Introduction

Cyanobacteria are an aged group of photosynthetic prokaryotes, their first molecular carbon skeletons can be identified in strata from around 2.75 billion years ago (Gould SB et al., 2008). The progenitors of current cyanobacteria originated oxygenic photosynthesis some 3. 6 billion years ago (Gould SB et al., 2008). Evaluative studies on firmly related cyanobacteria indicate rapid and highly variable gene fluxes in evolving microbial genomes (Walter JM et al., 2017). Cyanobacteria comprise both unicellular and colonial (including filamentous) forms. Taxonomically, cyanobacteria are classified into unicellular forms featuring binary fission (Order Chroococcales, or Bergey’s Subsection I) or multiple fission (Order Pleurocapsales, or Bergey’s Subsection II); and filamentous forms that are non-heterocystous (Order Oscillatoriales, or Bergey’s Subsection III) or show heterocysts in non-branching (Order Nostocales, or Bergey’s Subsection IV) or branching filaments (Order Stigonematales, or Bergey’s Subsection V). A sixth cyanobacterial order, Gloeobacterales, was proposed by Cavalier-Smith (2002)- to accommodate the genus Gloeobacter, formerly included in the Chroococcales (Komárek, 2013).

Cyanobacteria represent a difficult group for the microbiologists. Their conventional taxonomy, in view of morphogenesis traits, doesn't reflect the results of phylogenetic analyses (Howard-Azzeh M et al., 2014, Walter JM et al., 2017). The predominance of morphologic criteria assembled unrelated cyanobacteria under polyphyletic taxa which will require revisions later on (Komárek and Johansen., 2014). Morphologically similar strains might contrast extraordinarily at the molecular level and vice-versa. In a few occasions it will be not challenging distinguish cyanobacterial isolates to the genus level, especially where morphologic aspects are distinctive, e. g. Calothrix or Nostoc. However, to a number genera, including Oscillatoria, Lyngbya , and Phorrnidium, it may be frequently challenging to the non-expert to approach to convinced diagnoses. Identification problems expand further at the species level and little may be known about sub particular variability at the strain level. Despite these paramount traits and the expanding enthusiasm toward developing cyanobacterial strains for biotechnology, there is a shortage and disturbed dispensation of publicly accessible genomic data on Cyanobacteria. Improvements in the coverage of sequenced genomes will empower more accurate understanding of cyanobacterial niche-adaptation and evolution (Howard-Azzeh M et al., 2014, Sánchez-Baracaldo P et al., 2014, Schirrmeister BE et al., 2015, Shih PM et al., 2013, Uyeda JC et al., 2016).

Developments in molecular biology and bioinformatics permit mining the genome of an organism for the presence of unique sequences that can be used for recognizing a specific group of microorganism from its close relatives. Similar techniques have been developed based on primers that hybridize with repeated sequence structures present in bacterial DNA. These primers permit amplification of the DNA sequences between those adjacent repeated sequences happening in a suitable orientation and distance apart. Additionally, PCR-based techniques largely dependent on DNA polymorphism and fingerprinting of repetitive DNA fragments have been developed (Elhai J, 2015). RFLP (Restriction Fragment Length Polymorphism)(Iteman I et al., 2002), RAPD (Random Amplification of Polymorphic DNA) (Prabina BJ et al., 2005; Shishir et al., 2015), STRR (Short Tandemly Repeated Repetitive)(Akoijam C and Singh AK, 2011, Valerio E et al., 2009, Wilson KM et al., 2000), HIP1 (Highly Iterated Palindromes) (Neilan B et al., 2003, Orcutt K et al., 2002, Wilson AE et al., 2005, Zheng W et al., 2002) and ERIC (Enterobacterial Repetitive Interspersed Consensus) (Valério E et al., 2005) have been attempted with an overall aim to provide better resolution among closely related species. These repetitive sequences were diagnosed in several cyanobacterial taxa, up to now broadly in heterocystous cyanobacteria (Lyra C et al., 2005, Nilsson M et al., 2000, Prasanna R et al., 2006, Rasmussen U and Svenning MM, 1998, Teaumroong N et al., 2002, Wilson KM et al., 2000, Zheng W et al., 1999). but also in some non-heterocystous ones (Rasmussen U and Svenning MM, 1998). The conserved status of these repetitive sequences have made them ideal tools for diversity studies, and have brought forth a brand new PCR-based technique known as the rep-PCR technique that utilizes oligonucleotide-derived repetitive sequences present in bacterial strains to separate firmly related members of the same genus. This technique has been enormously productive in discriminating members of several eubacterial genera (Laguerre G et al., 1996, Rodriguez-Barradas MC et al., 1995). The genera Calothrix and Nostoc are filamentous cyanobacteria belonging to the Order Nostocales. Members of this order exhibit a high level of morphological complexity (Nowruzi B et al., 2012) and the incredible morphotype diversity observed in nature is typically underrepresented in cultures (Gugger MF and Hoffmann L, 2004).

