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Article

The CRISPR/Cas Machinery Evolution and Gene Flow in the Hot Spring Cyanobacterium Thermostichus

by
Eva Jahodářová
1,
Aloisie Poulíčková
2 and
Petr Dvořák
2,*
1
Department of Biology, Faculty of Education, Palacký University in Olomouc, Purkrabská 2, CZ-771 40 Olomouc, Czech Republic
2
Department of Botany, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(7), 502; https://doi.org/10.3390/d14070502
Submission received: 6 May 2022 / Revised: 10 June 2022 / Accepted: 18 June 2022 / Published: 21 June 2022
(This article belongs to the Section Microbial Diversity and Culture Collections)
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Abstract

:
Drivers of the speciation in bacteria, including geographical isolation and horizontal gene transfer, are still poorly understood. Here, we characterized a new lineage within an anciently diverged thermophilic cyanobacteria. We sequenced the whole genome of a strain Thermostichus vulcanus isolated from the Rupite spring (Bulgaria), which is closely related to Theromstichus strains JA-2-3Aa and JA-2-3B′a(2-13). We performed phylogenetic inference, horizontal gene transfer estimation, and CRISPR/Cas system characterization. We found that the Rupite strain is a distinct species from strains JA-2-3Aa, and JA-2-3B′a(2-13). Furthermore, the horizontal gene transfer seemed to be more frequent among the geographically distant species than between the two species within the same hot spring. The CRISPR/Cas system had variable complexity among the species of Thermostichus in terms of both the number of spacers and genes. The Rupite strain had the highest, and JA-2-3Aa the lowest number of spacers among the analyzed strains, and the CRISPR spacers were only rarely shared among the strains. We conclude that the CRISPR/Cas system size varied among the lineages as well as the gene flow.

1. Introduction

Mechanisms driving the diversification of the bacterial species are still far from being explained. The view of bacterial species as clonal populations that are changing via mutations and adaptation was challenged when homologous and non-homologous recombination (horizontal gene transfer—HGT) was discovered as abundant in many bacterial species. HGT may provide a channel for evolutionary adaptation [1] as well as genome diversification (reviewed by [2,3]). HGT events seem to be spread through most bacteria taxa, and thus, HGT played an important role in the evolution of bacteria [4]. Furthermore, HGT does not appear to be random, but it is more likely to happen among closely related species [5,6].
Geographical isolation has been traditionally recognized as a crucial driver of diversification in animals and plants because it represents a solid barrier to gene flow. However, with rapidly growing population genomic data, it has been shown that species can diverge while facing gene flow [2,7]. A view on geographical isolation in speciation of prokaryotes has taken an opposite course. Microorganisms, in general, were considered so small that they can disperse anywhere with no barriers [8]. This assumption seems to be working when only morphological characters are analyzed (e.g., [9]), but microorganisms possess relatively low morphological diversity in comparison with genome diversity. Molecular phylogeny provided evidence for the geographical structuring of microbial species in prokaryotes (e.g., [10,11]). Possible geographical isolation was also identified in cyanobacteria, e.g., in Microcoleus [12], and thermophilic cyanobacteria (see below).
Hot springs are isolated habitats inhabited by a specific group of microbes adapted to high temperatures. With a sufficient light intensity, oxyphototrophic bacteria (cyanobacteria) thrive in hot springs. The diversity of cyanobacteria in hot springs ranges from simple unicellular forms (e.g., Synechococcus [13]) to morphologically complex species characterized by cell differentiation, such as Mastigocladus laminosus [14].
Thermostichus was found in Yellowstone National Park in Octopus and Mushroom springs [15,16]. Thermostichus was derived from Synechococcus [17]. This lineage was identified as one of the earliest derived in the phylogeny of cyanobacteria [18,19]. There were two recognized lineages represented by two strains, JA-2-3Aa and JA-2-3B′a(2-13), whose full genome sequence is available. As mentioned above, hot springs are isolated biotopes and the distance between them can be thousands of kilometers. Thus, they are an ideal model system to study the importance of geographical isolation in the speciation of bacteria. Papke et al. [13] suggested that geographical isolation plays an important role in the speciation of Thermostichus and Synechococcus. Later, Ionescu et al. [20] analyzed a larger dataset of sequences which suggested that Thermostichus is much more diverse. It should be noted that mentioned analyses are limited to 16S rRNA obtained either from environmental samples or strains of Thermostichus. No genome of Thermostichus has been sequenced outside the Yellowstone hot spring. The purpose of this paper is to fill this gap.
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) is an immune system in prokaryotes with an adaptive ability. It provides immunity against phage infection (reviewed, e.g., in [21]). Although its potential in biotechnology and molecular biology has already been widely recognized [22], the role of CRISPR/Cas systems in evolution is still not understood [23]. It has been shown that sympatric species more likely share the same spacer rather than allopatric [24,25]. This suggests that CRISPR spacers evolve rapidly with the phage community [21]. Moreover, the immunity function of CRISPR/Cas systems suggests that it also provides a barrier to horizontal gene transfer, which potentially can play a significant role in genome diversification [26,27].
In this paper, we will focus on the whole-genome sequencing of a Thermostichus strain isolated from the Rupite hot spring in Bulgaria. We will perform phylogenomic reconstruction of the evolutionary history of our and related strains, we will compare their CRISPR/Cas system, and we will infer the intensity of the horizontal gene transfer among the strains.

