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The CIMS (Cyanobacterial ITS motif slicer) for molecular systematics
Nicolas A.
1*,
Callahan A.
1
, Aimee L.
1
, Anne C.
1, Marie R. 1 & Dale A. 1
1
Department of Biology, College of Arts and Sciences, University of North Florida, 1 UNF Drive, Jacksonville,
FL–32224, USA; *Corresponding author e–mail: nlab@fastmail.com
Abstract: The 16S–23S rRNA Internal Transcribed Spacer (ITS) is a commonly employed taxonomic marker
in cyanobacterial systematics. Due to numerous challenges in articulating phylogenetic relationships within this
ubiquitous, ancient lineage, a polyphasic approach including 16S rRNA sequence data, ecology, morphology,
and ITS secondary structure analysis has become the standard. In particular, the ITS motifs are being utilized
in the erection of novel and cryptic taxa. However, this is challenging as researchers must manually mine and
from using ITS motifs, may lead to inconsistencies, and is a rather dry, tedious enterprise. Thus, we present a
motifs (e.g., the Box–B, D1–D1´, tRNAs, etc.). After extensive testing, we note that the most common motifs
are recovered at ca. 97%. These motifs can then be easily exported into Mfold or other similar folding pack-
ages. We hope that this will both provide a valuable tool for researchers but will also facilitate new discoveries
and allow for greater consistency in publishing ITS comparisons. The tool can be accessed at www.phylo.dev.
Key words: Folding motifs, rRNA, secondary structures, taxonomy, 16S
Introduction
Cyanobacteria are an ancient lineage and describing their
evolutionary relationships have always been a challenge
( et al. in press). In order to avoid ambigu-
ous classifications/phylogenies that arise primarily from
phenotypic plasticity, cryptic diversity, and paucity of
informative morphological traits common in cyanobac-
teria, researchers employ a multi–faceted approach to
classification (e.g., et al. 2005;
2005; et al. 2018). This so–called
polyphasic approach includes molecular, ecological,
and morphological data, and has become crucial to the
accurate identification and description of cyanobacterial
relationships.
In addition to 16S rRNA sequence data, the RNA
secondary structures of conserved regions within the 16S
rRNA–23S rRNA internal transcribed spacer (ITS) region
have also been found to be phylogenetically informative
molecular characters (et al. 2000; &
2005) (Fig. 1). The cyanobacterial 16S–23S
ITS region contains up to two tRNA genes, as well as
several conserved sequence motifs and regions of RNA
secondary structure (et al. 2000). Among these
conserved domains are the transcriptional anti–termina-
tor sites Box–A, which is a short conserved sequence
domain, and Box–B, which is a stem–loop structure of
variable sequence (et al. 1995). Other secondary
structure motifs analyzed in the context of cyanobacte-
rial systematics include the D1–D1’, V2, and V3 helices
( et al. 2011; et al. 2007). Among
different taxa, these regions may differ in their nucleotide
sequence lengths, and their corresponding secondary
structures (Fig. 1) may vary in the length of the basal
stem, the relative size and position of unilateral and/or
bilateral bulges in the helix, and the relative size of the
terminal loop (et al. 2022).
While 16S rRNA sequence data is valuable for
resolving higher–level taxonomic relationships among
cyanobacteria (i.e., in determining orders and families),
high values of 16S rRNA sequence similarity among
closely related cyanobacterial strains has limited its
usefulness as a molecular character for distinguishing
species (et al. 2007). Compared to the 16S
rRNA gene, the 16S–23S ITS region exhibits a higher
degree of variation among species, and is thus useful
for determining relationships among closely related
strains characterized by highly similar 16S rRNA gene
sequences ( et al. 2011). ITS sequence and
secondary structure data have also been utilized in
phylogenetics research concerning other organisms,
including fungi ( et al. 2014) and green
Fottea, Olomouc, 24(1): 23–26, 2024 23
DOI: 10.5507/fot.2023.008
algae (et al. 2011; & 1997).
With increased employment of ITS motifs, a panoply of
these secondary folding structures has emerged for use
in species descriptions and identifications (
et al. 2020; et al. 2021; in press).
While phylogenetically informative, ITS folding pat-
terns are not without some challenges. First, there is the
issue of homology: bacteria are renown for potentially
possessing multiple operons ( et al. 2000;
2018), and reconstructing relationships relies
on comparing homologous, not analogous, regions.
Some cyanobacterial lineages possess multiple operons
( 2011). Second, not all of the mo-
tifs are necessarily present in all operons. For example,
some transcripts contain two tRNA genes (tRNAIle and
tRNAAla), while others may contain only one or neither.