The aim of the present investigation was to fingerprint these strains using PCR based on the repetitive DNA sequences and the 16S rRNA gene as molecular markers to resolve close cogeneric strains, also we used these markers together with the phylogenetic affinities, to differentiate seven heterocystous cyanobacteria sharing similar stress tolerance profiles.

Materials and methods

Isolation and maintenance of clonal and axenic cultures of heterocystous cyanobacteria Dry limestones harboring different Nostoc species have been observed
in the North-west Mountains of Iran. Such dry and sometimes cold conditions are suitable for Nostoc growths (Helm et al.,2000, Hill et al., 1994, Potts, 2000, Shirkey et al., 2003). Samples were collected from Cretaceous nodular chalk limestone rocks on the cliff face in the North-west Mountains of Khuzestan
province, Iran (34°25´04" N, 47°00´59" W). Rocks were collected from the upper greensand layer, where limestone is predominant, together with glauconitic inclusions. Nostoc inhabits the surface and interior of the rocks, forming a homogenous epilithic covering. For the exposure experiments, rocks were cut into blocks with an upper surface area of 5 cm². Additionally, soil samples with different textures (according to the pedological map of the Kermanshah province) were selected and collected from agricultural areas (34°24´32" N, 47°00´17" W). Samples were collected from the surface up to five cm deep with a sterilized
spatula after removing surface debris. Finally, saltwater samples (36°54´41? N, 54°47´25? W) were collected at ca. 30 cm depth and 1 m away from the shore using cone-shaped bottles. Samples were transferred to sterile Petri dishes with a suitable amount of BG11 nitrate-free liquid and solid media (Rippka et al., 1979). pH was adjusted to 7.1 after sterilization, and Petri dishes were incubated in a culture chamber at 28 °C, supplied with continuous artificial illumination (~1500–2000 lux) for two weeks (Kaushik et al., 2009). After 14 days, one or two colonies were isolated for purification,washed thrice with deionized water and transferred to fresh solid media. In order to keep bacteria-free cultures, the colonies were isolated and tested for bacterial contamination in dextrose-peptone
broth and caseinate-glucose agar media. Thereafter, bacteria free clones were selected and maintained on different agar slants. No further analysis of the cultures was done until pure clonal cultures were established and examined microscopically.

Morphological characterization

Cyanobacteria were characterized using the standard keys by Desikachary (1959) and Komárek (2013), considering only traditional morphological groups. Samples were analyzed in two stages: a first one in natural conditions and a second one when the growth phase begins under laboratory conditions. This ensures no differences between naturally occurring samples and laboratory-grown cultures. Phenotypic characters particularly observed were the shape and dimensions of vegetative and specialized cells (heterocysts, akinetes, baeocytes, arthrospores, hormocytes etc.), and some other features were observed depending on the type of cyanobacteria: division plane, branching and branching pattern, a polarity of trichomes, etc. Some samples of Nostocaceae were grown for a longer period so as to document life cycle changes if any.