2. Materials and Methods

2.1. Strain Information and Cultivation

We analyzed one strain of Thermostichus vulcanus isolated from the Rupite thermal spring. The hot spring is slightly mineralized (lat. 41°21′ N, long. 23°14′ E) with an inlet temperature of 71 °C [28]. It is located in the Sandanski-Petrich basin, Blagoevgrad Province, SW Bulgaria. The strain was isolated for a previous study [29]. Details of strain isolation and culture maintenance can be found there. The strain was identified as Synechococcus bigranulatus based on morphology in the light microscope. The BLAST search of the 16S rRNA sequence suggested its close relationship to Synechococcus strains JA-2-3Aa and JA-2-3B′a(2-13).

2.2. DNA Isolation and Sequencing

The genomic DNA was extracted by UltraClean Microbial DNA Isolation kit (Mobio, Carlsbad, CA, USA). The quality of DNA was assessed by 1.5% agarose electronic gel electrophoresis stained with ethidium bromide.
The sequencing library was prepared by Nextera DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA) and 50 ng of DNA was used. The selection of fragments was performed by Agilent High-Sensitivity DNA Kit (Agilent Technologies, Inc., Santa Clara, CA, USA) to obtain insert sizes of 1000 bp. The sequencing library concentration was evaluated by KAPA Library Quantification Kit for Illumina (Kapa Biosystems, Woburn, MA, USA). A sequencing run was performed using MiSeq Reagent Kit v3 (Illumina Inc., San Diego, CA, USA)—300 bp pair-end reads were generated. The sequencing was performed commercially at the Institute of Experimental Botany of the Czech Academy of Sciences (Olomouc, Czech Republic).
The raw reads were clipped and quality-filtered, and pair-ended reads were identified using Trimmomatic 0.36 [30]; by following settings: java -jar trimmomatic-0.36.jar PE -threads 24 -phred33 input.fq.gz output.fq.gz ILLUMINACLIP:Nextera-PE-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50. Filtered reads were assembled using Spades 3.10.1 [31]; with default settings. Spades 3.10.1 does not support metagenomics assembling with unpaired reads. Produced scaffolds were binned by Maxbin 2.2.4 [32]. The completeness of the genome was analyzed using CheckM v1.1.6 [33] and reached 100% with only 1.75% of contaminants. The scaffolds identified by BLASTn search as Thermostichus were annotated in RAST [34]. The annotation also included the identification of repeats and CRISPR spacer and repeats. The rest of the scaffolds were discarded. The genome assembly was submitted to GenBank (https://www.ncbi.nlm.nih.gov/ (accessed on 24 February 2021)) with the accession number JAFIRA000000000.

