nudix hydrolase genes are not
essential in Sulfolobus acidocaldarius
e genes encoding all four Nudix hydrolases in S. acidocaldarius,
SACI_RS00730, SACI_RS00060 (encodes Saci_NudT5), SACI_
RS02625 and SACI_RS00575 were individually targeted for deletion
using the double crossover method based on plasmid pSVA431
developed by Wagner etal., 2012. With this approach, all four genes
were removed from the genome without interrupting their partially
overlapping neighboring genes, indicating that none of the Nudix
hydrolases is essential for S. acidocaldarius. e absence of Nudix gene
transcripts was conrmed by RNA-sequencing
(Supplementary Figure S1). To account for a possible redundancy
between the two most similar genes (Supplementary Figure S2), a
double deletion mutant of SACI_RS00730 and SACI_RS00060 was
generated. In a phenotypical survey, the deletion strains were grown
in parallel to the wild-type strain under standard, nitrogen stress and
carbon stress conditions. All strains showed similar growth behavior
and no signicant deviation from wild-type growth was detected
(Figure2A). Similarly, no signicant deviation was observed between
the wild-type and deletion strains aer submission to 87°C heat shock
and cell growth was signicantly reduced in all strains except for
ΔSACI_RS02625 (Figure 2B). Here, heat-shocked cells showed
survival rates between 33 and 65% compared to their control samples,
while for ΔSACI_RS02625 80% of cells were still viable aer heat
shock exposure (Figure2C). In conclusion, the Nudix hydrolase genes
are not essential in S. acidocaldarius and their deletion did not elicit a
distinct phenotype compared to the wild-type strain under
conditions tested.
2.3. Each deletion strain exhibits a distinct
transcriptome profile
e Nudix deletion strains were grown in parallel to the wild-type
strain and their transcriptome proles were determined for the
mid-logarithmic and the early stationary phase using Illumina
RNA-seq. Dierential gene expression was analyzed using DEseq2 to
enable comparison of the strains’ transcriptome proles
(Supplementary Table S1). RT-qPCR was used to independently assess
the quality of RNA-seq data and veried gene expression proles of
selected genes (Supplementary Figure S3). Overall, the datasets exhibit
more dierentially regulated genes in the early stationary compared
to the mid-log phase. Both heatmaps show a similar clustering of the
strains according to the similarity of their transcriptome proles with
placement of ΔSACI_RS00730 and ΔSACI_RS00575 on opposite ends
of the neighborhood tree, clearly establishing these two transcriptomic
proles as most dissimilar (Figure 3). e highest number of
dierentially regulated genes is present in the ΔSACI_RS00730/SACI_
RS00060 double deletion strain and the ΔSACI_RS00575 strain, hence
establishing them to bemost impactful on the transcriptome. e
smallest transcriptomic impact is caused by the individual deletions
of SACI_RS00730 and SACI_RS00060 (Figure3) which were initially
believed to possess redundant activities due to high sequence
similarity. However, the presence of oppositely regulated gene clusters,
as well as distinct activities in the in vitro decapping assays refutes this
assumption (Gomes-Filho et al., 2022). In summary, clusters of
similarly aected genes are rarely shared between more than two
strains, giving each strain a unique transcriptomic prole and
suggesting unique roles for the enzymes in question. is agrees with
the occurrence of only four Nudix hydrolase genes in the genome, as
the likelihood of redundancies would beexpected to increase with the
number of Nudix genes.
To gain insight into the impact of the individual Nudix deletions,
the dierentially regulated genes of each dataset were assembled into

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 04 frontiersin.org
iModulons according to the iModulonDB database (Chauhan etal.,
2021; Rychel etal., 2021; Figure 4). An iModulon (independently
modulated signal) comprises a group of genes similarly expressed
under dierent (growth) conditions and is hence proposed to bethe
data-driven analog of a regulon without spatial restriction. ese
iModulons were identied by observing patterns in transcriptome
datasets using unsupervised machine learning and independent
component analysis (ICA) (Rychel etal., 2021). e genes encoding
the four Nudix family hydrolases are not assigned to any iModulons.
Notably, genes can beassigned to more than one iModulon and each
iModulon may encompass more genes than currently displayed.
Presumably, the iModulons are modulated by a common regulator or
related ones which must not necessarily bepart of its iModulon and
for many iModulons of S. acidocaldarius a common regulator has not
yet been identied (Chauhan etal., 2021). e DARC (Discovered
signal with Absent Regulatory Components) iModulon which consists
predominantly of poorly characterized genes, contains two
transcription factors, one of which (SACI_RS05830/saci_1223) is
upregulated in all Nudix deletion strains. A relation of this iModulon
to the cell membrane was proposed, however the large number of
uncharacterized genes impedes further predictions (Chauhan
etal., 2021).
Between the ve Nudix deletion strains, 24 genes were identied
to be upregulated in all strains in the early stationary phase
(Supplementary Table S2). No common genes were identied for the
mid-log phase nor downregulated genes in either growth phase.
Roughly half of these genes encode hypothetical proteins or proteins
with domains of unknown function (DUF). Furthermore, several
transcriptional regulators were aected in all deletion strains. Most of
these genes are attributed to the iModulons of transcriptional
regulation by XylR-SoxM or YtrA, as well as cold stress response
proteins (Supplementary Table S2).
