Ms1 RNA Interacts With the RNA Polymerase Core in Streptomyces coelicolor and Was Identified in Majority of Actinobacteria Using a Linguistic Gene Synteny Search

Abstract

Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.

Full text

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ORIGINAL RESEARCH
published: 11 May 2022
doi: 10.3389/fmicb.2022.848536
Edited by:
Damien Paul Devos,
Andalusian Center for Development
Biology (CSIC), Spain
Reviewed by:
Dagmara Jakimowicz,
University of Wrocław, Poland
Guoqing Niu,
Southwest University, China
*Correspondence:
Jarmila Hnilicová
hnilicova@biomed.cas.cz
Josef Pánek
panek@biomed.cas.cz
Specialty section:
This article was submitted to
Evolutionary and Genomic
Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 04 January 2022
Accepted: 22 February 2022
Published: 11 May 2022
Citation:
Va ˇ
nková Hausnerová V,
Marvalová O, Šiková M, Shoman M,
Havelková J, Kambová M,
Janoušková M, Kumar D, Halada P,
Schwarz M, Krásný L, Hnilicová J and
Pánek J (2022) Ms1 RNA Interacts
With the RNA Polymerase Core
in Streptomyces coelicolor and Was
Identified in Majority of Actinobacteria
Using a Linguistic Gene Synteny
Search. Front. Microbiol. 13:848536.
doi: 10.3389/fmicb.2022.848536
Ms1 RNA Interacts With the RNA
Polymerase Core in Streptomyces
coelicolor and Was Identified in
Majority of Actinobacteria Using a
Linguistic Gene Synteny Search
Viola Va ˇ
nková Hausnerová1, Olga Marvalová1, Michaela Šiková1, Mahmoud Shoman1,
Jarmila Havelková1, Milada Kambová1, Martina Janoušková1, Dilip Kumar1,
Petr Halada2, Marek Schwarz3, Libor Krásný1, Jarmila Hnilicová1*and Josef Pánek3*
1Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences,
Prague, Czechia, 2Laboratory of Structural Biology and Cell Signaling, Institute of Microbiology of the Czech Academy
of Sciences, Vestec, Czechia, 3Laboratory of Bioinformatics, Institute of Microbiology of the Czech Academy of Sciences,
Prague, Czechia
Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is
an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular
level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a
different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs
are widespread in the bacterial kingdom except for the industrially and medicinally
important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is
not clear whether Ms1 RNA is present also in other Actinobacteria species. Here,
using a computational search based on secondary structure similarities combined
with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In
S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with
an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates
with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we
applied the computational approach to other Actinobacteria and identified Ms1 RNA
candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of
RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational
approach to identify weakly conserved sRNAs in evolutionarily distant genomes.
Keywords: sRNA, Actinobacteria, Ms1 RNA, Streptomyces, gene synteny, Mycobacterium, 6S RNA
INTRODUCTION
Small non-coding RNAs (sRNAs) are important regulators of gene expression in bacteria.
A majority of sRNAs act by base-pairing to target mRNAs and change mRNA stability or translation
but a minor group of sRNAs directly regulates proteins through RNA-protein interaction (Svensson
and Sharma, 2016). The best-known example is 6S RNA that interacts with the RNA polymerase
(RNAP) holoenzyme (Wassarman and Storz, 2000;Klocko and Wassarman, 2009;Steuten et al.,
2013;Chen et al., 2017;Wassarman, 2018). The RNAP holoenzyme is composed of the catalytic
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core (E, subunits α2ββ0ω) and the primary σfactor that is
required to recognize promoters of housekeeping genes to initiate
transcription. All bacteria contain this housekeeping or primary
σfactor, termed σ70 in Escherichia coli,σAin Bacillus subtilis and
in Mycobacterium smegmatis (Gomez et al., 1998), or HrdB in
Streptomyces (Brown et al., 1992). In addition, bacterial species
contain different numbers of alternative sigma factors ranging
from zero in Mycoplasma genitalium (Fraser et al., 1995) to
almost 70 in Streptomyces coelicolor (Bentley et al., 2002).
6S RNA was first described in E. coli (Hindley, 1967) where
it interacts with the RNAP-σ70 holoenzyme (Eσ70) (Wassarman
and Storz, 2000) and regulates the expression of hundreds of
genes (Cavanagh et al., 2008;Neusser et al., 2010). 6S RNA
itself also serves as a template for transcription of pRNA, a
short RNA of ≤20 nucleotides (Wassarman and Saecker, 2006;
Beckmann et al., 2011;Hoch et al., 2016). Transcription of pRNA
rearranges the structure of 6S RNA, releasing Eσfrom 6S RNA
(Wurm et al., 2010;Beckmann et al., 2012;Cavanagh et al., 2012;
Panchapakesan and Unrau, 2012;Burenina et al., 2014).
6S RNA has a conserved secondary structure that is critical
for the interaction with Eσ70 (Barrick et al., 2005;Trotochaud
and Wassarman, 2005;Shephard et al., 2010). 6S RNA forms a
double-stranded hairpin like structure with a central, unpaired
bubble region which resembles an open promoter (Barrick et al.,
2005;Wassarman, 2018). Many 6S RNAs were predicted from
the genomic sequences based on secondary structure similarities
(Barrick et al., 2005;Trotochaud and Wassarman, 2005;Wehner
et al., 2014). Alternatively, 6S RNAs were directly identified as
abundant ∼180–200 nt RNAs in B. subtilis,Bordetella pertussis,
Pseudomonas aeruginosa or Caulobacter crescentus (Vogel et al.,
1987;Barrick et al., 2005;Trotochaud and Wassarman, 2005)
or 6S RNAs were discovered after the detection of their
complementary pRNAs (Sharma et al., 2010;Köhler et al., 2015).
The intracellular levels of 6S RNAs are high, similar to
those of essential non-coding RNAs, such as rRNAs, tRNAs,
RNAse P, tmRNA or SRP RNA. Furthermore, 6S RNAs have
been found in many bacterial species and are widespread in the
bacterial kingdom (Trotochaud and Wassarman, 2005;Faucher
et al., 2010;Sharma et al., 2010;Rediger et al., 2012;Wehner
et al., 2014;Köhler et al., 2015;Jones et al., 2016;Elkina et al.,
2017;Burenina et al., 2020) with one exception—the group of
Actinobacteria. This phylum includes serious human pathogens
(Mycobacterium tuberculosis,Mycobacterium leprae, and
Corynebacterium diphtheria), industrially important producers
of amino acids (Corynebacterium glutamicum) and antibiotics
(Streptomyces), bacteria involved in symbiotic nitrogen fixation
(Frankia), bacteria utilized for bioremediation (Rhodococcus),
probiotic bacteria (Bifidobacterium), and numerous other genera
(Nocardia,Micrococcus,Gardnerella).
In Actinobacteria, 6S RNAs had been undetected for a long
time. In Mycobacterium smegmatis, we identified a putative
6S RNA candidate by the computational suboptimal secondary
structure approach (Pánek et al., 2011). However, we discovered
that it interacted with the RNAP core (E) without the primary
sigma factor (Hnilicova et al., 2014) and, thus, by definition, was
not a 6S RNA and we therefore named it Ms1. In Mycobacterium
smegmatis, Ms1 accumulates during the stationary phase of
growth, regulates the RNAP level, and this facilitates cell
outgrowth from stationary phase (Sikova et al., 2019).
Ms1 RNA is longer than a typical 6S RNA (∼300 nt vs.
∼180 nt) and their secondary structures differ as well—in
addition to the central bubble, Ms1 has two additional short
hairpins at its 30and 50ends (Hnilicova et al., 2014). An Ms1
homolog was also found in M. tuberculosis (MTS2823 sRNA)
(Arnvig et al., 2011). Other Ms1 homologs were identified only
in closely related species within the Actinobacteria group (genera
Mycobacterium,Rhodococcus and Nocardia) (Hnilicova et al.,
2014;Behra et al., 2019). Currently, it is unclear whether Ms1
RNA is present only in the three above-mentioned bacterial
genera or is widespread throughout Actinobacteria.
In Streptomyces coelicolor, a putative 6S RNA (the gene
was named ssrS) was identified (Panek et al., 2008), but
the experimental proof that ssrS encodes 6S RNA was based
on an in vitro interaction between the purified RNAP-HrdB
holoenzyme and in vitro transcribed 6S RNA (Mikulík et al.,
2014). In addition, this interaction was detected only after UV
crosslinking (Mikulík et al., 2014). Moreover, in vitro transcribed
ssrS gene only partially overlaps with the in vivo detected scr3559
sRNA identified by RNA-seq (Vockenhuber et al., 2011;Moody
et al., 2013) which is expressed from the same genomic locus
(sco3558-sco3559 intergenic region) and thus it is unclear if the
putative 6S RNA sequence is expressed in vivo.
Recently, (Bobek et al., 2021) reported a new 6S RNA in
S. coelicolor, which was named “6S-like scr3559 RNA.” It is
expressed from the sco3558-sco3559 genomic locus but from the
minus strand (the previously published 6S RNA sequence was
expressed from the plus strand), with its transcription start site
located at position 3,934,888 in the genome (Bobek et al., 2021).