2.3. Phylogenetic Inference and Horizontal Gene Transfer Detection

We downloaded all proteomes of species that were related based on BLAST search to our genome and strains Thermostichus JA-2-3Aa and JA-2-3B′a(2-13). It was a total of nine genomes plus Thermostichus sp. PCC 7336 as an outgroup. The average nucleotide identity (ANI) represents the similarity of all homologous regions which are shared among the genomes. The BLAST (ANIb) and mummer (ANIm) algorithms were estimated using Jspecies 1.2.1 [35]. Orthologs were identified using OrthoFinder 2.3.1 [36]. The search yielded 1513 ortholog groups. The best model was identified using ModelFinder [37]; based on BIC (LG+F+G4). Subsequently, ML phylogenetic reconstruction was performed in IQ-TREE 1.6.5 [38]. The tree topology was tested by 2000 ultrafast bootstrap re-samplings. The relationships between the analyzed strains were also visualized by Densitree 2.2.5 [39]. The 1513 individual trees were constructed based on ortholog sets from the previous step using IQ-TREE with option -m TEST, which selected the most appropriate maximum likelihood model for each tree. The ortholog sets were aligned in MUSCLE 3.8.31 [40] with default settings.
The 16S rRNA phylogeny was performed in IQ-TREE 1.6.5. [38] using GTR+I+G (identified by ModelFinder) and it was tested by 2000 ultrafast bootstrap re-samplings. Sequences were identified using BLAST search. The 16S rRNA of Thermostichus (extracted from annotated ribosomal sequences of the draft genome) was used as a query. Sequences of 90% similarity were downloaded and aligned using MUSCLE 3.8.31 [40].
Horizontal gene transfer was analyzed using T-REX web server version 3.4 [41] with the following settings: consensus HGT tool, HGT detection mode—several HGTs by iteration, and optimization criterion—bipartition dissimilarity. The tree used for HGT analyses was obtained in the previous step.

3. Results

We obtained a draft genome of a strain of Thermostichus isolated from the Rupite hot spring in Bulgaria. The quality of genomes was provided by CheckM. Completeness of the genome was 100% and contamination was only 1.75%. N50 was 59 kb and the assembly was composed of 120 scaffolds. The estimated genome size based on the total length of scaffolds was 3.7 Mb and G+C content was 54.9%. The annotation revealed 3832 protein-coding regions and 44 RNAs.
The evolutionary history of the Thermostichus Rupite strain was reconstructed using phylogenomic analysis of 1513 single-copy orthologs. It revealed that Thermostichus Rupite is the sister group to the Yellowstone lineages represented by strains JA-2-3B′a(2-13) and JA-3-3Ab, but the node had low bootstrap support (Figure 1). Moreover, we estimated average nucleotide identity (ANI) using both BLAST and mummer algorithms. The ANI values for Thermostichus Rupite and the rest of the strains were below the species threshold (95–96% [35])—ANIb = 67–77% and ANIm = 83–87%. Similarly, the strain JA-2-3B′a(2-13) and the cluster of JA-3-3Ab had ANI values below the species threshold—ANIb = 84–85% and ANIm = 87–88%. The strains within the cluster of JA-3-3Ab had ANIb and ANIm between 98–99%, which is indicative that they belong to the same species (Supplementary Table S1). Further, we used densely sampled 16S rRNA phylogeny to clarify the relationship of Thermostichus Rupite to the other related taxa. The result confirmed the independent origin of the Thermostichus Rupite strain. Moreover, it is distantly related to Synechococcus group T1 and Synechococcus group A/B (Supplementary Figure S1).