2.4. Nudix gene deletions indirectly aect
transcription via transcriptional regulators
The transcriptional activator XylR regulates genes involved
in xylose/arabinose uptake and its respective degradation
pathway in S. acidocaldarius, while SoxM constitutes a terminal
oxidase complex (Komorowski etal., 2002; van der Kolk etal.,
2020). The respective iModulon is activated under nutrient-
limited conditions, suggesting that it contains genes related to
cell growth and starvation (Bischof etal., 2019; Chauhan etal.,
2021). A general upregulation of numerous genes responsive to
nutrient limitation and environmental stress might explain the
lack of a distinct phenotype when the Nudix deletion strains were
grown under carbon, nitrogen or heat stress conditions
(Figure2). Indeed, deletion of the Nudix hydrolases, especially
SACI_RS00730, Saci_NudT5 and SACI_RS02625, elicits a
transcriptome response highly similar to environmental stressors.
Accordingly, genes attributed to several of these iModulons are
significantly affected in all Nudix deletion strains and found to
bemostly upregulated (Figure4).
FIGURE2
Phenotypical characterization of S. acidocaldarius Nudix gene deletion strains under standard and stress conditions. (A) Growth curves performed
under standard conditions, nitrogen, and carbon stress. (B) Cell viability of each knockout strain relative to the wild-type with and without heat stress.
(C) The relative cell viability after heat treatment was normalized by cell viability of non-heat-treated control samples. Each experiment was performed
with triplicates, and error bars depict their standard deviation. Asterisks (*) denote Student’s t-test value <0.05.

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 05 frontiersin.org
e Nudix deletion strains exhibit several upregulated
transcriptional regulators, predominantly from the MarR family and
four regulators which are present in all strains. e MarR family
belongs to the super-group of transcriptional regulators present in
bacteria and archaea predating the divergence of the domains (Pérez-
Rueda and Collado-Vides, 2001). Its members can act as repressors or
activators and their targets comprise genes involved in diverse cellular
processes, such as antibiotic resistance, stress response, virulence and
catabolism of aromatic compounds (Perera and Grove, 2010; Contursi
etal., 2013). Hence, the eect of the Nudix gene deletions is possibly
transmitted via interconnected transcriptional regulators. Notably, the
highest number of dierentially regulated transcriptional regulators is
found in the datasets that contain the highest number of dierentially
regulated genes overall. On the same note, the number of dierentially
regulated genes also correlates with the number of upregulated
thermosome subunits and Type II toxin-antitoxin (TA) system genes:
one in ΔSACI_RS00730 and ΔSACI_RS00060, four in ΔSACI_
RS00575, and ve in ΔSACI_RS02625 and the double knock-out. In
Saccharolobus solfataricus, the toxin VapC6 is a heat-dependent
ribonuclease which is inactivated by VapB6 binding. e
ribonucleolytic activity is suggested to aid in the repression of protein
synthesis during heat shock response (Maezato etal., 2011). Another
study in Sa. solfataricus found vapBC locus expression to beheat-
induced, however some vapBC loci were also expressed under normal
growth conditions, suggesting roles beyond heat stress response for
this system (Tachdjian and Kelly, 2006). e upregulation of a putative
vapBC locus (encoded by SACI_RS10050/saci_2079 and SACI_
RS10055/saci_2080) in two Nudix deletion strains corroborates this
idea. Interestingly, the same study not only found a large number of
MarR family transcriptional regulators upregulated in the heat-
shocked Sa. solfataricus samples, but also upregulation of sso_3167
which encodes a Nudix family hydrolase (Tachdjian and Kelly, 2006).
e homolog of sso_3167 in S. acidocaldarius is SACI_RS02625, whose
deletion strain did not show signicantly reduced growth following
the 87°C heat shock (Figure2). Another study on heat shock response
in S. acidocaldarius revealed high upregulation of the repressor YtrA
(encoded by SACI_RS08880/saci_1851) upon heat stress (Baes etal.,
2020). ough YtrA itself is not aected in the Nudix deletion strains,
its iModulon is predominantly upregulated in ΔSACI_RS02625,
ΔSACI_RS00575 and the double KO strain (Figure4). Notably, YtrA
FIGURE3
Transcriptomes of Nudix gene deletions. Heatmap of the log2 (FoldChange) of the Nudix gene deletion strains’ transcriptome relative to the wild-type
strain S. acidocaldarius MW001in the mid-log and early stationary growth phases and clustered according to Euclidean distances. Individual datasets
can befound in Supplementary Table S1. Green: upregulated genes, blue: downregulated genes, yellow: unaected.

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 06 frontiersin.org
does not regulate known heat shock proteins but two putative
membrane proteins of unknown function and though its connection
to thermal stress remains unclear, a recent study links transcriptional
regulators such as YtrA and Type II TA systems with thermal stress
response in thermophilic archaea (Lemmens etal., 2019; Cooper etal.,
2023). While the exact nature of the connection between Nudix
hydrolases, specically SACI_RS02625, and the heat stress response
system remains unclear, our results suggest it to be more than
coincidental. Notably, a denite transcription factor regulating heat
shock response in the Sulfolobales remains to beidentied.
e LysM iModulon represents lysine biosynthesis in
S. acidocaldarius and contains specically (though not exclusively) the
genes of the lysWXJK operon (Chauhan etal., 2021). is operon
encodes bi-functional enzymes involved in lysine and arginine
biosynthesis and is activated by LysM. In turn, LysM is inactivated by
lysine and excess amounts of lysine were shown to inactivate the
expression of the operon, leading to a shutdown of arginine synthesis
(Brinkman etal., 2002; Ouchi etal., 2013). e LysM iModulon is
downregulated in the early stationary phase in the ΔSACI_RS00575
strain and the double KO strain (Figure4). However, downregulation
of this operon was also observed under nutrient limitation (Bischof
etal., 2019). Notably, the lysine biosynthesis operon is downregulated
in the double KO strain but not in the respective single deletion strains
ΔSACI_RS00730 and ΔSACI_RS00060 (Figure4).