This 6S-like RNA is processed and its 50end corresponds to
position 3,934,820 (Bobek et al., 2021). However, the processed
6S-like RNA was almost undetectable in wild type S. coelicolor
by Northern blotting (Bobek et al., 2021). In addition, the 6S-like
RNA does not correspond to the scr3559 sRNA that is transcribed
from the plus strand with the transcription start site at position
3,934,693 (Moody et al., 2013;Jeong et al., 2016). Both sequences
of the putative 6S RNAs (Mikulík et al., 2014;Bobek et al.,
2021) differ from scr3559, although scr3559 is the main transcript
derived from the sco3558-sco3559 intergenic region as detected
by RNA-seq (Vockenhuber et al., 2011;Moody et al., 2013). The
function of scr3559 is unknown.
To elucidate whether Ms1 RNA is conserved in the
phylogenetic group of Actinobacteria, we combined a homology
search based on evolutionary conservation of RNAs with a
bioinformatic linguistic search for genomic context (synteny) of
RNA genes to identify Ms1 candidate genes. First, we applied
this approach to Streptomyces coelicolor. Our search identified
a Ms1 candidate—the scr3559 sRNA. We validated this result
experimentally, demonstrating that scr3559 RNA binds the
RNAP core without the primary σfactor HrdB in vivo and, thus,
is an Ms1 but not a 6S RNA homolog. Subsequently, we applied
our linguistic search to the phylum of Actinobacteria, revealing
that Ms1 is present in most orders of Actinobacteria. Ms1 is thus
a new type of regulatory RNA associated with RNA polymerase,
in addition to 6S RNA, B2 RNA (regulates RNA polymerase
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II in humans) (Allen et al., 2004;Espinoza et al., 2004), or
svRNAs (regulates the Influenza A virus RNA-dependent RNA
polymerase) (Perez et al., 2010;Perez et al., 2012).
MATERIALS AND METHODS
Computational Homology Search for
New sRNAs
For the search, 10 selected Streptomyces well annotated genomes
were used (GenBank assembly IDs shown in parenthesis
following organism name): S. coelicolor A3(2) (GCA_000203835.
1_ASM20383v1), S. ambofaciens ATCC 23877 (GCA_00126788
5.1_ASM126788v1), S. nodosus (GCA_000819545.1_ASM81954
v1), S. reticuli (GCA_001511815.1_TUE45), S. avermitilis MA
4680 (GCA_000009765.2_ASM976v2), S. griseus NBRC (GCA_0
00010605.1_ASM1060v1), S. scabies 87 22 (GCA_000091305.1_
ASM9130v1), S. cattleya NRRL8057 (GCA_000237305.1_ASM
23730v1), S. lincolnensis (GCA_001685355.1_ASM168535v1,
S. pristinaespiralis ATCC 25486 (GCA_000154945.1_ASM
15494v1).
The search was performed in the following steps:
(i) Intergenic regions (IGRs) were identified in the 10 selected
Streptomyces genomes, according to GenBank genomic
annotations. IGRs in both DNA strands were searched,
hence, an IGR in one strand may overlap with an open
reading frame encoded by the other strand.
(ii) The IGR sequences were sampled with approximate
nucleotide lengths of 180 and 300 nucleotides that are
specific for 6S and Ms1 RNAs, respectively. The sampling
was done in a sliding window, moving sequence window by
5 nucleotides a step through IGR sequences.
(iii) 10 suboptimal secondary structures were predicted for each
sampled sequence by UNAfold (Markham and Zuker, 2008)
with parameters P= 1000, W= 1 and X= 10.
(iv) The suboptimal structures were matched to secondary
structures of B. subtilis 6S RNA (Trotochaud and
Wassarman, 2005;Beckmann et al., 2012;Burenina et al.,
2014) (for 6S RNA-length sequences) and M. smegmatis
Ms1 RNA (Hnilicova et al., 2014) (for Ms1 RNA-
length sequences) that were used as structural templates
to get their pairwise structural similarity scores using
RNAdistance (Lorenz et al., 2011).
(v) The average of three best scores for each sampled sequence
was compared to the structural similarity thresholds of
80 and 135 for 6S RNA and Ms1 RNA, respectively. The
thresholds were chosen such that we were able to identify
known 6S and Ms1 RNAs by the search. To be considered
as a candidate for one of the searched for sRNAs, the
candidate sequence had to have the averaged similarity
score of three most similar suboptimal structures better
than the thresholds.
To increase reliability of the search, the candidates had to
fulfill the following additional criteria derived from genomic
properties of known 6S and Ms1 RNAs that were: (1) existence
of similar sequences in evolutionarily close species identified
by BLAST with BLAST E-values <10−20, and (2) conserved
genomic position in the ten searched Streptomyces genomes.
Computational Prediction of Full
Sequences of New Streptomyces sRNAs
The full-length sequences were predicted using the approximate
length of the transcripts detected by Northern blotting and the
genomic positions of oligonucleotide sequences used for sRNA
verification by Northern blotting (Figure 1). The sequences of
approximate length were constructed using the oligonucleotides
positioned at the 50end, center, and 30end. The three
constructed sequences were BLASTed against genomes of the ten
Streptomyces species listed in the previous paragraph. As sRNAs
in general are conserved in evolutionarily related species, here
in Streptomyces species, the constructed sequences had to be
identified by BLAST in multiple Streptomyces species to ensure
that they were constructed correctly. Using the superposition of
constructed sequences according to their conservation identified
by BLAST, we reconstructed probable sRNA sequences in
Streptomyces coelicolor. The probable sequences are shown in
Supplementary Table 1 for expressed Streptomyces sRNAs.
Linguistic Search for Ms1 RNAs in
Actinobacteria
The search was based on text phrases in genomic annotations
of the genes flanking potential Ms1 RNA genes and sequence
similarity search based on BLAST for verification. The search was
possible as sRNAs including Ms1 RNA are known to be conserved
in evolutionarily close bacteria (Hnilicova et al., 2014). The search
has several steps:
(i) Synteny analysis. Specific words in annotations of 50and
30end flanking genes of known and newly discovered
IGR containing Ms1 RNAs are identified. Among them,
most repeating specific words are identified. This step is
explained in detail in the following section.
(ii) Synteny phrases. Text phrases, comprising of at least one
most repeating specific word, that are specific for genomic
annotation of genes flanking Ms1 RNAs are generated.
Synteny phrases used in this work are shown in Tables 1–3.
(iii) Text search for Ms1 synteny phrases in genomic
annotations of Actinobacteria genera which had >4
annotated genomes and in which Ms1 RNA has not
yet been discovered. The search is implemented as
a sequence of grep LINUX searches, followed by
additional text processing implemented in MATLAB
computational environment.
(iv) Synteny hit. An IGR with Ms1 synteny containing a
putative Ms1 RNA gene.
(v) Evolutionary conservation of the synteny hit. The hit was
considered as evolutionarily conserved when there existed
IGRs with similar sequence and Ms1 synteny for at least one
of the flanking genes.
The IGRs with similar sequences were identified by BLAST
of the sequence of the synteny hit in NCBI’s nt database
with sensitive setting for cross-species exploration (with
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parameters -r 1 -q 1 -G 1 -E 2 -W 7 (Korf et al., 2003) with
BLAST E-value threshold set to 0.05).
Annotations of flanking genes of the identified IGRs
were checked for Ms1 synteny phrases. The annotations
contained either Ms1 phrases, or their synonyms, or
contained new phrases. When contained new phrases, only
one of the flanking genes was allowed to contain them
and at the same time they had to repeat in annotations
of multiple IGRs. Both new phrases and synonyms were
used to update old phrases for the next synteny search
iteration (Figure 5).
If the synteny hit was not found to be evolutionarily
conserved, it was not used.
(vi) Flanking genes annotations of both synteny hit and IGRs
homologous to it in new, related species. They were
analyzed to get their specific words [step (ii)] and to update
old synteny phrases [step (iii)].
Synteny Analysis
Annotations of flanking genes were first split into single words
and non-specific words were removed. The non-specific words
were (allowing for grammar errors): ‘hypothetical’, ‘protein’, ‘type’,
‘family’, ‘domain’, ‘putative’, ‘precursor’, ‘component’, ‘subunit’,
‘subfamily’, ‘conserved’, ‘chain’, ‘or’, ‘superfamily’, ‘unknown’,
‘function’, ‘of’, ‘like’, ‘containing’, ‘II’, ‘and’, ‘short’, ‘dependent’,
‘probable’, ‘associated’, ‘to’, ‘that’, ‘the’, ‘predicted’, ‘uncharacterized’,
‘production’, ‘proteases’, ‘fold’, ‘in’, ‘by’, ‘universal’, ‘pathway’,
‘involved’, ‘related’, ‘general’, ‘group’, ‘sequence’, ‘class’, ‘cluster’,
‘accepting’, and ‘determining’.
We also removed the so-called questionable non-specific
words whose (non)specificity depended on the analyzed
context. They were: ‘box’, ‘enzyme’, ‘factor’, ‘secreted’, ‘release’,
‘neighborhood’, ‘solute’, ‘accessory’, ‘peptide’, ‘biosynthesis’,
and ‘substrate’.
Specific words were left. They characterized specific aspects
of gene function. An example is the ‘Fic/Doc family protein’
annotation contained two non-specific words, ‘family’ and
‘protein’, and one specific word ‘Fic/Doc’.