Genomic DNA isolation and PCR conditions 

DNA was isolated from 8-10 days old cultures using the EZNA^® SP Plant DNA kit (Omega Bio-Tek). Microtubes containing 100 mg wet cells were filled with 300 mg of two differently-sized acid-washed glass beads (180 and 425–600 µm in diameter, Sigma-Aldrich), adding lysis buffer and RNase solution as provided by the kit. In order to ensure proper disruption of the cells, tubes were homogenized three times for 20 s at 6.5 ms^-1 with a FastPrep homogenizer (Savant Instruments). The extraction procedure continued following the manufacturer protocol. DNA was quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc). 16S rRNA gene amplifications were done using the primers pA (5'-AGAGTTTGATCCTGGCTCAG-3') and B23S (5'-CTTCGCCTCTGTGTGCCTAGGT-3') (Taton A et al., 2003). The PCR reaction starts with 1× Buffer solution (DyNAzyme^TM PCR buffer, Finnzymes), 0.5 µM of forward primer, 0.5 µM of reverse primer, 0.5 U of Taq polymerase (DyNAzyme^TM II DNA polymerase, Finnzymes), and 1 µL of template DNA, with sterile water until a total volume of 20 µL. Amplification reactions were conducted in a thermocycler (iCycler, Bio-Rad) with the following program: initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and  at 72 °C for 30 s, and a final annealing phase at 72 °C for 5 min. In order to amplify repetitive DNA fragments, reactions were performed in 25 µL aliquots containing 10-20 ng of DNA template, 0.5 µM of each ERIC, STRR1a and HIP primers, 1.5 mM of MgCl₂, 200 µM of dNTPs and 1U/µL of Taq DNA polymerase. ERIC1A (5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC1B (5'-AAGTAAGTGACTGGGGTGAGCG-3') were used as ERIC primers (Valério E et al., 2005). The first cycle at 95 °C for 7 min was followed by 30 cycles at 94 °C for 1 min, at 52 °C for 1 min, at 65 °C for 8 min, one cycle at 65 °C for 16 min, and a final incubation at 4 °C for 30 min (Valério E et al., 2005). For the STRR1a primer (5'-CCARTCCCCARTCCCC-3'), cycles were as follows: initial denaturation at 95 °C for 6 min, 30 cycles at 94 °C for 1 min, at 56 °C for 1 min, and at 65 °C for 5 min, with a subsequent extension at 65 °C for 16 min and a final incubation at 4 °C for 30 min (Rasmussen U and Svenning MM, 1998). For all the HIP variants (HIP-TG: 5'-GCGATCGCTG-3', HIP-GC: 5'-GCGATCGCGC-3' and HIP-CA: 5'-GCGATCGCCA-3'), thermal cycling conditions began with an initial denaturation at 95 °C for 5 min, 30 cycles at 95 °C for 30 s, at 30 °C for 30 s, at 72 °C for 60 s, and a final cycle at 72 °C for 5 min (Smith J et al., 1998).

PCR products were checked by electrophoresis on 1% agarose gels (SeaPlaque® GTG®, Cambrex Corporation) at 100 V, followed by 0.10 µg mL^-1 EtBr (Bio-Rad) staining. PCR products were visualized in the gel by UV light using a Molecular Imager® Gel Doc^TM XR system (Bio-Rad). A digital image was obtained utilizing the QUANTITY ONE® 1-D V 4.6.7 analysis software. The size of the products was estimated by comparison with marker DNA (?/HinfIII + fx/HaeIII, Finnzymes). The products were purified using the Geneclean® Turbo kit (Qbiogene, MP Biomedicals) and quantified with a Nanodrop^TM ND-1000 spectrophotometer (Thermo Scientific). Sequencing of the partial 16S rRNA genes was subsequently performed using a BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Life Technologies) (Elhai J, 2015, Valerio E et al., 2009).

Phylogenetic analyses of partial 16S rRNA gene

BLAST searches (http://www.ncbi.nlm.-nih.gov/BLAST) for the partial 16S rRNA gene was performed to identify similar sequences deposited in the NCBI GenBank™ database. The 16S rRNA gene sequences obtained in this study, as well as reference sequences retrieved from GenBank, were first aligned with MUSCLE (Edgar RC, 2004) and maximum likelihood phylogenetic trees were inferred in IQ-Tree (Nguyen L-T et al., 2014). The robustness of the tree was estimated by bootstrap percentages using 1000 replications. The root of the tree was determined using the 16S rRNA of Aquifex aeolicus and Chloroflexus aurantiacus as outgroups (Fig. 4). To prevent in group monophyly, 16S rRNA sequences of Escherichia coli, Chloroflexus aurantiacus, and Agrobacterium tumefaciens were included in the alignment.