In S. acidocaldarius, the archaellum formation operon is
controlled by the inducers ArnR and ArnR1 and the repressors ArnA
and ArnB and represented in the datasets by the ArnRAB iModulon
(Reimann et al., 2012; Lassak et al., 2013). ArnR (SACI_
RS05625/saci_1180) and the archaellum formation iModulon are
downregulated in ΔSACI_RS00575, in contrast to upregulation of
FIGURE4
Dierentially regulated genes in Nudix gene deletion strains. Dot plot of dierentially regulated genes for all Nudix knockout strains in the early
stationary (A) and mid-log phase (B). Genes were assembled into clusters of similarly regulated genes based on the iModulonDB database (Chauhan
etal., 2021; Rychel etal., 2021). Green: upregulated genes, blue: downregulated genes, yellow: unaected.

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 07 frontiersin.org
ArnR1 (SACI_RS05580/saci_1171) and the archaellum formation
iModulon in ΔSACI_RS00730 (Figure4). e archaellum repressors
ArnA and ArnB are not aected in any of the Nudix deletion strains.
ArnR was observed to be induced under tryptone starvation
conditions and is suggested to becontrolled by a still unidentied
superior transcription factor (Lassak et al., 2013). ArnR1 is
upregulated in the double knock-out while the remaining archaellum
formation iModulon is downregulated (Figure4). ArnR and ArnR1
both promote motility to a dierent extent, as deletion mutants of
arnR and arnR1 exhibited a strongly and mildly diminished motility
phenotype, respectively (Lassak et al., 2013). Several studies in
dierent archaeal organisms report archaellum formation to beeither
repressed or stimulated under dierent nutrient limitation conditions
(Mukhopadhyay etal., 2000; Szabó etal., 2007; Hendrickson etal.,
2008; Xia etal., 2009). Arguably, intracellular nutrient limitations in
ΔSACI_RS00575 and the double knock-out strain may cause the cells
to shut down energy-consuming archaellum production. Hence,
downregulation of the archaellum formation iModulon may
beindicative of the cell’s energy state in consequence of Nudix gene
deletion(s).
Furthermore, ΔSACI_RS02625 and the double KO strain
exhibit upregulation of nadA (a putative quinolinate synthase) and
nadB (a putative L-aspartate oxidase). In the genome, nadB is
located directly upstream of SACI_RS02625, however its
upregulation in the double KO strain refutes a locational eect.
Both gene products presumably catalyze the initial two steps in the
NAD+ de novo biosynthesis pathway, as inferred from homology to
T. kodakarensis. Upregulation of the de novo synthesis pathway is
possibly elicited by a disturbance in the NAD
+
salvage pathway,
which involves recycling NAD
+
from nicotinamide and ADPR
following its thermal degradation (Hachisuka etal., 2018). Other
genes involved in these pathways were not detected to
beco-regulated in the respective datasets.
2.5. Concluding remarks
e genome of S. acidocaldarius encodes four Nudix domain-
containing proteins, which were shown to benot essential to the
organism under conditions tested. While preliminary phenotypical
screenings did not exhibit a distinct deletion phenotype compared to
the wild type strain, RNA-seq revealed unique transcriptome proles
for each Nudix deletion strain. eir transcriptomes were shown to
behighly aected, especially regarding iModulons which assemble
genes responsive to nutrient limitation and heat stress. Considering
the high number of aected transcriptional regulators, the Nudix
hydrolases might beinvolved in the pathways of metabolites that act
as eector molecules to the transcriptional regulators, leading to an
altered transcriptomic state as a consequence of altered metabolite
composition. Indeed, the transcriptomes of the Nudix deletion strains
exhibit remarkable similarity to the transcriptome in response to
nutrient limitation stress (Bischof etal., 2019). Alternatively, Nudix
hydrolases could also aect transcription via direct regulation of the
levels of (capped) transcripts. Weexpect that these results stimulate
the characterization of the Nudix hydrolases from S. acidocaldarius,
which is proposed to include metabolomic proling of the Nudix
deletion strains.
3. Materials and methods
3.1. Generation of Nudix deletion strains
is work uses the uracil auxotrophic strain Sulfolobus
acidocaldarius DSM639 MW001 (Wagner etal., 2012). Cultures
were grown aerobically at 120 rpm and 75°C in Brock media at pH
3.5 (Brock etal., 1972). e media was supplied with 0.1% (w/v)
NZ-amine, 0.2% (w/v) dextrin and 10 μg/ml uracil. Cell growth
was determined by measuring the optical density (OD) at 600 nm
with a cell density meter (Amersham Biosciences). e generation
of markerless Nudix deletion strains was conducted using the
deletion plasmid pSVA431, as described in Wagner etal. (2012).
Briey, this plasmid carries a dual marker system consisting of the
uracil cassette pyrEF and the lacS gene from Saccharolobus
solfataricus plus two multiple cloning sites, harboring part of the
gene of interest and its upstream and downstream anking regions,
respectively. e entire cassette is transformed as a linear fragment
and integrated into the genome via homologous recombination.