The first synteny phrases were obtained from annotations
of homologs of M. smegmatis Ms1 RNA identified by
sequence similarity by BLAST in 498 species of 8 genera—
Mycobacterium,Mycolicibacterium,Rhodococcus, Nocardia,
Gordonia,Mycobacteroides,Hoyosella, and Tsukamurella
(Supplementary Figure 1). Specific words were selected
as those occurring in annotations of flanking genes above
the average (Supplementary Figure 1). The specific words
were ‘oxidoreductase’, ‘hydrolase’, ‘HAD’, ‘IB’, ‘morphological’,
‘differentiation’, ‘inhibition’, ‘phosphoserine’ and ‘phosphatase’.
The occurrence in genera was used rather than the occurrence
in species as there were genera with many species with repeating
annotations with repeating specific words causing bias of an
occurrence of certain words.
Phrases were generated from the most frequent specific
words according to their co-occurrence in the annotations
from which they were extracted and which defined their
semantic binding. For example, specific words ‘hydrolase’, ‘HAD’,
‘IB’, ‘morphological’, ‘differentiation’, ‘inhibition’, ‘phosphoserine’
and ‘phosphatase’ (identified in Supplementary Figure 1 as
most occurring) formed the following phrases: ‘IB HAD’,
‘HAD hydrolase’, ‘inhibition morphological differentiation’ and
‘phosphoserine phosphatase’. A complete list of first phrases is in
Table 1.
Synteny phrases were repeatedly updated after identification
of Ms1-containing IGRs in new species (Supplementary
Figures 2–4). New annotations either contained synonyms to
old phrases, e.g., oxidoreductase, whose synonym was Fic that
also has oxidoreductase activity or contained new annotation
phrases. The synonyms were identified using information found
on the internet, most often in Wikipedia and/or many various
public protein databases/knowledgebases. New annotations were
considered as Ms1-syntenous when they repeated for one
flanking gene of newly identified Ms1 IGRs in many species, while
annotation of the other flanking gene must have contained the
known Ms1 phrase. New annotations were analyzed to get new
phrases out of them that were merged with old phrases as can be
seen in Tables 2,3.
Bacterial Strains, Growth Conditions
Mycobacterium smegmatis mc2155 cells ATCC 700084 (wt,
LK865) were grown at 37◦C in Middlebrook 7H9 medium with
0.2% glycerol and 0.05% Tween 80, and harvested in exponential
(OD600 ∼0.5) or early stationary phase (OD600 ∼2.5–3, 24 h of
cultivation). S. coelicolor A3(2) spore stock expressing HA-tagged
HrdB was thawed and inoculated to 2YT medium. Germination
was carried out at 30◦C for 5 h as described previously (Moody
et al., 2013;Šmídová et al., 2019). The germinated spores
were harvested by centrifugation (3200 ×g, 25◦C, 5 min),
inoculated into Na-glutamate medium supplemented with trace
element solution and TMS1 as described previously (Šmídová
et al., 2019), cultivated at 30◦C and harvested by centrifugation
(3200 ×g, 4◦C, 5 min) at different time points after germination.
Rhodococcus erythropolis CCM259 (Strnad et al., 2014) (LK1556)
was cultivated at 26◦C in 2x TY medium to exponential phase
(OD600 ∼2) or stationary phase (OD600 ∼11, 24 h of cultivation),
Corynebacterium glutamicum ATCC 13032 (LK1557) was grown
in 2xTY at 30◦C and harvested in exponential (OD600 ∼1) or
stationary phase (OD600 ∼8, 24 h of cultivation). Bacillus subtilis
168 strain was grown in LB medium at 37◦C to exponential phase
(OD600 ∼0.3) or stationary phase (OD600 ∼4).
RNA Isolation and Staining, Northern
Blotting
Each frozen cell pellet was resuspended in 240 µl TE (pH 8.0)
plus 60 µl LETS buffer (50 mM Tris–HCl pH 8.0, 500 mM
LiCl, 50 mM EDTA pH 8.0, 5% SDS) and 600 µl acidic
phenol (pH∼3):chloroform (1:1). Lysates were sonicated in a
fume hood, centrifuged, the aqueous phase extracted three more
times with acidic phenol (pH∼3): chloroform and precipitated
with ethanol. RNA was dissolved in water, treated with DNase
(TURBO DNA-free Kit, Ambion) and visualized on a 7 M urea
7% polyacrylamide gel by staining with GelRed (Labmark).
RNAs were resolved on a 7% polyacrylamide gel and
transferred onto an Amersham Hybond-N membrane according
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to the protocol described in Pánek et al. (2011). 50biotinylated
oligonucleotide probes were hybridized to the membrane and
detected with BrightStar BioDetect Kit (Ambion) according to
manufacturer’s instructions. For Northern blot probes sequences,
see the Supplementary Material.
50RACE
The protocol was adopted from Martini et al. (2019). An
adapter oligo CTGGAGCACGAGGACACTGACATGGACTG
AAGGAGTagaaa (lower case letters are ribonucleotides, upper
case letter deoxyribonucleotides) was ligated to RNA 50ends. 5
µg DNase-treated RNA was treated with RppH (NEB). Treated
and untreated RNA samples (8 µl) were mixed with 1 µl of
1µg/µl adapter oligo and incubated at 65◦C for 10 min, then
ligation reaction was set up including 10 µl 50% PEG8000,
3µl 10X ligase buffer, 3 µl 10 mM ATP, 3 µl DMSO, 1 µl
Murine RNase inhibitor (NEB), and 1 µl T4 ligase (NEB).
Ligation was performed at 20◦C overnight and RNA cleaned
using RNA Clean and Concentrator 25 kit (Zymo). RNA was
reverse-transcribed into cDNA (SuperScript III, Invitrogen)
with random hexamers. PCR was done using a forward
primer CTGGAGCACGAGGACACTGA and reverse (gene
specific) primers (Str11, 50-AGCCGCTCCCCTGGTCTGGG-
30, Ms1 candidate 50-GGTGTCCATGCTCGGTCC-30). The
nucleotide position of the TSS was taken from EMBL/GenBank
Accession No. AL645882.
30RACE
The protocol was adopted from Sedlyarova et al. (2017).
550 pmol of 50-phosphorylated RNA adaptor (50P -
AAUGGACUCGUAUCACACCCGACAA-30) was ligated to
6µg of total DNase treated RNA using T4 RNA Ligase 1 (ssRNA
Ligase, New England Biolabs) according to the manufacturer’s
protocol overnight at 16◦C. RNA was purified with RNA Clean
and Concentrator 25 kit (Zymo) and reverse-transcribed
into cDNA (SuperScript III, Invitrogen) with 30RACE
specific primer (50-TTGTCGGGTGTGATACGAGTCCATT-
30). The same primer was used as reverse primer for
PCR together with gene specific forward primer (50-
GATCACCTTAAACACGCATATGG-30).
Immunoprecipitation and RT-qPCR
Streptomyces coelicolor cells expressing HA-tagged HrdB were
pelleted and resuspended in lysis buffer (20 mM Tris–HCl pH
7.9, 150 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT),
0.5 mM phenylmethylsulfonyl fluoride (PMSF), supplemented
with Calbiochem Protease Inhibitor Cocktail Set III protease
inhibitors), sonicated 15 ×10 s with 1 min pauses on ice and
centrifuged. 500 µg (protein) of lysates were incubated for 16–
18 h at 4◦C with 20 µl of Protein G plus agarose beads (Santa
Cruz) coated with 5 µg mouse monoclonal anti-βsubunit of
RNAP antibody [clone 8RB13] (BioLegend), 2.5 µg of anti-σ70
antibody [clone 2G10] (BioLegend), 1.25 µg of anti-HA antibody
[clone HA-7] (Sigma-Aldrich) or 5 µg of mouse non-specific
IgG (Sigma-Aldrich) used as a negative control, respectively. The
captured complexes were washed 4 times using 20 mM Tris–
HCl pH 7.9, 150 mM KCl, 1 mM MgCl2, finally resuspended in
300 µl 20 mM Tris–HCl pH 7.9, 150 mM KCl, 1 mM MgCl2and
divided into two parts. One third of the beads were incubated
in SDS sample buffer for 5 min at 95◦C and eluted proteins
were detected by Coomassie staining and Western blotting.
The remaining two thirds of the beads were resuspended in
200 µl 1% SDS, 150 mM KCl, 20 mM Tris–HCl pH 7.9, 1 mM
MgCl2and incubated on a rotating platform with 200 µl acidic
phenol (pH∼3):chloroform (1:1) for 15 min. Eluted RNA was
precipitated with ethanol, dissolved in water and DNase treated
(TURBO DNA-free Kit, Ambion). RNA was reverse transcribed
into cDNA (SuperScript III, Invitrogen) using random hexamers
and amplified by quantitative reverse transcription PCR (RT-
qPCR) in a LightCycler 480 System (Roche Applied Science) in
duplicate reactions containing LightCycler 480 SYBR Green I
Master and 0.5 µM primers (each). Primers were designed with
Primer3 software and their sequences are in the Supplementary
primer list. Negative controls (no template reactions and
reactions with RNA as a template to control for contamination
with genomic DNA) were run in each experiment, the quality of
the PCR products was determined by dissociation curve analysis,
and the efficiency of the primers determined by standard curves.