We identified 1149 CRISPR spacers in all strains, and the number of spacers largely varied among them. The largest number of spacers was found in the Rupite Bulgaria strain (390; Figure 1). The second largest was identified in the non-thermophilic outgroup strain PCC 7336–286. Strains JA-2-3B′a(2-13) and JA-3-3Ab had 124 and 88, respectively. The strains clustering with JA-3-3Ab had between 21 and 72 spacers (Figure 1). The CRISPR spacers carry information about historical viral infections. They serve as a memory; therefore, if the same spacers are present in the genome, the strains may have been infected by the same virus. Moreover, a number of CRISPR spacers are indicative of the complexity of the CRISPR/Cas system within a strain [23]. Interestingly, all the strains had between four (63AY4M1) and 11 (65AY6Li; Table 1) CRISPR arrays. Thus, although the number of the CRISPR spacer was lower in the group of JA-3-3Ab, the number of the CRISPR arrays was not lower or was larger.
We found that the strains shared only a minority of the spacers; at most 14, in the case of 65AY6A5 and 60AY4M2 (Table 2). These two strains belong to the cluster of JA-3-3Ab and they were isolated from the same locality. The Rupite Bulgaria strain shared no spacers with the rest of the strains, and JA-2-3B′a(2-13) shared only one spacer with JA-3-3Ab (Table 2). All spacers were submitted to the BLAST search, which revealed no significant hit with any sequenced viruses stored in the NCBI GenBank.
The number of CRISPR spacers has varied during the evolution of Thermostichus. The Rupite Bulgaria strain had the most complex CRISPR system indicated by the number of genes. The CRISPR system was severely reduced in JA-2-3B′a(2-13) and again expanded in JA-3-3Ab and its related strains. All investigated strains possess class 1 [23] cas genes.
It should be also noted that the Rupite Bulgaria strain had a larger genome (estimated, 3.7 Mbp) than the rest of the thermophilic strains, which had genome sizes of approximately 3 Mbp. Besides the larger number of cas genes, RAST functional gene annotation revealed 81 genes with annotated functions that were not present in the JA-2-3B′a(2-13) genome (Supplementary Tables S2–S4). In total, JA-2-3B′a(2-13) had 727 fewer genes, but most of them were identified with a hypothetical function. The genes with identified functions were involved in the metabolism of amino acids, carbohydrates, cofactors and vitamins, DNA, membrane transport, nitrogen, nucleotides, phosphorus, protein, RNA, respiration, stress response, sulfur, and defense.
A number of horizontal gene transfers were used as a proxy for gene flow intensity between the investigated strains. A visual inspection of the Densitree visualization of 1513 individual phylogenetic trees revealed that the species tree topology (Figure 2) is violated by many gene trees in the case of Thermostichus Rupite and Synechococcus JA-2-3B′a(2-13), which are geographically distant. Further entanglement of gene trees appeared among Synechococcus sp. JA-3-3Ab and its related strains that were found at the same place, and they seem to belong to the same species. To provide statistical evidence for this pattern, we used T-Rex inference of HGT. Indeed, we found that HGT events were the most frequent within the clade of Synechococcus sp. JA-3-3Ab and its related strains (4741). Moreover, HGT events were more frequent between Thermostichus Rupite and Synechococcus JA-2-3B′a(2-13) (347) than between Synechococcus JA-2-3B′a(2-13) and Synechococcus sp. JA-3-3Ab and its related strains (52).