Phylogenetic analysis of fingerprints

The generated HIP profiles were run on agarose gels with the same concentration in order to differentiate strong and doubtful signals/bands. Presence/absence of distinct and reproducible bands in each of the individual DNA fingerprinting pattern generated by HIP-AT, HIP-CA, HIP-GC, HIP-TG, ERIC, and STRR1a PCR profiles were converted into binary data (Selvakumar G and Gopalaswamy G, 2008), and the pooled binary data was used to construct a composite dendrogram (Abony et al., 2018). The BioDiversity Pro software (vers. 2) was used to perform the hierarchical analyses using the Jaccard cluster analysis option. All reactions were repeated three times.

Nucleotide accession numbers

Studied strains named Calothrix spp. R11 andR42, and Nostoc spp. FA1, FA3, FA5, F4, and F3 were registered in the DNA Data Bank of Japan (DDBJ) based on their partial 16S rDNA gene and under accession numbers MG356332, MG356333, MG385055, MG385056, MG385057, MG549315 and MG549314, respectively, and deposited at herbarium ALBORZ in Cyanobacteria Culture Collection (CCC) of the Science and Research Branch (Islamic Azad University, Iran) with herbarium numbers R11, R42, FA1, FA3, FA5, F4 and F3, respectively.

Results

Morphological characterization

Calothrix spp. R11 and R42

Trichomes in both strains always with basal, more or less spherical or hemispherical heterocysts (A and D) (Fig. 1), yellow-brownish colored. Cells cylindrical or barrel-shaped. In Calothrix sp. R11, there is an immediate intercalary heterocysts near the basal heterocysts (E) (Fig. 1a), while it is absent in Calothrix sp. R42. A distinctive feature of R42 is the presence of more swollen cells at the base of mature trichomes, with very thick sheaths (B) (Fig. 1b), whereas trichomes in sp. R11 have a distinct basal-apical polarity and display a high degree of tapering. Trichomes ending in hair-like apical structures, composed of narrow, hyaline cells (C) (Fig. 1a), are characteristic in Calothrix sp. R11. No obvious macroscopic colonies were visible on the agar plates for both strains. Microscopic colonies were light to dark green in mature Calothrix sp. R42, but light to dark brown in Calothrix sp. R11 (Fig. 1).

Figure 1: photomicrograph of Calothrix sp. R11 (Left) and Calothrix sp. R42 (right). Bars, 10 µm. Spherical or hemispherical heterocysts (A and D). Thick sheaths (B). Trichomes ending in hair-like apical structures, composed of narrow, hyaline cells (C)

Nostoc spp. FA1, FA3, and FA5

The three Nostoc species collected from limestone did not exhibit remarkable morphological differences. Trichomes were isodiametrical throughout, composed of cylindrical and uniforms cells, 2.5-5 µm wide, 6-7 µm long, light blue-green or olive, heterocysts spherical or oblong, 4-6.5 µm wide, 7-9.5 µm long, spores ellipsoidal to oblong, 4-6.5 µm wide, 9-11 µm long (Fig. 2).

Figure 2: Photomicrograph of Nostoc sp. A3 (left), Nostoc sp. A5 (right) and Nostoc sp. FA1 (middle). Bars, 10 µm. (Heterocysts are shown by arrows).

Nostoc spp. F4 and F3

Cell dimensions were very similar in both strains, although in Nostoc sp. F4 filaments flexuously twisted. Vegetative cells were spherical or slightly oblong (3-5 µm broad, 4.5-7 µm long), olive and heterocysts were oval (4.5-6.5 µm broad, 4-7.5 µm long). Akinetes were rarely found (Fig. 3).

Figure 3: Photomicrograph of Nostoc sp. F4 (left) and Nostoc sp. F3 (right). Bars, 10 µm. (Heterocysts are shown by arrows).

Phylogenetic analysis of the 16S rRNA gene

A section of the 16S rRNA gene was successfully amplified by the PCR technique. The resulting phylogenetic tree showed that, among available 16S rRNA sequences, studied strains formed three closely related clusters and were strictly separated from other members of the Nostocales clade (Fig. 4).

Figure 4: Maximum-likelihood tree (IQ-Tree) based on the partial 16S rRNA gene sequenced in this study or taken from the GenBank. The studied strains are shown with red color. The scale bar represents 0.03 base substitutions per 1000 nucleotide position. Bootstrap percentages calculated from 1000 resembling are indicated at nodes.