The relative amounts of co-immunoprecipitated RNAs were
quantified on the basis of the threshold cycle (Ct) for each PCR
product that was normalized to input values according to the
formula 2∧[Ct(immunoprec)–Ct(input)].
Western Blotting
Proteins were detected by Western blotting using a rat
monoclonal antibody recognizing the HA tag conjugated with
HRP [clone BMG-3F10] or a mouse monoclonal antibody
recognizing βsubunit of RNA polymerase [clone 8RB13] in a
combination with secondary antibody conjugated with HRP.
RESULTS
Computational Search for 6S RNA and
Ms1 RNA Candidates in Streptomyces
First, we conducted a computational homology search for
putative 6S RNA/Ms1 RNA in the Streptomyces genus.
Using this computational search, we identified 12 candidate
genes for 6S/Ms1 RNAs in Streptomyces (see Supplementary
Material). Interestingly, flanking genes of one of them (Str11,
Supplementary Table 1) displayed annotations syntenous to
6S-1 RNA in Firmicutes (Wehner et al., 2014), making it a
prime candidate for Streptomyces 6S RNA. Nevertheless, none
of the putative identified sRNA genes had the synteny of
mycobacterial Ms1 RNAs. We suspected that the search might
have not identified all candidate genes (for reasons see section
“Discussion”). Therefore, prior to the experimental validation,
we extended the homology search with linguistic synteny
analysis approach.
The linguistic approach was based on search for text phrases
that were specific to Ms1 RNA synteny and could be found in
Ms1 flanking genes annotations. Note that meaning of the term
‘synteny’ in this work is ‘conserved genomic context’. Synteny
annotations are annotations of conserved flanking genes of Ms1
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TABLE 1 | Synteny phrases for linguistic search for Ms1 RNA in Streptomyces.
‘oxidoreductase’
‘inhibition morphological differentiation’
‘IB HAD’
‘HAD hydrolase’
‘phosphoserine phosphatase’
The phrases were extracted from synteny annotations of Mycobacteria Ms1 RNA
homologs (Supplementary Figure 1). Columns contains phrases for individual
flanking genes. Rows indicate pairing of phrases for both flanking genes. Cells
contain semantic synonyms (different phrases for the same protein function). Italics
indicates functional synonyms (phrases denoting different aspects of proteins with
the same function).
RNAs. The phrases were used to identify new IGRs containing
putative Ms1 RNAs in other species.
Synteny was rarely employed previously for identification of
sRNAs (Sridhar and Gunasekaran, 2013), mostly because the
sRNAs and their synteny occurred only in certain phyla, e.g.,
in Enterobacteriaceae (Sridhar and Rafi, 2007) and were not
widespread in bacteria. Nevertheless, 6S RNA is an example of a
sRNA identified throughout the bacterial kingdom with synteny
conserved in specific taxons—for example in Enterobacteriaceae
(γ-Proteobacteria) (Wehner et al., 2014). Although no 6S RNA
syntenic pattern is valid for all bacteria, some proteins frequently
occur in the syntenic regions of the 6S RNA throughout
the bacterial kingdom. For example, ygfA, which encodes 5-
formyltetrahydrofolate cyclo-ligase, is found adjacent to 6S
RNA gene in α-, γ-Proteobacteria, and some species from β-
Proteobacteria,δ-Proteobacteria, or Firmicutes (Barrick et al.,
2005;Wehner et al., 2014). Therefore, as Ms1 and 6S RNAs
are structurally and functionally similar, we assumed that
Ms1 flanking genes would be at least partially conserved in
Actinobacteria similarly to 6S RNA in γ-Proteobacteria.
Annotations of flanking genes of Ms1 RNAs identified
previously in Mycobacterium,Rhodococcus, and Nocardia were
conserved (Hnilicova et al., 2014). We speculated that the
conservation would also be kept in those Actinobacteria where
Ms1 RNA had not been identified. To verify this assumption,
we first identified homologs of M. smegmatis Ms1 RNA by
sequence similarity using BLAST in 498 species of 8 genera and
analyzed annotations of their flanking genes. As expected, we
found it conserved (Supplementary Figure 1). Therefore, we
extracted synteny text phrases (Table 1) from the annotations and
used them to search genomic annotations for their occurrence
indicating putative Ms1 RNAs.
Intergenic regions with flanking genes with Ms1 RNA-
specific synteny phrases (synteny hits) were found in
numerous Streptomyces species for both ‘HAD hydrolase’
and ‘inhibition morphological differentiation’ phrases paired
with the ‘oxidoreductase’ phrase. For example, in the first of
the Streptomyces species, S. actuosus, a total of six hits of ‘HAD
hydrolase’ were obtained. Only one of them fulfilled the other
criteria of the synteny search, which was Ms1 synteny phrase
(‘oxidoreductase’) in annotation of the other flanking gene
and the length of IGR between flanking genes larger than 300
nucleotides. This IGR was considered as an IGR containing a
putative Streptomyces Ms1 RNA.
The next criterion was an evolutionary conservation of the
candidate Ms1 IGR in related species, i.e., in other Streptomyces
species, analogously to Ms1 RNA from M. smegmatis conserved
in other Mycobacteria. Therefore, the sequence of the S. actuosus
Ms1 IGR was BLASTed against the NCBI nt database, which
produced ∼250 BLAST hits in 188 different Streptomyces species
(Supplementary Figure 6) with E-values <1×10−24, i.e.,
strong sequence similarity and with a similar position in the
middle of the linear Streptomyces chromosome (Figure 2A).
Both sequence similarity and similar genomic loci indicated
evolutionary conservation thus suggesting that the IGRs
contained Streptomyces Ms1 RNAs.
Among the BLAST hits, a 419 nucleotides long IGR in
S. coelicolor A3(2) (ENA ID AL645882.2) were identified at
genomic locus 3934559: 3934978. To find out where within this
IGR a putative Ms1 RNA was, the 419 nt sequence was sampled
with 200–300 nt subsequences in 5 nt steps for which suboptimal
secondary structures were predicted using UNAfold (Markham
and Zuker, 2008) and compared to the secondary structure of
M. smegmatis Ms1 RNA used as a structural template. This way
we aimed at identification of a subsequence of the IGR able
to adopt a Ms1 RNA-like secondary structure, thus identifying
a local position of Ms1 RNA. The Ms1 RNA-like secondary
structures were obtained with 220–235 nt subsequences at
positions 126–141 downstream of 50end of the IGR sequence.
Note that this sequence of the putative S. coelicolor Ms1 RNA
had no similarity detectable by cross-species exploration BLAST
to the sequence Ms1 RNA from M. smegmatis.
To summarize this part, we identified a total of 13 (12 + 1)
potential 6S/Ms1 RNA candidate genes in S. coelicolor.
Expression of Potential 6S/Ms1 RNAs
Next, we used Northern analysis to determine expression of these
putative sRNA, probing their expression from both strands. The
analysis revealed that several of them were expressed, to various
degrees, in exponential and stationary phases in S. coelicolor
(Figures 1A–F,2C). Expression of the remaining six sRNAs was
not detected. Genomic loci of the new sRNAs are depicted in
Figure 1 and their basic characteristics are described in detail
in the next two sections and summarized in Supplementary
Table 1 (including predicted sequence, position in the S. coelicolor
genome, annotations of flanking genes, and location at the
chromosome in the Streptomyces genus).
Characterization of the Expressed
sRNAs: Str1, Str3, Str5, Str8, and Str10
Str1 sRNA is an antisense RNA (as1791, Figure 1A) to the
tetratricopeptide repeat protein gene (sco1791) and it was found
in two forms (90 and 120 nt). Additionally, for Str1 we detected a
short transcript (100 nt) from the opposite strand, a fragment of
the sco1791 (1260 nt) mRNA.
Str3 localizes to the sco1761 gene and similarly to Str1,
Str3, is also an antisense RNA (130 nt, AS1711, Figure 1B).
Furthermore, we also detected two short transcripts (110 and
80 nt, respectively) from the opposite strand, fragments of the
1086 nt long sco1761 mRNA. The antisense nature of both Str1
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A B C D E
F
G H
FIGURE 1 | (A–F) Expression of predicted sRNAs. Total RNA was isolated from S. coelicolor at 35 (exponential phase of growth), 55 and 65 h after germination and
the expression was detected by Northern blotting. 65 hours of growth represents stationary phase of growth. Orientation and the flanking genes are schematically
shown for each sRNA. For as1791 and as1761 RNAs, fragments of sco1791 and sco1761 transcripts detected by Northern blotting are also shown in (A,B). For
scr1506, 50ends was detected by 50RACE (G,H).
and Str3 sRNAs suggests that these sRNAs might be cis-acting
antisense RNAs and the detected transcripts are fragments of the
respective regulated mRNAs.
Str5 (scr5401, Figure 1C), Str8 (scr3567, Figure 1D), and
Str10 (scr5145, Figure 1E) were expressed from intergenic
regions and their lengths ranged from 120 to 130 nt.
We concluded that the identified genes encode bona fide
sRNAs that are expressed in S. coelicolor but their short
length (<150 nt) excluded them as potential 6S/Ms1 RNAs. 6S
RNA/Ms1 RNA must adopt specific secondary structures and one
of the shortest known 6S RNAs is from Aquifex aeolicus, which is
∼160 nt long (Köhler et al., 2015).