Plasmids were constructed using Gibson Assembly (Gibson etal.,
2009) with the primers listed in Supplementary Table S3. e genes
encoding all four Nudix proteins SACI_RS00730, Saci_NudT5
(SACI_RS00060), SACI_RS02625 and SACI_RS00575 were
individually deleted from the genome of S. acidocaldarius DSM639
MW001 without interrupting their partially overlapping
neighboring genes. To generate the double deletion strain,
competent cells from the Saci_NudT5 deletion strain were
transformed with the linear marker cassette targeting SACI_
RS00730. e successful removal of the Nudix genes was
subsequently veried by Sanger sequencing of PCR products
amplied from the deletion loci and RNA-sequencing. For PCR
analysis of S. acidocaldarius cells, 20 μl cell culture was lysed in
20 μl 0.2 M NaOH for 5 min at RT, neutralized by addition of 80 μl
0.2 M Tris–HCl pH 6.5 and 5 μl suspension was used in a 20 μl
reaction using DreamTaq DNA Polymerase (ermo Scientic).
Genomic DNA of S. acidocaldarius was isolated from 2 mL late
logarithmic-phase cultures using the NucleoSpin® Tissue Kit
(Macherey-Nagel), according to the manufacturer’s instructions
for cultured cells.
3.2. Preparation of electrocompetent
Sulfolobus acidocaldarius cells
Cells were grown in 50 mL Brock medium supplied with 0.1%
(w/v) NZ-Amine, 0.2% (w/v) dextrin and 10 μg/ml uracil, pH 3.5,
at 75°C and 120 rpm to OD
600
= 0.3–0.7. A calculated amount of
culture was subsequently transferred into 50 ml fresh medium,
grown to OD600 = 0.2–0.3 and then incubated on ice for 10–15 min.
Cells were harvested by centrifugation for 15–20 min at 2500 × g and
4°C and the pellet was washed three times with each 30 ml of
ice-cold 20 mM sucrose. Next, the pellet was resuspended in 1 ml of
ice-cold 20 mM sucrose, transferred to a 1.5 ml aliquot and
centrifuged for another 5 min at 2500 × g, 4°C. Finally, the pellet was
resuspended in 20 mM ice-cold sucrose to a theoretical OD600 = 20
and 50 μl aliquots were stored at −80°C without the use of liquid
nitrogen until further use.

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 08 frontiersin.org
3.3. Transformation of Sulfolobus
acidocaldarius
Prior to transformation into S. acidocaldarius, all plasmids or
linearized DNA fragments were methylated to circumvent the
activity of the restriction endonuclease SuaI (Berkner etal., 2007).
To this end, plasmids were transformed into the strain Escherichia
coli ER1821 (New England Biolabs) carrying the plasmid
pM.ESABC4I. e methylated deletion plasmids were digested with
NotI-HF (New England Biolabs) to yield linear fragments and
electroporated in 1 mm Gene Pulser® electroporation cuvettes (Bio-
Rad) with a constant time protocol using the input parameters
1.5 kV, 25 μF and 600 Ω on a Gene Pulser® II electroporation system
(Bio-Rad). Recovery was performed for 30 min at 75°C, 300 rpm, in
Brock Recovery Medium (Brock medium supplied with 0.1% (w/v)
NZ-Amine, no pH adjustment), before plating cells on uracil-
lacking rst selection plates. e plates were wrapped in wet paper
towels, placed in plastic boxes to avoid drying out and incubated for
7 days at 75°C. For blue-white screening, plates were sprayed with
25 mg/mL X-gal in DMF diluted 1:5in 20% (w/v) dextrin and
incubated for 30 min at 75°C.
3.4. Isolation of total RNA
Sulfolobus acidocaldarius DSM639 and Nudix deletion strains
were grown in duplicates in Brock media supplied with 0.1% (w/v)
NZ-Amine, 0.2% (w/v) dextrin and 10 μg/ml uracil at 75°C, 120 rpm,
to an OD
600
= 0.3 and 0.7, corresponding to mid-log and early
stationary growth phases, respectively. 2 mL culture samples were
pelleted by centrifugation for 15 min at max. Speed, RT, and total RNA
was isolated using the mirVana™ miRNA Isolation Kit (Invitrogen)
according to the manufacturer’s instructions. Subsequently, total RNA
extractions were digested with 1 U DNaseI/μg RNA (New England
Biolabs) for 2 h at 37°C and cleaned up using the Monarch® RNA
Cleanup Kit (50 μg) (New England Biolabs). Quantitation of RNA
samples was performed using the Qubit™ RNA High Sensitivity
Assay (Agilent Technologies) and Qubit® 2.0 uorometer (ermo
Fisher Scientic GmbH).
3.5. Library preparation for
RNA-sequencing
Ribosomal RNAs were depleted using the Pan-Archaea
riboPOOL probes (siTOOLs Biotech) and streptavidin-coated
magnetic beads (siTOOLs Biotech) according to the
manufacturer’s instructions. Depleted RNA samples were cleaned
up with the Monarch
®
RNA Cleanup Kit (10 μg) (New England
Biolabs) and successful rRNA depletion was verified with the
RNA 6000 Pico Assay for the Agilent Bioanalyzer (Agilent
Technologies) according to the manufacturer’s instructions. The
preparation of cDNA libraries from rRNA-depleted total RNA
was performed using the NEBNext® Ultra™ II Directional RNA
Library Prep Kit for Illumina® (New England Biolabs), AMPure
XP SPRI beads (Beckman Coulter) and NEBNext® Multiplex
Oligos for Illumina® (New England Biolabs), following the
instructions of the NEBNext Library Prep Kit. Quality control
and size distribution of the cDNA libraries was assessed with the
High Sensitivity DNA Assay for Bioanalyzer (Agilent
Technologies). Sequencing was performed as 150 nt single reads
on an Illumina® NextSeq550 at the Genomics Core Facility of the
Philipps University Marburg.