4. Discussion

Horizontal gene transfer (HGT) plays an important role in the evolution of bacterial species. Barriers to HGT can be raised by a geographical distance and genome defense mechanisms—CRISPR/Cas system or various restriction-modification systems (e.g., [2,12,23,42]). Here, we shed some new light on the patterns of HGT and the CRISPR/Cas evolution in the Thermostichus.
The Thermostichus strain isolated from the Rupite hot spring formed a distinct lineage from both Yellowstone strains in the phylogeny (Figure 1) and based on ANI values. The divergence is so deep that all three lineages (represented by strains Rupite, JA-2-3B′a(2-13), and JA-3-3Ab) can be considered as three species based on the criteria of [35] (ANI < 95–96). These three lineages together with Synechococcus PCC 7336 (it can also be identified as Thermostichus as in [17]) form the earliest diverged lineage in the photosynthetic cyanobacteria [19,43,44]. Although metagenomic studies and isolation efforts suggest that there are many cyanobacterial strains related to the Yellowstone Thermostichus in the other hot springs [20], this is the first genome of thermophilic strain from this clade that has been sequenced outside Yellowstone. It provides a unique source of information that can bring important insights into patterns in the evolution of the thermophilic cyanobacteria.
The Rupite strain had a one Mb larger predicted genome in comparison with the rest of the strains, which suggests that its genome is more complex. The genes with known annotation based on homology belonged to various metabolic pathways (Supplementary Table S2). We focused on the CRISPR/Cas system, which varied with respect to both genes and CRISPR spacers. Rupite strain had three times more spacer than JA-2-3B′a(2-13) and four times more than JA-3-3Ab and associated strains. Rupite strain also possessed more CRISPR-associated genes (Figure 1). However, they had almost the same number of CRISPR arrays, suggesting that the reduction happened relatively recently. The larger CRISPR spacer diversity may be suggestive of the adaptation to wider phage diversity (reviewed in [45]). Moreover, the CRISPR/Cas system has been shown to provide an effective barrier to HGT in Bacillus cereus [46]. Thus, diversification and adaptation of species of Thermostichus may have affected the intensity of the phage predation in the investigated species.
We identified over 1000 different spacers, but they were only rarely shared by the investigated strains and were predominantly in the clade of JA-3-3Ab. This evidences that the three hot spring Thermostichus species recently encountered different viruses, because the evolution of the spacers seems to be quite rapid [47]. It contradicts patterns observed in the thermophilic bacterium Thermus. Lopatina et al. [48] found that the Thermus strains which were isolated from the springs several hundred meters distant from each other have similar spacer sets. With an increasing distance, the spacers were more different. Furthermore, the BLAST search did not reveal any significant hit with any virus in the GenBank database. It suggests that the Thermostichus cyanobacteria are infected by unknown viruses.
The role of geographical isolation in the speciation of the microbes has been discussed for a long time with contrasting results. Thermophilic cyanobacteria were thought to have been geographically isolated due to scarce dispersal routes and because the hot spring has a very specific environment (e.g., [13,14]). These studies were based on several conserved markers, especially 16S rRNA, and had small taxon sampling. A larger dataset by Ionescu et al. [20] showed that the geographical isolation among the hot spring is less likely. We provide further evidence for the gene flow across long distances using the whole-genome data. We found that there was a more frequent horizontal transfer between the Rupite strain and JA-2-3B′a(2-13) than among the species originating from the same locality in the Yellowstone National Park (Figure 2). The two species JA-2-3B′a(2-13) and JA-3-3Ab showed very low HGT intensity.
The two species JA-2-3B′a(2-13) and JA-3-3Ab were suggested to represent two separate putative ecotypes inhabiting the same hot spring [49,50,51]. Their temperature optima concur with that—JA-2-3B′a(2-13) prefers 58−65 °C and JA-3-3Ab 51−65 °C [52]. Strunecký et al. [52]; showed that the Thermostichus Rupite strain occurs in temperatures of about 62 °C. Thus, the temperature optima lie between the putative ecotypes JA-2-3B′a(2-13) and JA-3-3Ab. This suggests that Thermostichus Rupite may be adapted to a different temperature regime than the rest of the strains.
Taken together, there have evolved at least three species of Thermostichus. Their evolution has been affected by an HGT and there seemed to be local barriers to HGT. CRISPR spacer diversity suggests that the trajectory of the evolution of the three species may have been directed by the different intensities of the virus predation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d14070502/s1, Figshare with https://doi.org/10.6084/m9.figshare.14423189, https://doi.org/10.6084/m9.figshare.14423195.v1, https://doi.org/10.6084/m9.figshare.14386688.v1, and https://doi.org/10.6084/m9.figshare.14386661.v1. Table S1: Average nucleotide values estimated by both Mummer and BLAST for all strain combinations. The third matrix summarizes Tetra values. https://doi.org/10.6084/m9.figshare.14423189. Table S2: A table of comparison of gene function between the Rupite strain and JA-2-3B′a(2-13). https://doi.org/10.6084/m9.figshare.14423195. Table S3: A table with a list of CRISPR repeats for each investigated strain. https://doi.org/10.6084/m9.figshare.19710835. Table S4: A list of investigated strains with assembly accession numbers in GenBank and their finishing level according to the GenBank nomenclature. https://doi.org/10.6084/m9.figshare.19710841. Figure S1: A phylogenetic reconstruction based on 16S rRNA using IQ-TREE inference. Thermostichus Rupite is marked with an arrow. Orange color represents strains from Synechococcus group A and B. Green color represents members of group T1. Supports at the nodes represent only significant values (>99 ultrafast bootstraps). Dataset S1: Multiple sequence alignment of the protein sequences used for the phylogenomic reconstruction. Fasta format. Size: 10 sequences, 492,086 amino acid positions. https://doi.org/10.6084/m9.figshare.14386661. Dataset S2: Phylogenetic trees, which were used for the horizontal gene analysis by T-Rex. The trees are in the Newick format. https://doi.org/10.6084/m9.figshare.14386688.