Third, accurate annotation of a given ITS motif may be
impeded by the presence of multiple sequence regions
which resemble or are identical to the expected sequence
of the motif’s conserved flanking regions. For example,
it is possible to obtain very similar Box–B motifs even
though the leading sequences are very different from each
other ( et al. 2022). Fourth, when assembling
large ITS plates, it can be both tedious and error–prone to
use Microsoft Word (commonly employed to manually
highlight/annotate regions) in these endeavors.
To address these issues, we present CIMS (the
Cyanobacterial ITS Motif Slicer). This web–based ap-
plication has been created to find the most commonly
used ITS folding motifs (e.g., D1–D1´, V2, etc.), both
tRNAs, and other pertinent nucleotide regions (e.g., the
leader, D2–D3, etc.) (Fig. 1). The CIMS script is also
available for users to download and run on MacOS/
Linux and Windows systems. To help researchers ensure
they are using homologous operons (e.g., containing the
same number of tRNAs) when comparing ITS second-
ary structures between taxa, the script can be executed
with a flag (–t) that counts tRNAs in each ITS region
and returns taxa and their corresponding tRNA count.
Researchers may also choose to output only certain mo-
tifs (e.g., D1–D1´, Box–B, etc.) by using the flag (–s).
Both the web–based CIMS application and the script
as input. CIMS then returns the sequences and lengths
of all identified ITS motifs.
The program leverages the Biopython library
(et al. 2009) both to query the Entrez database and
to parse the FASTA file. CIMS searches for the nucleotide
sequence which marks the end of the 16S rRNA gene
(CCTCCTT or CCTCCTA) in order to identify where
the ITS region begins; the ITS region is then sliced off
from the full sequence and the program begins looking
for the conserved flanking regions of each element in
consecutive order (Fig. 1).
CIMS will return the following features (if
present in the transcript) in order: ITS leader, D1–D1´,
spacer D2–D3 spacer, tRNA
Ile
, V2, tRNA
Ala
, Box–B,
Box–A, D4, and V3. With every successful find it slices
off the substring of the motif and stores it in a dictionary
where the key is the motif name, and the value is an ar-
ray of all the possible sequences for that motif. Once the
program is finished, it either downloads the output in the
form of a text file to the user’s computer (web–based)
or prints the output to the terminal and saves the output
as a JSON file in the working directory (if running the
script directly). The program follows Entrez guidelines
query, but the script does not store or transmit email
addresses in any other way.
Results and Discussion
we performed ex-
tensive testing from all cyanobacterial orders except the
data available) employing ca. 300 taxa (Table 1). For full
(or identifying that no tRNAs were present). Both D1–D1´
and Box–B motifs (the most commonly folded motifs in
manuscripts) were recovered at >95%, but with some het-
between the orders are likely less a result of the software
more sequence data available) (Table S1). A preliminary
review of publications containing predicted V3 secondary
structures for cyanobacterial taxa revealed that, in contrast
to the D1–D1´ and Box–B regions, the V3 region had highly
secondary structure (e.g., Box–B and D1–D1´) by searching
rate in accurately identifying the V3 region has not yet been
24 et al.: The CIMS (Cyanobacterial ITS motif slicer) for molecular systematics
-
ployed motifs by cyanobacterial order based on ca. 300 test cases of
transcripts that represent full ITS regions. tRNAs were always reco-
vered from ITS regions that contained them. Actual strains employed
are in Suppl. Table 1.
Order D1-D1´BoxB tRNAs
Synechococcales 96.49% 92.98% 100%
Chroococcales 100% 97.87% 100%
Nostocales 93.90% 92.68% 100%
100% 100% 100%
Chroococcidiopsidales 93.75% 87.50% 100%
Spirulinales 94.44% 94.44% 100%
Oscillatoriales 100% 98.39% 100%
Cyanobacteria (all orders) 96.94% 94.84% 100%
Potential limitations/confounding factors
-
logenetic assessments, we realize that there exist some
limitations with any script.
1.
before and after the Box–B or D1–D1´, etc.); it is
deterministic for any given sequence.
2.
the search parameters, e.g., if you want to modify
particular strain has a single nucleotide polymor-
phism), you have to go to the code itself to initiate
3.
concerning sequence quality, and CIMS cannot
evaluate the quality of the sequence (e.g., is the
sequence data accurate).
4.
The CIMS script has been optimized for cyanobacteria;
5. There is no way of verifying if the motif or folded
secondary structure is “correct” (no quality score).
We suggest that users compare their structures with
those from the literature.
6.
CIMS may output several motif sequences when
We suggest comparing the lengths and predicted
secondary structures of these sequences to those of
other cyanobacterial ITS regions presented in the
literature; if one is similar in length and structure
to others in the literature while another is radically
our explorations.