3.6. Analysis of Illumina RNA-Seq data
Raw reads were adapter and quality trimmed using Cutadapt
(v2.8) and checked with FASTQC (v0.11.9) (FastQC, 2015). Processed
reads (≥18 nt) were mapped to the reference genome of
S. acidocaldarius DSM639 (NC_007181.1) using Hisat2 (v2.2.1) (Kim
etal., 2019). Multiple mapped reads with the exact match score were
randomly distributed. Aer the strand-specic screening, HTSeq
(v2.0.2) was used to count gene hits (Anders etal., 2015). Statistical
and dierential expression analyses were performed with DESeq2
(v1.36.0) and genes with a log2 fold-change (≤ −1 or ≥ 1), value of p
<0.05, and adjusted value of p <0.1 were considered dierentially
expressed (Love etal., 2014). Next, genes were classied according to
previously established iModulons (Chauhan etal., 2021) and their
general expression proles were analyzed. e Integrative Genomics
Viewer (IGV, v2.13.2) was used for data inspection (Robinson
etal., 2011).
3.7. RT-qPCR analyses
Total RNA samples (0.04 ng/μl) were used as template for
RT-qPCR analysis using the KAPA SYBR fast one-step qRT-PCR kit
(Merck) following the manufacturer’s instructions. e reactions were
carried out in a CFX384 Touch real-time PCR detection system (Bio-
Rad). e gene SACI_RS06385 was used as an internal control. All
primers used for RT-qPCR analysis are listed in the
Supplementary Table S3. Each RNA sample was tested in triplicates,
and data was analyzed using CFX Manager soware (Bio-Rad).
3.8. Growth curves
Sulfolobus acidocaldarius DSM639 and Nudix deletion strains
were grown as pre-cultures in 25 ml Brock media supplied with 0.1%
(w/v) NZ-Amine, 0.2% (w/v) dextrin and 10 μg/ml uracil at 75°C,
120 rpm. Upon reaching the stationary phase, a calculated volume of
each strain was transferred into 50 ml fresh medium corresponding to
a starting OD
600
= 0.01. Each strain was grown in triplicates in Brock
media + NZ-Amine + Dextrin + Uracil or Brock + Dextrin + Uracil or
Brock + NZ-Amine + Uracil. At the indicated time points, 200 μl from
each culture were transferred into a 96 well plate and adsorption at
600 nm was measured using a CLARIOstar® Plus microplate reader
(BMG Labtech).
3.9. Heat shock spotting assays
Heat shocking spotting assays were modied from Baes etal.
(2020). S. acidocaldarius DSM639 and Nudix deletion strains were
grown in triplicates in Brock media supplied with 0.1% (w/v)

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 09 frontiersin.org
NZ-Amine, 0.2% (w/v) dextrin and 10 μg/ml uracil at 75°C,
120 rpm, until reaching mid-logarithmic phase. Samples were
transferred into pre-warmed aliquots and incubated for 15 min at
75°C, 300 rpm, in a thermomixer (StarLab GmbH) (“adaption
period”). Aer removing a heat shock-control sample from each
tube, tubes were covered with gas-permeable sealing membrane
(Breathe-Easy, Diversied Biotech) and heat shock was
administered for 30 min at 87°C, 300 rpm, on the thermomixer,
using a digital pocket thermometer with a K-type probe (Traceable
®
Products) to monitor the temperature inside the liquid. Heat shock
and control samples were diluted down to OD600 = 0.1, followed by
the preparation of a 10
−1
to 10
−6
dilution series in Brock Recovery
medium [Brock medium supplied with 0.1% (w/v) NZ-Amine, no
pH adjustment]. Finally, 3 μl of each dilution of each sample were
spotted onto a solid Brock Gelrite plate supplied with 0.1% (w/v)
NZ-Amine, 0.2% (w/v) dextrin and 10 μg/ml uracil and plates were
incubated in a plastic box lined with wet paper towels at 75°C. Aer
5 days, plates were photographed and cell viability was determined
by measuring spot density from the 10
−4
dilution step using the oval
selection and area measurement tools from Fiji (Schindelin
etal., 2012).
Data availability statement
e datasets presented in this study can be found in the online
repository European Nucleotide Archive (ENA) under the accession
number PRJEB60684.
Author contributions
RB, JG-F, and LR designed the experiments. RB, JY, and JG-F
performed the experiments. RB, JY, and JG-F analyzed the data. RB
wrote the manuscript with input from JG-F and LR. All authors
reviewed and edited the manuscript.
Funding
is work was funded by the German Research Foundation
(DFG) (Grant RA 2169/8–1 to LR). Open Access funding was
provided by the Open Access Publishing Fund of Philipps-Universität
Marburg with support of the Deutsche Forschungsgemeinscha
(DFG, German Research Foundation).
Acknowledgments
We thank Sonja-Verena Albers and Marleen van Wolferen for
providing strain S. acidocaldarius DSM639 MW001 and plasmid
pSVA431 and for teaching us genetic manipulation of S. acidocaldarius.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer is not guaranteed or endorsed by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1197877/
full#supplementary-material
References
Abdelraheim, S. R., Spiller, D. G., and McLennan, A. G. (2003). Mammalian NADH
diphosphatases of the Nudix family: cloning and characterization of the human
peroxisomal NUDT12 protein. Biochem. J. 374, 329–335. doi: 10.1042/bj20030441
Alm, E. J., Huang, K. H., Price, M. N., Koche, R. P., Keller, K., Dubchak, I. L., et al.