Strain differentiation by Rep-PCR generated fingerprint profile

In the present investigation, the maximal amplification was observed for the nontolerant cyanobacterial Calothrix sp. R11 culture. 28 amplified products with the size 400–1500 bp were obtained using the HIP TG primer (Fig. 5C). 29 amplified products with the size 300–1500 bp were obtained using HIP GC primer (Fig. 5C). Salt-tolerant Nostoc sp. F4 and F3 cultures produced the maximal number of amplified fragments with these primers. Finally, 33 amplified products with the size 300–1500 bp were obtained using STRR primer (Fig. 5D). Salt-tolerant and limestone cultures produced the maximal number of amplified fragments with these primers (Table 1).

Table 1: Number of amplified products of studied strains by Rep-PCR generated fingerprint profile

Stress tolerant Nostoc sp. F4 and Nostoc sp. F3 cultures represented unique banding patterns (Fig. 3). A total of 13 amplified products with the size 400–3000 bp were obtained by using ERIC1A primer (Figs. 6E and 6F), whereas 43 amplified products (200–3000 bp in size) were obtained with the ERIC1B primer (Figs. 6E and 6F). ERIC1A led to the lowest number and the broadest range of bands. The largest number of amplified fragments was obtained for Nostoc sp. F4 with this primer. HIP primers used in the study were decamers with a common consensus sequence (5’GCGATCGC3’) followed by a two-base tail of either AT, TG, GC or TC. A total of 30 amplified products with the size 200–1500 bp were obtained by using HIP CA primer (Figs. 6E and 6F). A total of 33 amplified products with the size 200–1500 bp were obtained using HIP AT primer (Figs. 6E and 6F).

Figure 5. PCR amplification pattern of the cyanobacterial cultures with the primer ERIC1A Lane (1-5) and ERIC1B Lane (6-10) (A). HIP-CA (1-5) and HIP-AT (6-10) (B). HIP-TG (1-5) and HIP-GC (6-10) (C). STRR1a (D). Lane M Molecular weight marker (100 bp Plus); Complete names are Nostoc sp. FA5; Nostoc sp. FA1; Nostoc sp. FA3; Calothrix sp. R11; Calothrix sp. R42; Nostoc sp. FA1; Nostoc sp. FA3; Nostoc sp. FA5; Calothrix sp. R11; Calothrix sp. R42.

Figure 6. PCR amplification pattern of the Nostoc sp.F3 (E) and Nostoc sp.F4 (F) with the primer HIP-CA and HIP-AT, HIP-GC, HIP-TG, STRR, ERIC1A, ERIC1B and STRR1. Lane M Molecular weight marker (100 bp Plus).

Clustering of the PCR profiles revealed the presence of three major groups (S. Figs. 7-10). The two salt-tolerant cultures (Nostoc spp. F4 and F3) formed a first cluster for the ERIC1A and ERIC1B primers, sharing a 100% similarity (Fig. 7), while they exhibited the lowest similarity (50%) with the HIP-AT primer. The two nontolerant cultures (Calothrix spp. R11 and R42) formed the third cluster for the all primers (except HIP-AT) and were found to be distinct from the stress-tolerant isolates, sharing a 100% similarity with ERIC1A and HIP-CA primers but the lowest similarity (50%) with the STRR primer. Limestone cultures were found to share a 100% similarity with HIP-GC and STRR primers; however, they had little similarity between themselves using other primers. For instance, Nostoc sp. FA3 and FA5 were found to share a 100% similarity, while Nostoc sp. FA1 shared a 60% similarity with both strains. Interestingly, the stress-tolerant Nostoc strains (saltwater and limestone cultures) did not share any clustering (S. Fig. 8- 10).

Conclusion

From the present investigation, it is concluded that the repetitive sequences found in the genomes of Cyanobacteria are very useful in exploring genomic relationships among different strains, which supports their use for Cyanobacterial discrimination and identification in different climatic/geographic Regions and habitats.

Author Contributions

BN designed the study, analyzed the data and drafted the manuscript; RS, HF and SB did meticulous revision of the manuscript.

Acknowledgment

No acknowledgment.

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