Characterization of the Expressed
sRNAs: Str11 and Str13
Str11 (scr1506), the candidate with the same synteny as 6S-1
RNA in B. subtilis, was long enough (∼220 nt long, Figure 1F)
to be considered as a 6S RNA candidate. We identified
the exact Str11 50end by 50RACE (Figures 1G,H). Str11
also had a 6S-like predicted consensus secondary structure
(Supplementary Figure 5).
Str13, the Ms1 candidate identified by the linguistics search,
partially overlaps with the previously discovered ssrS gene (Panek
et al., 2008), which was proposed to encode a 192 nt long 6S
RNA (Mikulík et al., 2014). The Str13 sequence also overlaps
with the scr3559 sRNA identified by RNA-seq (Vockenhuber
et al., 2011;Moody et al., 2013). In S. coelicolor, both scr3559
and ssrS are located between the sco3558 and sco3559 genes
(Vockenhuber et al., 2011;Moody et al., 2013). The flanking genes
and positions of Str13 in S. coelicolor and Ms1 in M. smegmatis
and MTS2823 in M. tuberculosis are shown in Figure 2B;Arnvig
et al. (2011),Hnilicova et al. (2014).
To start deciphering whether Str13 (Ms1 candidate) or ssrS
(putative 6S RNA) is expressed from the sco3558 -sco3559
intergenic region, we performed Northern blot analysis. We used
a probe that could hybridize to both Str13 and ssrS/6S RNA
and we detected a signal that corresponded to a ∼230 nt RNA
(Figure 2C). This could represent the previously reported 244 nt
long ssrS/6S RNA unprocessed transcript (Mikulík et al., 2014).
Although we also detected a shorter transcript (Figure 2C), the
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D
FIGURE 2 | S. coelicolor Ms1 RNA candidate. (A) Histogram of relative genomic position of a Ms1 candidate, Str13, in 188 Streptomyces species. x-axis shows a
relative genomic position with “0” and “1” corresponding to the terminal arms of the linear Streptomyces genome and with “0.5” corresponding to the middle of
Streptomyces linear genome. y-axis shows percentage of 188 Streptomyces species in which Str13 homologs were identified using sequence similarity. The figure
indicates that the Str13 relative genomic position is conserved in the middle of the linear Streptomyces genomes. (B) Ms1 candidate in S. coelicolor, Ms1 in
M. smegmatis and MTS2823 Ms1 homolog in M. tuberculosis and their flanking genes. In Streptomyces coelicolor, positions of the previously published sRNAs are
also included. The position of 6S-Like scr3559 was adopted from the 6S-Like scr3559 sequence reported in Figure 2B of Bobek et al. (2021). The position of
ssrS/6S RNA was adopted from Mikulík et al. (2014), from the sequences of primers that were used to generate DNA template carrying T7 promoter for in vitro
transcription of 6S RNA. Experiments showing 6S RNA—RNAP interaction were performed with this in vitro transcribed RNA. scr3559 position was adopted form
available S. coelicolor dRNA-seq data (Romero et al., 2014;Jeong et al., 2016;Kim et al., 2020) and RNA-seq data (Vockenhuber et al., 2011;Moody et al., 2013).
(C) ∼230 nt long RNA was detected by Northern blotting with the probe specific to Ms1 candidate/scr3559/(probe 2796). The 50end of Ms1 candidate was
determined by 50RACE (D), 30end by 30RACE (E) and corresponds to the scr3559 sRNA (F). 6S RNA expression was not detected by Northern blotting (G).
(H) Structure of scr3559. (I) Structure of Ms1 RNA from M. smegmatis.
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major isoform was the ∼230 nt long RNA and not the 192 nt
ssrS/6S RNA. The size of the RNA detected by the Northern blot
(∼230 nt) corresponded to the previously published lengths of
scr3559: 235 bp (Vockenhuber et al., 2011) or 227 bp, respectively
(Moody et al., 2013).
Next, we mapped the 50and 30ends of Str13 by 50RACE.
The 50end was identified at position 3,934,693 (Figure 2D) that
is 52 nucleotides downstream from the 50end of the previously
annotated ssrS/6S RNA (Mikulík et al., 2014;Figure 2F) and
134 nt downstream from 50end of IGR. This position agrees
with the computationally predicted genomic locus of the putative
S. coelicolor Ms1 (Str13) RNA - predicted Ms1 starts 126–141 nt
downstream from 50end of IGR. This 50RACE result matches
the 50end of scr3559 as determined by RNA-seq (Vockenhuber
et al., 2011;Moody et al., 2013) and dRNA-seq (Romero et al.,
2014;Jeong et al., 2016;Kim et al., 2020) in S. coelicolor. We
also searched for additional transcription start sites in previously
published data and found position 3,933,713 (Kim et al., 2020)
that was 20 nucleotides upstream of the 50end of scr3559
but also did not correspond to the 50end of ssrS/6S RNA
that is 52 nucleotides upstream. The 30end of Str13 was then
determined by 30RACE (Figure 2E) at the position 3,934,920,
which corresponds to the 30end of scr3559 (Vockenhuber et al.,
2011;Moody et al., 2013) but not ssrS/6S RNA. The 30end in
the same position (3,934,920) was identified also by Term-seq
(Lee et al., 2020).
To determine whether scr3559, along with ssrS/6S RNA is
perhaps expressed, we used three different probes for Northern
blot analysis. Probe “A” should specifically hybridize to ssrS/6S
RNA, probe “B” to both sr3559 and ssrS/6S RNA, and probe “C”
only to the sr3559 (Figure 2F). We detected the ∼230 nt band
only with probes B and C that hybridized to sr3559 (Figure 2G).
No signal specific for ssrS/6S RNA was detected at 35 and 65 h
after germination, indicating that the putative 6S RNA (ssrS gene)
is not expressed in detectable amounts in these growth phases.
We cannot exclude that ssrS/6S RNA is expressed under
unknown conditions but the main sRNA transcript derived from
the sco3558-sco3559 genomic locus starts at position 3,934,693
and differs from the ssrS/6S RNA sequence that was used to
experimentally test the interaction of the putative 6S RNA with
the RNAP-HrdB holoenzyme in vitro (Mikulík et al., 2014).
As we identified the full sequence of the Ms1 candidate by
50and 30RACE, we used suboptimal structure folding to search
for Ms1-like secondary structure motifs. We folded the Ms1
candidate sequence by UNAfold (with parameters P= 5000,
W= 2 and X= 100) that predicted 78 suboptimal structures. The
structures were clustered into 5 clusters based on their mutual
structure similarity to find structurally representative folds. The
clusters represented structural variations of a typical Ms1 fold.
The most representative fold was identified in a cluster with most
mutually similar suboptimal structures that contained typical
Ms1-like structures which resembled Ms1 from M. smegmatis
(Figure 2I), revealing its potential to interact with RNAP. An
example of the secondary structure from that cluster is shown in
Figure 2H.
We concluded that both Str11 (scr1506) and Str13 (scr3559)
satisfied the criteria for potential 6S/Ms1 candidates and we
selected them for further analysis.
Str13 (scr3559) Binds the RNAP Core
in vivo
To answer whether Str11 (scr1505) and/or Str13 (scr3559) are
homologs of 6S RNAs or Ms1, we wanted to immunoprecipitate
the primary σfactor, HrdB, and RNAP from S. coelicolor. As the
commercially available antibody against the primary σ70 (clone
2G10) interacted with HrdB only weakly (Figure 3A, lanes 2 and
6), we used a strain with an HA-tagged S. coelicolor hrdB gene
(Šmídová et al., 2019) and immunoprecipitated HA-HrdB with
an anti-HA antibody from exponentially growing (42 h post-
germination) and stationary (66 h post-germination) cells. The
anti-HA antibody pulled down HA-HrdB and α,β,β0subunits
of RNAP, especially at 42 h post-germination (Figure 3A, lane
3, protein band identities were verified by mass spectrometry).
Thus, the anti-HA antibody interacted both with HA-HrdB alone
and also with the RNAP-HrdB complex, which binds 6S RNA
in many bacterial species. Then, we immunoprecipitated RNAP
with the antibody against the RNAP βsubunit (Figure 3A lanes
1 and 5). This antibody preferentially recognizes the RNAP core
without the primary σfactor (Figure 3A). We subsequently
isolated co-immunoprecipitated RNAs and measured their
relative amounts by RT-qPCR (Figures 3B,C).
Str11 (scr1505) associated neither with the RNAP core nor
with the RNAP-HrdB holoenzyme, similar to four control RNAs
that also did not interact with RNAP: 16S rRNA, sco3552,
sco3710 encoding membrane proteins, and sco2013 encoding
response regulator PdtaR.
Importantly, ∼2% of Str13 (scr3559) was bound to the RNAP
core at 42 h post-germination in S. coelicolor (Figure 3B, the
input represents the total amount of scr3559 isolated from the
cell lysates), and it increased to ∼5% at 66 h post-germination
(Figure 3C). scr3559 bound neither HrdB alone nor the HrdB-
RNAP complex. As a control, we performed immunoprecipitation
with the same antibody from stationary phase M. smegmatis cells
and ∼6% of Ms1 co-immunoprecipitated with the RNAP core
(Figure 3D). As we noticed that only a low amount of HrdB-HA
was immunoprecipitated at 66 h post-germination (Figure 3A,
lane 7) compared to 42 h (Figure 3A, lane 3), we compared the
relative levels of HrdB-HA and RNAP in S. coelicolor. The amount
of HrdB-HA significantly decreased at 66 h post-germination
compared to 42 h (Figure 3E) while the level of the RNAP β
subunit was almost unchanged. This suggests that the level of
HrdB and subsequently, the level of the RNAP-HrdB complex
is low in the late phase of growth in S. coelicolor, similar to
previous observations in M. smegmatis (Hnilicova et al., 2014).