(2005). e MicrobesOnline web site for comparative genomics. Genome Res. 15,
1015–1022. doi: 10.1101/gr.3844805
Anders, S., Pyl, P. T., and Huber, W. (2015). HTSeq—a Python framework to work
with high-throughput sequencing data. Bioinformatics 31, 166–169. doi: 10.1093/
bioinformatics/btu638
Baes, R., Lemmens, L., Mignon, K., Carlier, M., and Peeters, E. (2020). Dening heat
shock response for the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius.
Extremophiles 24, 681–692. doi: 10.1007/s00792-020-01184-y
Berkner, S., Grogan, D., Albers, S. V., and Lipps, G. (2007). Small multicopy, non-
integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and
Sulfolobus solfataricus, model organisms of the (cren-)archaea. Nucleic Acids Res. 35, e88–e12.
doi: 10.1093/nar/gkm449
Bessman, M. J. (2019). A cryptic activity in the Nudix hydrolase superfamily. Protein
Sci. 28, 1494–1500. doi: 10.1002/pro.3666
Bessman, M. J., Frick, D. N., and O’Handley, S. F. (1996). e MutT proteins or
“Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.
J. Biol. Chem. 271, 25059–25062. doi: 10.1074/jbc.271.41.25059
Bischof, L. F., Haurat, M. F., Homann, L., Albersmeier, A., Wolf, J., Neu, A., et al.
(2019). Early response of Sulfolobus acidocaldarius to nutrient limitation. Front.
Microbiol. 9, 1–17. doi: 10.3389/fmicb.2018.03201
Brinkman, A. B., Bell, S. D., Lebbinkt, R. J., De Vos, W. M., and Der Van Oost, J.
(2002). e Sulfolobus solfataricus Lrp-like protein LysM regulates lysine biosynthesis
in response to lysine availability. J. Biol. Chem. 277, 29537–29549. doi: 10.1074/jbc.
M203528200
Brock, T. D., Brock, K. M., Belly, R. T., and Weiss, R. L. (1972). Sulfolobus: a new genus
of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Mikrobiol. 84,
54–68. doi: 10.1007/BF00408082
Cahová, H., Winz, M. L., Höfer, K., Nübel, G., and Jäschke, A. (2015). NAD captureSeq
indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature 519, 374–377.
doi: 10.1038/nature14020
Chauhan, S. M., Poudel, S., Rychel, K., Lamoureux, C., Yoo, R., Al Bulushi, T., et al.
(2021). Machine learning uncovers a data-driven transcriptional regulatory network for
the crenarchaeal thermoacidophile Sulfolobus acidocaldarius. Front. Microbiol.
12:753521. doi: 10.3389/fmicb.2021.753521
Chen, Y. G., Kowtoniuk, W. E., Agarwal, I., Shen, Y., and Liu, D. R. (2009). LC/MS
analysis of cellular RNA reveals NAD-linked RNA. Nat. Chem. Biol. 5, 879–881. doi:
10.1038/nchembio.235
Contursi, P., Fusco, S., Limauro, D., and Fiorentino, G. (2013). Host and viral
transcriptional regulators in Sulfolobus: an overview. Extremophiles 17, 881–895. doi:
10.1007/s00792-013-0586-9
Cooper, C. R., Lewis, A. M., Notey, J. S., Mukherjee, A., Willard, D. J., Blum, P. H.,
et al. (2023). Interplay between transcriptional regulators and VapBC toxin-antitoxin
loci during thermal stress response in extremely ermoacidophilic Archaea. Environ.
Microbiol. 25, 1200–1215. doi: 10.1111/1462-2920.16350

Breuer et al. 10.3389/fmicb.2023.1197877
Frontiers in Microbiology 10 frontiersin.org
Deana, A., Celesnik, H., and Belasco, J. G. (2008). e bacterial enzyme RppH triggers
messenger RNA degradation by 5′ pyrophosphate removal. Nature 451, 355–358. doi:
10.1038/nature06475
Dunn, C. A., O’Handley, S. F., Frick, D. N., and Bessman, M. J. (1999). Studies on the
ADP-ribose pyrophosphatase subfamily of the Nudix hydrolases and tentative
identication of trgB, a gene associated with tellurite resistance. J. Biol. Chem. 274,
32318–32324. doi: 10.1074/jbc.274.45.32318
Frick, D. N., and Bessman, M. J. (1995). Cloning, purication, and properties of a
novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic
domain in MutT-like enzymes. J. Biol. Chem. 270, 1529–1534. doi: 10.1074/
jbc.270.4.1529
Frindert, J., Zhang, Y., Nübel, G., Kahloon, M., Kolmar, L., Hotz-Wagenblatt, A., et al.
(2018). Identication, biosynthesis, and Decapping of NAD-capped RNAs in B. subtilis.
Cell Rep. 24, 1890–1901.e8. doi: 10.1016/j.celrep.2018.07.047
Gabelli, S. B., Bianchet, M. A., Xu, W. L., Dunn, C. A., Niu, Z. D., Amzel, L. M., et al.
(2007). Structure and function of the E. coli Dihydroneopterin triphosphate
pyrophosphatase: a Nudix enzyme involved in folate biosynthesis. Structure 15,
1014–1022. doi: 10.1016/j.str.2007.06.018
Gibson, D. G., Young, L., Chuang, R., Venter, J. C., Hutchison, C., and Smith, H. O.