Author Contributions

Conceptualization, P.D.; methodology, P.D. and E.J.; data analysis, P.D. and E.J.; writing—original draft preparation, P.D., E.J. and A.P.; writing—review and editing, P.D. and E.J.; visualization, P.D. and E.J.; supervision, A.P.; project administration, P.D.; funding acquisition, P.D. and A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the project Internal Fund of Faculty of Education VaV_PdF_2021_04 and the Internal Grant Agency of the Palacký University Prf-2022-002.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets and supplements are available at Figshare with https://doi.org/10.6084/m9.figshare.14423189, https://doi.org/10.6084/m9.figshare.14423195.v1, https://doi.org/10.6084/m9.figshare.14386688.v1, and https://doi.org/10.6084/m9.figshare.14386661.v1. The genome was stored at the GenBank with an accession number: JAFIRA000000000.

Acknowledgments

We would like to thank Jaromír Lukavský (Institute of Botany, Czech Academy of Sciences) who provided the strain of Thermostichus.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Maximum likelihood phylogenomic inference based on 1513 orthologous genes. The studied strain is in bold. Asterisk represents 100 ultrafast bootstraps. Each taxon has a CRISPR spacer number and a list of CRISPR genes annotated within the respective genome.
Figure 1. Maximum likelihood phylogenomic inference based on 1513 orthologous genes. The studied strain is in bold. Asterisk represents 100 ultrafast bootstraps. Each taxon has a CRISPR spacer number and a list of CRISPR genes annotated within the respective genome.
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Figure 2. The Densitree visualization of the 1513 maximum likelihood phylogenetic trees. The thickened line represents the root canal—consensus tree with the highest support. The HGT frequency and direction estimated by T-Rex for each species are visualized by arrows and numbers.
Figure 2. The Densitree visualization of the 1513 maximum likelihood phylogenetic trees. The thickened line represents the root canal—consensus tree with the highest support. The HGT frequency and direction estimated by T-Rex for each species are visualized by arrows and numbers.
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Table
Table 1. The table summarizes basic CRISPR/Cas system features for each strain.
Table 1. The table summarizes basic CRISPR/Cas system features for each strain.
StrainCRISPR ArraysCRISPR SpacersCRISPR Genes
Synechoccus sp. Rupite Bulgaria639016
Synechocccus sp. JA-3-3Ab78814
Synechocccus sp. JA-2-3B′a(2-13)71242
Synechocccus sp. 60AY4M25336
Synechocccus sp. 63AY4M14216
Synechocccus sp. 63AY4M28455
Synechocccus sp. 65AY6A59566
Synechocccus sp. 65AY6Li117213
Synechocccus sp. 65AY6407346
Synechocccus sp. PCC7336828612
Table
Table 2. The matrix of common identical spacers among the investigated strains.
Table 2. The matrix of common identical spacers among the investigated strains.
Rupite BulgariaJA-3-3AbJA-2-3B′a(2-13)60AY4M263AY4M163AY4M265AY6A565AY6Li
Rupite Bulgaria
JA-3-3Ab0
JA-2-3B′a(2-13)01
60AY4M2000
63AY4M100010
63AY4M2000813
65AY6A500014813
65AY6Li0000010
65AY64001000000
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Jahodářová, E.; Poulíčková, A.; Dvořák, P. The CRISPR/Cas Machinery Evolution and Gene Flow in the Hot Spring Cyanobacterium Thermostichus. Diversity 2022, 14, 502. https://doi.org/10.3390/d14070502

AMA Style

Jahodářová E, Poulíčková A, Dvořák P. The CRISPR/Cas Machinery Evolution and Gene Flow in the Hot Spring Cyanobacterium Thermostichus. Diversity. 2022; 14(7):502. https://doi.org/10.3390/d14070502

Chicago/Turabian Style

Jahodářová, Eva, Aloisie Poulíčková, and Petr Dvořák. 2022. "The CRISPR/Cas Machinery Evolution and Gene Flow in the Hot Spring Cyanobacterium Thermostichus" Diversity 14, no. 7: 502. https://doi.org/10.3390/d14070502

APA Style

Jahodářová, E., Poulíčková, A., & Dvořák, P. (2022). The CRISPR/Cas Machinery Evolution and Gene Flow in the Hot Spring Cyanobacterium Thermostichus. Diversity, 14(7), 502. https://doi.org/10.3390/d14070502

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