Str13 (scr3559) was associated in vivo with the RNAP core but
not with the HrdB-RNAP holoenzyme.
Therefore, we concluded that Str11 (scr1505) is neither 6S
RNA nor Ms1 but a sRNA of unknown function. To the contrary,
Str13 (scr3559) is a bona fide homolog of Ms1 in S. coelicolor and
we propose to rename Str13 (scr3559) as Ms1.
Relative Amounts of Ms1 in Selected
Actinobacteria Species
In Mycobacterium smegmatis and Rhodococcus erythropolis,
Ms1 is an abundant RNA in stationary phase, prominently
visible in the gel, similarly to 6S RNA in Bacillus subtilis
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% INPUT
0
4
2
6
8
10
anti-RNAP E
anti-IgG
Ms1 RpoB RpoC
Mycobacterium smegmatis
stationary phase (24 hrs)
RNAP E
HrdB-HA
66 hrs
42 hrs
anti-V70 (HrdB)
anti-RNAP E
anti-IgG
anti-HA (HrdB)
anti-RNAP E
anti-IgG
anti-HA (HrdB)
EE´ RNAP
HA-HrdB
160
110
60
50
40
MW
[kDa]
42 hrs 66 hrs
anti-V70 (HrdB)
D RNAP
1 2 3 4 5 6 7 8
Streptomyces coelicolor 66 hrs
16S rRNA
sco3352
sco3710
sco2013
Str11
% INPUT
anti-HA (HrdB)
anti-RNAP E
anti-IgG
0
2
4
6
anti-HA (HrdB)
anti-RNAP E
anti-IgG
% INPUT
0
2
4
6
16S rRNA
sco3352
sco3710
sco2013
Str11
scr3559
(Str13)
Streptomyces coelicolor 42 hrs
scr3559
(Str13)
AB
C
DE
FIGURE 3 | Immunoprecipitation of S. coelicolor RNAP βand HA-HrdB (A–C) and M. smegmatis RNAP β(D). Lysates from S. coelicolor cells carrying HA-tagged
HrdB (42 and 66 h after germination, exponential and stationary phase of growth, respectively) were incubated with antibodies against RNAP β, sigma 70 and HA
tag and immunoprecipitated proteins were resolved on SDS PAGE and stained with Coomassie (A). RNA that co-immunoprecipitation with RNAP or HrdB was
isolated, cDNA was reverse transcribed and the amount of Ms1 and 6S RNA candidates were determined by qRT-PCR (B,C).InS. coelicolor, 16S rRNA and RNAs
expressed from sco3352, sco3710, and sco2713 genes were selected as controls that should not bind to RNAP/HrdB. In M. smegmatis, the amount of Ms1
associated with RNAP is shown as a positive control, RpoB and RpoC mRNAs do not co-immunoprecipitate with RNAP (D). The error bars show ±SEM from at
least three independent experiments. The amount of RNAP βand HrdB was measured by western blotting in A3(2) hrdB-HA 42 h (exponential phase) and 66 h after
germination (stationary phase) (E), the same amount of proteins (15 µg) was loaded.
(Figure 4). In Streptomyces coelicolor, we detected a weak
∼230 nt long RNA visible in stationary phase RNA, which
might be Ms1 RNA identified in this study. However, in
Corynebacterium glutamicum, a species that is relatively
evolutionarily close to Mycobacteria (both are in one order
-Corynebacteriales), there are no prominent bands in the
∼200–300 nt range, suggesting that it might not contain
an Ms1 RNA or its expression is below the detection
limit of the staining. Therefore, we decided to extend
the linguistic search to the whole group of Actinobacteria
to reveal how widespread Ms1 RNA is within other
Actinobacteria species.
Linguistic Search for Ms1 RNA in
Actinobacteria
After we identified S. coelicolor Ms1 RNA using the linguistic
search, we applied it to other Actinobacteria. A flowchart of the
search procedure is shown in Figure 5.
We started with the synteny phrase identified in
Mycobacterium, Rhodococcus, Nocardia,Gordonia,
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100 nt
200 nt
300 nt
400 nt
M. smegmatis
5S rRN
A
tRNA
ex st ex st ex st ex stst
C. glutamicum
R. erythropolis
S. coelicolor
B. subtilis
6S RNA
*Ms1 RNA
*
**
FIGURE 4 | Total RNA isolated from Mycobacterium smegmatis mc2155,
Corynebacterium glutamicum,Rhodococcus erythropolis CCM2595,
Streptomyces coelicolor A3(2), and Bacillus subtilis from exponential (“ex”)
and stationary (“st”) phase of growth, resolved on polyacrylamide gel
electrophoresis and stained with GelRed. Ms1 RNAs in M. smegmatis and
R. erythropolis and a band corresponding to the size of Str13/Ms1 in
S. coelicolor are labelled with *. Two forms of B. subtilis 6S-1 RNA are marked
by arrow.
Mycobacteroides, Hoyosella, and Tsukamurella (Table 1,
step ii. in Figure 5) which had been used to discover Str13 (Ms1
homolog) in Streptomyces coelicolor.
For the search, Actinobacteria genera with more than four
annotated species available in GenBank, according to NCBI
Taxonomy (Schoch et al., 2020), were used. In total, there were 40
including Streptomyces. Synteny hits (step iv. in Figure 5) were
obtained in five of them, namely in Cellulomonas, Williamsia,
Actinospicaceae, Actinopolyspora, and Streptomyces (Str13). Note
that sequences of the synteny hits, i.e., IGRs containing putative
Ms1 RNAs, may be dissimilar to each other and therefore could
not be identified by sequence similarity searches.
Based on sequence similarity to synteny hit from Streptomyces
actuosus, we identified IGRs containing Ms1 in 158 species
from 46 Actinobacteria genera other than Streptomyces (step v.
in Figure 5). Based on occurrence of specific words from the
annotations of the identified IGRs (Supplementary Figure 2 step
vi. in Figure 5), we generated the new synteny phrases (step i. in
Figure 5) and added them to the original phrases (this returned
us to the step ii. in Figure 5 and new Table 2 was generated with
updated synteny phrases).
Using the updated synteny phrases and the second iterative
synteny search, a synteny hit in Cellulomonas gilvus was found.
TABLE 2 | Synteny phrases for linguistic search after 1st update using synteny
annotations of Streptomyces Ms1 RNA homologs (Supplementary Figure 2).
For description of the table, see legend of Table 1.
‘beta acetylhexosaminidase’ ‘inhibition morphological
differentiation’
‘beta glycosyl glucosidase’
‘glycoside hydrolase’
‘oxidoreductase’
‘Fic’
‘IB HAD’
‘HAD hydrolase’
‘phosphoserine phosphatase’
‘CpaE’
Here, semantic synonyms are either underlined or in italics or in bold. Note, that
specific words were used to text search in a case-sensitive manner. Higher diversity
of the phrases than in Table 1 was given by higher phylogenetic diversity of species
bringing a higher diversity of annotations.
TABLE 3 | Synteny phrases for linguistic search after 2nd update using synteny
annotations of Cellulomonas Ms1 RNA homologs (Supplementary Figure 3).
‘beta acetylhexosaminidase’‘inhibition morphological
‘beta glycosyl glucosidase’differentiation’
‘glycoside hydrolase’
‘oxidoreductase’ ‘IB HAD’
‘PH’ ‘HAD hydrolase’
‘transcriptional regulator’ ‘phosphoserine phosphatase’
‘Fic’ ‘CpaE’
‘chromosome partitioning’
For description of the table, see legend of Tables 1,2.
Based on sequence similarity, Ms1 IGRs in 708 species from
109 Actinobacteria genera were identified. Specific words and
a histogram of their occurrence in synteny annotations of the
708 Ms1 IGRs are shown in Supplementary Figure 3. The
subsequently updated synteny phrases are shown in Table 3.
The phrases in Table 3 yielded synteny hits into another
three genera: Williamsia,Actinopolyspora, and Actinospica whose
synteny annotations did not produce any new synteny phrases.
Sequence similarity of the Williamsia synteny hit was limited
to the Williamsia genus and sequence similarity of the both
Actinopolyspora and Actinospicaceae synteny hits identified with
mostly already known Ms1 RNA candidates in species of
evolutionarily closed genera.
Interestingly, in Catenulispora (Catenulisporales),
Brevibacterium (Micrococcales), Corynebacterium
(Corynebacteriales), Actinomyces (Actinomycetales), and
Bifidobacterium (Bifidobacteriales) we found no IGRs that
could contain Ms1 RNA (Figure 5A, labeled in gray). We
found IGRs with the Ms1 RNA synteny in the species of these
taxonomic groups but they were too short to accommodate Ms1
RNA. For example, in Corynebacterium bovis, the Ms1-syntenous
IGR was only 6 bps long and in species of the other groups there
were Ms1 RNA-syntenous IGRs between only 20 and 100
nucleotides long.