(2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat.
Methods 6, 343–345. doi: 10.1038/nmeth.1318
Gomes-Filho, J. V., Breuer, R., Morales-Filloy, H. G., Pozhydaieva, N., Börst, A.,
Paczia, N., et al. (2022). Identication of NAD-RNAs and ADPR-RNA decapping in the
archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. bioRxiv. doi:
10.1101/2022.11.02.514978
Grudzien-Nogalska, E., Wu, Y., Jiao, X., Cui, H., Mateyak, M. K., Hart, R. P., et al.
(2019). Structural and mechanistic basis of mammalian Nudt12 RNA deNADding. Nat.
Chem. Biol. 15, 575–582. doi: 10.1038/s41589-019-0293-7
Hachisuka, S., Sato, T., and Atomi, H. (2018). Hyperthermophilic archaeon
ermococcus kodakarensis utilizes a four-step pathway for NAD+ salvage through
nicotinamide deamination. J. Bacteriol. 200:e00785–17. doi: 10.1128/JB.00785-17
Hendrickson, E. L., Liu, Y., Rosas-Sandoval, G., Porat, I., Söll, D., Whitman, W. B.,
et al. (2008). Global responses of Methanococcus maripaludis to specic nutrient
limitations and growth rate. J. Bacteriol. 190, 2198–2205. doi: 10.1128/JB.01805-07
Höfer, K., Li, S., Abele, F., Frindert, J., Schlotthauer, J., Grawenho, J., et al. (2016).
Structure and function of the bacterial decapping enzyme NudC. Nat. Chem. Biol. 12,
730–734. doi: 10.1038/nchembio.2132
Hori, M., Fujikawa, K., Kasai, H., Harashima, H., and Kamiya, H. (2005). Dual
hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by
Orf17 (NtpA), a MutT-type enzyme. DNA Repair 4, 33–39. doi: 10.1016/j.
dnarep.2004.07.010
Hudeček, O., Benoni, R., Reyes-Gutierrez, P. E., Culka, M., Šanderová, H.,
Hubálek, M., et al. (2020). Dinucleoside polyphosphates act as 5′-RNA caps in bacteria.
Nat. Commun. 11, 1–11. doi: 10.1038/s41467-020-14896-8
Jiao, X., Doamekpor, S. K., Bird, J. G., Nickels, B. E., Tong, L., Hart, R. P., et al. (2017).
5′ end nicotinamide adenine dinucleotide cap in human cells promotes RNA decay
through DXO-mediated deNADding. Cells 168, 1015–1027.e10. doi: 10.1016/j.
cell.2017.02.019
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021).
Highly accurate protein structure prediction with AlphaFold. Nat. Cell Biol. 596,
583–589. doi: 10.1038/s41586-021-03819-2
Kim, D., Paggi, J. M., Park, C., Bennett, C., and Salzberg, S. L. (2019). Graph-based
genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol.
37, 907–915. doi: 10.1038/s41587-019-0201-4
Komorowski, L., Verheyen, W., and Schäfer, G. (2002). e archaeal respiratory
supercomplex SoxM from S. acidocaldarius combines features of quinole and
cytochrome c oxidases. Biol. Chem. 383, 1791–1799. doi: 10.1515/BC.2002.200
Lassak, K., Peeters, E., Wróbel, S., and Albers, S. (2013). e one-component system
ArnR: a membrane-bound activator of the crenarchaeal archaellum. Mol. Microbiol. 88,
125–139. doi: 10.1111/mmi.12173
Lemmens, L., Tilleman, L., De Koning, E., Valegård, K., Lindås, A. C., Van
Nieuwerburgh, F., et al. (2019). YtrA
Sa
, a GntR-family transcription factor, represses two
genetic loci encoding membrane proteins in Sulfolobus acidocaldarius. Front. Microbiol.
10:2084. doi: 10.3389/FMICB.2019.02084/BIBTEX
Li, Y., Song, M., and Kiledjian, M. (2011). Dierential utilization of decapping
enzymes in mammalian mRNA decay pathways. RNA 17, 419–428. doi: 10.1261/
rna.2439811
Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change
and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550–521. doi: 10.1186/
s13059-014-0550-8
Maezato, Y., Daugherty, A., Dana, K., Soo, E., Cooper, C., Tachdjian, S., et al. (2011).
VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus
solfataricus. RNA 17:1381. doi: 10.1261/rna.2679911
McLennan, A. G. (2006). e Nudix hydrolase superfamily. Cell. Mol. Life Sci. 63,
123–143. doi: 10.1007/s00018-005-5386-7
McLennan, A. G. (2013). Substrate ambiguity among the nudix hydrolases:
biologically signicant, evolutionary remnant, or both? Cell. Mol. Life Sci. 70, 373–385.
doi: 10.1007/s00018-012-1210-3
Mukhopadhyay, B., Johnson, E. F., and Wolfe, R. S. (2000). A novel P(H2) control on
the expression of agella in the hyperthermophilic strictly hydrogenotrophic
methanarchaeaon Methanococcus jannaschii. Proc. Natl. Acad. Sci. U. S. A. 97,
11522–11527. doi: 10.1073/pnas.97.21.11522
Ouchi, T., Tomita, T., Horie, A., Yoshida, A., Takahashi, K., Nishida, H., et al. (2013).
Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.
Chem. Biol. 9, 277–283. doi: 10.1038/nchembio.1200
Perera, I. C., and Grove, A. (2010). Molecular mechanisms of ligand-mediated
attenuation of DNA binding by MarR family transcriptional regulators. J. Mol. Cell Biol.