7.
may present some operons more com-
monly than others (e.g., et al. 2022).
This issue is broader than merely being a feature of
CIMS but included here for completeness.
With those caveats, the useful aspects of
this package are that it: 1) makes it easier to potentially
variants (i.e., how many tRNA’s are present to look
at homologous regions), 3) allows researchers easy
access to potentially phylogenetically informative
data (e.g., leader length, total bp, etc., all of which
are being increasingly employed in manuscripts),
and 4) enables consistent and automated annotation.
Future directions
This project is open source so that the cyanobacterial
contribute to the future growth of this application. As
more researchers employ it, we will continue to modify
the code for improved functionality.
As a tool for scientists, we always welcome
feedback on positive and negative aspects of the script.
-
vide feedback. We are also planning on several possible
future features. First, HTML output of the ITS region
color coded by motif (which is how most researchers
visualize the sequence). Second, u
regions for motifs may be useful for lineages whose
evolutionary trajectories have created novel or unique
sequence variants. Third, we hope to be able to connect
Fig. 1. ITS region of Anagnostidinema visii LHM–M between the 16S gene and the 23S gene and its motifs labeled in order. Secondary struc-
tures of motifs D1–D1`, Box–B, and V3 shown.
Fottea, Olomouc, 24(1): 23–26, 2024 25
DOI: 10.5507/fot.2023.008
their 16S rRNA. – Frontiers in Microbiology 9: 1232.
M. (2000): Comparison of conserved structural and
regulatory domains within divergent 16S rRNA–23S
rRNA spacer sequences of cyanobacteria. – Microbiology
146: 1275–1286.
(2005): Recognizing di-
versity through adoption of a new species paradigm. –
Algological Studies 117: 71–93.
(2011). Utility of 16S–23S ITS sequence
and secondary structure for recognition of intrageneric
and intergeneric limits within cyanobacterial taxa:
Leptolyngbya corticola
Cyanobacteria). – Nova Hedwigia, 92: 283–302.
(2020):
Description of novel species of Aliinostoc, Desikacharya
and Desmonostoc using a polyphasic approach. –
International Journal of Systematic and Evolutionary
Microbiology 70: 1–9.
(2014): Internal transcribed spacer 1 secondary
structure analysis reveals a common core throughout
one 9: e91928. DOI: https://doi.org/10.1371/journal.
pone.0091928
The internal transcribed
spacer 2 exhibits a common secondary structure in
Evolution 44: 258–271. DOI: https://doi.org/10.1007/
pl00006143
(2018): Revision of the Synechococc
ales () through recognition of four
families including Oculatellaceae fam. nov. and
Trichocoleaceae fam. nov. and six new genera contain-
. (2007): Morphological and molecular
characterization of selected desert soil cyanobacteria:
three species new to science including Mojavia pulchra
and fold motifs in a more streamlined fashion.
Future work on the CIMS tool will also include
improving its capability to identify the V3 region. The
the basal portion of the V3 stem–loop structure, were
found to be more variable than those of the D1–D1´ and
Box–B regions. Due to this variability, the CIMS script is
not currently optimized to identify the V3 region across
a wide range of taxa. In contrast, regions D1–D1´ and
Box–B are characterized by more conserved 5´ and 3´
ITS sequences tested. While further work is required in
order for CIMS to successfully identify V3 across a wide
range of cyanobacterial taxa, the current CIMS script
in several ITS sequences of interest, and expects that
remaining ITS sequences of interest (for instance, when
the user is annotating ITS sequences obtained from a
group of very closely related strains or from multiple
clones derived from a single strain).
The web–based tool can be accessed at
www.phylo.dev. The CIMS script and documenta-
tion are available at: https://github.com/nlabrad/
CIMS–Cyanobacterial–ITS–motif–slicer.
References
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dolomitestris – two new Nostocaceae (Cyanobacteria)
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(2011): Internal transcribed
spacer 2 (nu ITS2 rRNA) sequence–structure phylo-
genetics: towards an automated reconstruction of the
10.1371/journal.pone.0016931
(2005): Molecular and morphological characterization of
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lecular biology and bioinformatics. – Bioinformatics
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Control of
rRNA transcription in Escherichia coli. – Microbiological
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operons in Bacteria: their concerted evolution and
potential consequences on the rate of evolution of
© Czech Phycological Society (2024)
Received October 27, 2022
Accepted February 2, 2023
Supplementary material
The following supplementary material is available for this article:
Table S1. All taxa used as test cases for CIMS (Box-B and D1-
D1’).
Table S2.
This material is available as part of the online article (http://
fottea.czechphycology.cz/contents)
26 et al.: The CIMS (Cyanobacterial ITS motif slicer) for molecular systematics