In summary, the linguistics gene synteny search identified Ms1
RNA homologs in 824 Actinobacteria species (Supplementary
Table 2 and Supplementary Figure 5 for specific words in
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FIGURE 5 | Scheme of the linguistic gene synteny search for Ms1 RNA in Actinobacteria. Titles of analyzed data and information are in boxes, while analytic steps
are shown as plain text. Arrows indicate the data and information flow. Synteny analysis (step i.) generates Synteny phrases (Tables 1–3, step ii.). For the first
synteny analysis, annotations of Ms1 flanking genes from Mycobacterium, Rhodococcus, Nocardia,Gordonia, Mycobacteroides, Hoyosella, and Tsukamurella were
used and Table 1 generated. Synteny phrases are used to search for new putative Ms1 candidates (iv. Synteny hits). If the putative Ms1 candidates are evolutionary
conserved in related species (step v.), annotations of their flanking genes (step vi.) are added to the Synteny analysis (step i.) to generate updated Synteny phrases
(step ii., Tables 2,3) and the whole procedure is repeated.
their synteny annotations) belonging to 146 genera and 14
Actinobacteria orders (Figure 6A, labeled in red and Table 4).
Thus, Ms1 RNA is widespread among Actinobacteria and Ms1
RNA interaction with the RNA polymerase core is conserved both
in Mycobacterium smegmatis and Streptomyces coelicolor.
DISCUSSION
The presented study reveals the ubiquitous presence of Ms1 RNA
in Actinobacteria (exceptions might exist, see below), identifying
this sRNA as a major class of protein-interacting RNAs. Ms1 RNA
associates with the RNAP core as previously demonstrated in
Mycobacteria (Hnilicova et al., 2014) and here in Streptomyces
(Figure 3). In addition, our linguistic gene synteny search proved
to be a potent tool to identify sequentially unrelated RNAs in
evolutionarily distant species.
Ms1 RNA Binds RNAP in Streptomyces
We bioinformatically identified Str13 as the Ms1 RNA
candidate in S. coelicolor. Str13 overlaps with scr3559
sRNA (Figure 2B), an sRNA with unknown function. We
showed that Str13/scr3559 is an Ms1 homolog in S. coelicolor.
Str13/scr3559 had a similar predicted secondary structure
with the mycobacterial Ms1 (cf. Figures 2H,I) and both
RNAs bind the RNAP core (Figure 3). Ms1 RNA is thus
conserved in evolutionarily distant and morphologically
divergent Mycobacterium and Streptomyces.Mycobacteria
are unicellular rod-shaped bacteria, while Streptomyces have
a complex life cycle, which starts with the germination
of a spore that prolongs into filamentous tubes of highly
branched vegetative (primary) mycelium, then secondary
mycelium is formed and eventually spores (Trotochaud and
Wassarman, 2004, 2006;Faucher et al., 2010;Cavanagh
and Wassarman, 2013;Hoch et al., 2015). Despite the
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Actinobacteria (Actinomycetia)
Kineosporiales
Bifidobacteriales
Actinomycetales
Microbacteriaceae
Dermatophilaceae
Dermacoccaceae
Intrasporangiaceae
Brevibacteriaceae
Micrococcaceae
Cellulomonadaceae
Beutenbergiaceae
Ruaniaceae, Sanguibacteriaceae
Promicromonosporaceae
Micrococcales
0
8
2
10
4
12
6
Corynebacterium (126)
Gordonia (39)
Mycobacterium (106)
Nocardia (95)
Rhodococcus (35)
Tsukamurella (8)
Williamsia (7)
genome size [Mb]
Corynebacteriales
Ms1 synteny and Ms1 RNA identified
Ms1 synteny identified, no Ms1 RNA found
no Ms1 synteny, no Ms1 RNA
0
8
2
10
4
12
6
genome size [Mb]
Actinomycetales (92)
Bifidobacteriales (100)
Actinopolysporales (10)
Frankiales (17)
Geodermatophilales (42)
Glycomycetales (16)
Jiangellales (18)
Kineosporiales (11)
Micrococcales (705)
Micromonosporales (187)
Nakamurellales (9)
Propionibacteriales (199)
Pseudonocardiales (211)
Streptomycetales (535)
14
Streptosporangiales (210)
Nocardia
Rhodococcus
Tsukamurella
Williamsia
Corynebacterium
Mycobacterium
Frankiales
Streptosporangilales
Gordonia
Pseudonocardiales
Actinopolysporales
Nakamurellales
Geodermatophilales
Glycomycetales
Micromonosporales
Cryptosporangium
Corynebacteriales
Streptomycetales
Jiangellales
Propionibacteriales
A B
C
FIGURE 6 | (A) Ms1 homologs were identified in Actinobacteria orders (in italics) or families (narrow italics) labeled by red. Actinobacteria groups with no identified
Ms1 homologs and Ms1 flanking genes are in black, groups with identified Ms1 flanking genes but no Ms1 homologs in grey. The tree was adopted from Nouioui
et al. (2018).(B) Genome sizes of the Corynebacteriales and (C) Actinobacteria. Only NCBI reference genomes are shown, number of genomes is indicated in
brackets.
completely different life cycles, both bacterial species have
maintained Ms1 sRNA.
Genomic Position of Ms1
In circular genomes of Mycobacterium,Nocardia, and
Rhodococcus, Ms1 RNA is located close to the ori (replication
start site) with the direction of transcription oriented toward
it (Hnilicova et al., 2014). This is similar to Ms1 RNAs
in Streptomyces that is positioned in the middle of the
linear genome, close to ori (Figure 2A). The Streptomyces
genomes have a core region of about 4.9 Mb containing
essential genes and left and right arms with 1.5 Mb and
2.3 Mb, respectively (Hopwood, 2006), carrying mostly non-
essential and species-specific genes. The position of Ms1
RNA genes in the core region indicates that this sRNA
belongs among conserved genes in Streptomyces, consistent
with our findings that identified Ms1 RNA candidates in
188 Streptomyces species (see Supplementary Figure 6). We
further found Ms1 RNA in 145 other Actinobacteria genera
(Table 4). Note that the number of newly identified Ms1 RNAs
was limited by the availability of annotated Actinobacteria
genomes as there were relatively many genera with only
one or two genomes available or/and with a single species
classified per genus.
Ms1 and Genome Size
In some genera, such as Corynebacterium, Bifidobacterium,
and Actinomyces, we detected the Ms1 synteny but the IGR
was too short to accommodate Ms1. The missing Ms1 RNA-
containing IGR in Corynebacterium was consistent with the
absence of a prominent band in the 200–300 nt range in RNA
gels from Corynebacterium (Figure 4). The Corynebacterium
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TABLE 4 | List of Actinobacteria orders, families, and genera with species with predicted Ms1 RNAs.
Order Family Genus
Actinopolysporales Actinopolysporaceae Actinopolyspora, Halopolyspora
Catenulisporales Actinospicaceae Actinospica, Actinocrinis
Corynebacteriales Gordoniaceae Gordonia
Mycobacteriacea Mycobacterium, Mycobacteroides, Mycolicibacterium
Nocardiaceae Nocardia, Rhodococcus, Williamsia
Tsukamurellaceae Tsukamurella
Corynebacteriales incertae sedis Fodinicola
Cryptosporangiales Cryptosporangiaceae Cryptosporangium
Sporichthyales Sporichthyaceae Sporichthya
Jiangellales Jiangellaceae Jiangella
Kineosporiales Kineosporiaceae Angustibacter, Kineosporia
Micrococcales Beutenbergiaceae Beutenbergia
Cellulomonadaceae Cellulomonas, Actinotalea, Oerskovia
Dermacoccaceae Allobranchiibius, Barrientosiimonas, Calidifontibacter, Demetria, Flexivirga, Leekyejoonella,
Luteipulveratus, Metallococcus, Piscicoccus, Rudaeicoccus, Yimella
Dermatophilaceae Austwickia, Mobilicoccus
Intrasporangiaceae Humibacillus, Janibacter, Intrasporangium, Knoellia, Lapillicoccus, Ornithinicoccus, Oryzihumus,
Pedococcus, Phycicoccus, Segeticoccus, Tetrasphaera
Micrococcaceae Arthrobacter, Ornithinimicrobium
Ornithinimicrobiaceae Ornithinimicrobium
Promicromonosporaceae Cellulosimicrobium, Isoptericola
Ruaniaceae Occultella, Ruania
Sanguibacteriaceae Sanguibacter
Micrococcales incertae sedis Luteimicrobium
Micromonosporales Micromonosporaceae Actinocatenispora, Actinoplanes, Allocatelliglobosispora, Allorhizocola, Asanoa, Catellatospora,
Catelliglobosispora, Catenuloplanes, Couchioplanes, Dactylosporangium, Hamadaea,
Krasilnikovia, Mangrovihabitans, Phytohabitans, Pilimelia, Planosporangium,
Pseudosporangium, Rhizocola, Rugosimonospora, Spirilliplanes, Virgisporangium
Nakamurellales Nakamurellaceae Nakamurella
Propionibacteriales Nocardioidaceae Actinopolymorpha, Aeromicrobium, Kribbella, Nocardioides, Marmoricola, Pimelobacter
Propionibacteriaceae Auraticoccus, Friedmanniella, Microlunatus
Pseudonocardiales Pseudonocardiaceae Actinoalloteichus,Actinokineospora, Actinophytocola, Actinopolyspora, Actinosynnema,
Alloactinosynnema, Amycolatopsis, Allokutzneria, Crossiella, Goodfellowiella, Haloechinothrix,
Herbihabitans, Kibdelosporangium, Kutzneria, Labedaea, Lentzea, Longimycelium, Prauserella,
Pseudonocardia, Saccharomonospora, Saccharopolyspora, Saccharothrix, Streptoalloteichus,
Tamaricihabitans, Thermocrispum, Thermobispora, Umezawaea
Streptomycetales Carbonactinosporaceae Carbonactinospora
Streptomycetaceae Embleya, Kitasatospora, Streptomyces
Streptosporangiales Nocardiopsaceae Lipingzhangella, Marinactinospora, Marinitenerispora, Nocardiopsis, Spinactinospora,
Streptomonospora, Thermobifida
Streptosporangiaceae Acrocarpospora, Bailinhaonella, Herbidospora, Microbispora, Microtetraspora, Non-omuraea,
Planobispora, Planomonospora, Planotetraspora, Sinosporangium, Sphaerimonospora,
Sphaerisporangium, Spongiactinospora, Streptosporangium, Thermoactinospora,
Thermocatellispora, Thermopolyspora
Thermomonosporaceae Actinoallomurus, Actinocorallia, Actinomadura, Spirillospora, Thermomonospora,
Thermostaphylospora
One hit was obtained in unspecified species annotated as ’Actinobacteria bacterium’ (not included in the table).