2, 243–254. doi: 10.1093/jmcb/mjq021
Pérez-Rueda, E., and Collado-Vides, J. (2001). Common history at the origin of the
position–function correlation in transcriptional regulators in Archaea and Bacteria. J
Molec Evol 53, 172–179. doi: 10.1007/s002390010207
Reimann, J., Lassak, K., Khadouma, S., Ettema, T. J. G., Yang, N., Driessen, A. J. M.,
et al. (2012). Regulation of archaella expression by the FHA and von Willebrand
domain-containing proteins ArnA and ArnB in Sulfolobus acidocaldarius. Mol.
Microbiol. 86, 24–36. doi: 10.1111/j.1365-2958.2012.08186.x
Richards, J., Liu, Q., Pellegrini, O., Celesnik, H., Yao, S., Bechhofer, D. H., et al. (2011).
An RNA Pyrophosphohydrolase triggers 5′-exonucleolytic degradation of mRNA in
Bacillus subtilis. Mol. Cell 43, 940–949. doi: 10.1016/j.molcel.2011.07.023
Robinson, J. T., orvaldsdóttir, H., Winckler, W., Guttman, M., Lander, E. S.,
Getz, G., et al. (2011). Integrative genomics viewer. Nat Biotechnol 29, 24–26. doi:
10.1038/nbt.1754
Rodionov, D. A., De Ingeniis, J., Mancini, C., Cimadamore, F., Zhang, H., Osterman, A. L.,
et al. (2008). Transcriptional regulation of NAD metabolism in bacteria: NrtR family of Nudix-
related regulators. Nucleic Acids Res. 36, 2047–2059. doi: 10.1093/nar/gkn047
Ruiz-Larrabeiti, O., Benoni, R., Zemlianski, V., Hanišáková, N., Schwarz, M.,
Brezovská, B., et al. (2021). NAD+ capping of RNA in Archaea and mycobacteria.
BioRxiv. doi: 10.1101/2021.12.14.472595
Rychel, K., Decker, K., Sastry, A. V., Phaneuf, P. V., Poudel, S., and Palsson, B. O.
(2021). iModulonDB: a knowledgebase of microbial transcriptional regulation derived
from machine learning. Nucleic Acids Res. 49, D112–D120. doi: 10.1093/nar/gkaa810
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., et al.
(2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9,
676–682. doi: 10.1038/nmeth.2019
Song, M.-G., Bail, S., and Kiledjian, M. (2013). Multiple Nudix family proteins possess
mRNA decapping activity. RNA (New York, N.Y.) 19, 390–399. doi: 10.1261/rna.037309.112
Song, M.-G., Li, Y., and Kiledjian, M. (2010). Multiple mRNA decapping enzymes in
mammalian cells. Mol. Cell 40, 423–432. doi: 10.1016/j.molcel.2010.10.010
Srouji, J. R., Xu, A., Park, A., Kirsch, J. F., and Brenner, S. E. (2017). e evolution of
function within the Nudix homology clan. Proteins 85, 775–811. doi: 10.1002/prot.25223
Szabó, Z., Sani, M., Groeneveld, M., Zolghadr, B., Schelert, J., Albers, S. V., et al.
(2007). Flagellar motility and structure in the hyperthermoacidophilic archaeon
Sulfolobus solfataricus. J. Bacteriol. 189, 4305–4309. doi: 10.1128/JB.00042-07
Tachdjian, S., and Kelly, R. M. (2006). Dynamic metabolic adjustments and genome
plasticity are implicated in the heat shock response of the extremely thermoacidophilic
archaeon Sulfolobus solfataricus. J. Bacteriol. 188, 4553–4559. doi: 10.1128/JB.00080-06
van der Kolk, N., Wagner, A., Wagner, M., Waßmer, B., Siebers, B., and Albers, S. V.
(2020). Identication of XylR, the activator of arabinose/xylose inducible regulon in
Sulfolobus acidocaldarius and its application for homologous protein expression. Front.
Microbiol. 11:1066. doi: 10.3389/FMICB.2020.01066
Varadi, M., Anyango, S., Deshpande, M., Nair, S., Natassia, C., Yordanova, G., et al.
(2022). AlphaFold protein structure database: massively expanding the structural
coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50,
D439–D444. doi: 10.1093/nar/gkab1061
Wagner, M., van Wolferen, M., Wagner, A., Lassak, K., Meyer, B. H., Reimann, J., et al.
(2012). Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius. Front.
Microbiol. 3, 1–12. doi: 10.3389/fmicb.2012.00214
Walters, R. W., Matheny, T., Mizoue, L. S., Rao, B. S., Muhlrad, D., and Parker, R.
(2017). Identication of NAD+ capped mRNAs in Saccharomyces cerevisiae. Proc. Natl.
Acad. Sci. U. S. A. 114, 480–485. doi: 10.1073/pnas.1619369114
Weixler, L., Feijs, K. L. H., and Zaja, R. (2022). ADP-ribosylation of RNA in
mammalian cells is mediated by TRPT1 and multiple PARPs. Nucleic Acids Res. 50,
9426–9441. doi: 10.1093/nar/gkac711
Xia, Q., Wang, T., Hendrickson, E. L., Lie, T. J., Hackett, M., and Leigh, J. A. (2009).
Quantitative proteomics of nutrient limitation in the hydrogenotrophic methanogen
Methanococcus maripaludis. BMC Microbiol. 9, 1–10. doi: 10.1186/1471-2180-9-149