genus belongs to the Corynebacteriales order, which also includes
Mycobacterium,Nocardia,Rhodococcus [where Ms1 RNA has
been already described (Hnilicova et al., 2014)], Williamsia,
Gordonia, and Tsukamurella, where we identified Ms1 RNAs
using the linguistic gene synteny search (Figure 6A). Within
the Corynebacteriales order, Corynebacteria have the smallest
genome (Figure 6B). Ms1 thus might have been lost from
Corynebacterium due to the evolutionary pressure to maintain a
reduced genome. Alternatively, Ms1 RNA could be essential for
Actinobacteria with the larger genomes.
A comparison of genome sizes of the main Actinobacteria
orders with the occurrence of predicted Ms1 RNAs
(Figures 6A,C) reveals a trend where Ms1 is lost in bacteria
with smaller genomes while the Ms1 synteny is still present.
Examples are Bifidobacteriales and Actinomycetales where the
respective IGRs were too short to accommodate Ms1 RNA;
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these orders have the smallest genomes within Actinobacteria
(mostly <3.0 Mbp).
However, in Frankiales,Geodermatophilales, and
Glycomycetales, where neither the Ms1 synteny nor Ms1 RNA
were detected, genome sizes were comparable to Actinobacteria
orders with identified Ms1 RNAs. Frankia (Frankiales) genome
sizes vary between 4.3 and 10 Mb and this variability is due to the
degree of host dependence. Frankia are N2-fixing filamentous
plant symbiotic bacteria that can either survive independently
in the soil or be entirely dependent on their host plants (Benson
et al., 2011). The high diversity of Frankia genomes might be
a reason why Ms1 RNA was not detected in our search (the
gene synteny on which our search depends, might be too low
in Frankia). Glycomycetales are aerobic bacteria that produce
branched vegetative mycelia and aerial mycelia with chains
of square-ended conidia (Labeda and Kroppenstedt, 2004).
Glycomyces were isolated from soil, hypersaline habitats, and
seawater (Han et al., 2014;Xing et al., 2014;Nikitina et al.,
2020). Geodermatophilales create pigmented (very often black)
colonies with the individual cubic cells and extracellular matrix
forming cauliflower-like aggregates and have been reported
to be highly resistant to stresses such as gamma-radiation,
UV, and desiccation (Hezbri et al., 2016). Species from both
Glycomycetales and Geodermatophilales can adapt to extreme
stress conditions and thus might have evolved specific regulatory
pathways independent of Ms1 RNA.
To summarize this section, the genome size is not the only
indicator of the Ms1 RNA presence. Alternatively, Ms1 RNA may
be present in these species but the Ms1 RNA gene synteny was
lost and therefore we were unable to detect Ms1 RNA using our
gene-synteny based approach.
Gene Linkage of Ms1 With HAD
Hydrolase
The Ms1 RNA synteny itself, especially the “HAD hydrolase” gene
is of interest. HAD hydrolase is annotated also as “inhibition
of morphological differentiation protein” or “phosphoserine
phosphatase” (see Tables 1–3) and it was found in the Ms1
RNA synteny of most species. In Mycobacterium smegmatis, HAD
hydrolase is MSMEG_6173, a 293 amino acid long protein with a
predicted transmembrane domain at its C-terminus. SCO3558,
a 50end flanking gene of scr3559, that shares 60% identity with
MSMEG_6173, is homologous to CicA (Bellier et al., 2006),
which encodes a phosphotransferase in Caulobacter crescentus.
An increased concentration of CicA in Caulobacter crescentus
causes a loss of the normal rod shape, an almost 10-fold increase
of the bacteria’s cell volume and a cell division block (Fuchs
et al., 2001). As Ms1 regulates the RNAP amount in stationary
phase (Sikova et al., 2019) it is tempting to speculate that the
conserved association of the HAD hydrolase gene with the Ms1
RNA gene indicates a link between transcription regulation and
cell size and shape.
Computational Approaches Revisited
From the perspective of computational biology, our work
demonstrated limits of the use of secondary structure in
computational homology searches for homologs of known sRNAs
in bacterial genomes. Homology searches use similarity of
secondary structure between potential homologs and known
sRNAs either solely [e.g., (Pánek et al., 2011)] or in combination
with sequence similarity [e.g., (Barquist et al., 2016)]. Structure
similarity increases the efficiency and capability of these searches
to find homologs as the sRNA secondary structures are more
evolutionarily conserved than sRNA sequences.
Nevertheless, still the efficiency of homology searches
decreases substantially with the increasing evolutionary distance.
This was demonstrated here in the extremely diverse group of
Actinobacteria by the search for Ms1 RNA homologs, which
was not successful. Also Rfam (Kalvari et al., 2017), an RNA
database that employs the infernal software (Barquist et al.,
2016) for computational search for homologs of known RNAs,
provides Ms1 RNA candidates only from species closely related
to Mycobacteria.
A weak point of homology searches is the limited reliability of
secondary RNA structure prediction, which decreases especially
for sequences >100 nt. Furthermore, a correct RNA sequence,
i.e., a sequence with both a correct position in the genome
and a correct length, is required for the prediction. But it
is not always available in the homology search as (1) the
length of the sequence can vary substantially between species
or genera, and (2) genomic position of the sequence can
be only approximated. In the presented work, the length
difference between the known M. smegmatis Ms1 RNA and the
identified S. coelicolor Ms1 RNA homolog was 70 nucleotides
and therefore predicted secondary structures of potential
S. coelicolor Ms1 RNA homologs were wrong and structure
similarity to known Ms1 RNA could not be detected. Thus, the
unavailability of correct sequences of potential homologs could
be another reason why homology search was not successful in
the presented work.
To overcome this problem, we employed a genomic synteny
search. We adopted a linguistics approach based on similarity
of genome annotations rather than similarity of nucleotide or
amino acid sequences of flanking genes. Ms1 RNA has conserved
synteny with a highly specific phrase in one of its flanking
genes, the ‘HAD IB hydrolase’. This phrase is relatively rare and
occurs only a few times in well annotated genomes (e.g., 5 ×in
S. coelicolor). The annotation helped us to identify the IGRs
that might contain Ms1 RNA even in extremely distant species
as represented here by the Actinobacteria genera with predicted
Ms1 RNA, in which flanking nucleotide/amino acid sequences
might be dissimilar.
We designed the most parsimonious form of a linguistic
search for conserved synteny based on text searches for exact
words or phrases. Once the search had identified the first possible
Ms1 homolog in a genus (synteny hit), it then proceeded in
an iteratively progressive manner within the genus and also
in evolutionarily close genera using sequence similarity of the
synteny hits. The obtained information was subsequently applied
to other genera, expanding the list of identified candidates. Even
in this simple form, the computational text search was able
to identify Ms1 homologs in Streptomyces and other distantly
related Actinobacteria genera. Its versatility and ease of use make
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it a convenient tool that can be, in principle, applied to searches
for other RNAs/genes, allowing their fast identification across a
wide range of organisms.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding authors.
AUTHOR CONTRIBUTIONS
OM, MK, DK, and MJ validated sRNAs expressions. MŠ and
JH performed 50and 30RACE. VVH and MŠ performed
immunoprecipitation experiments. PH identified proteins by
mass spectrometry. JP and MSc performed the computations.
MSc, LK, JHn, and JP wrote the manuscript. JHn and JP designed
the study. All authors contributed to the article and approved the
submitted version.
FUNDING
This research was funded by the grants (20-07473S) to
JH and (20-12109S) to LK from Czech Science Foundation
(www.gacr.cz), ELIXIR CZ research infrastructure project (MEYS
Grant No. LM2018131) including access to computing and
storage facilities, and European Regional Development Fund
(project BIOCEV CZ.1.05/1.1.00/02.0109).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2022.848536/full#supplementary-material
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