First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

Introduction

Correct taxonomic identification and proper estimates of bacterial diversity are very important issues, due to wide environmental distribution, ecological functions, pathogenic potential and biotechnological applications of this domain. For more than a century, the identification/classification of bacteria has been done exclusively through culture-dependent methods, based on a series of morphological, physiological and biochemical tests after growth in appropriate media (Janda & Abbott, 2002). With the advances in molecular genetics, a criterion of DNA-DNA hybridization (DDH) showing a reassociation of 70% or more was established to define genospecies (Stackebrandt & Goebel, 1994). In the genomic era, various concepts and approaches have been debated as biologically meaningful systems for bacterial species definition (Konstantinidis, Ramette & Tiedje, 2006).

Sequencing of the 16S ribosomal RNA gene in prokaryotes has been widely used to determine taxonomic and phylogenetic relationships (Clarridge, 2004; Armougom & Raoult, 2009; Manaka, Tokue & Murakami, 2017). Bacterial strains with ≥70% DNA-DNA reassociation usually have >97% identity in their 16S rRNA gene sequence; on the other hand, less than 70% DNA-DNA hybridization, even with almost identical 16S rDNAs, may indicate different species (Janda & Abbott, 2007). This is especially relevant when ecological niches are included in the comparative analyses (Gevers et al., 2005; Konstantinidis, Ramette & Tiedje, 2006). Multi-locus sequencing analysis (MLSA) is also used for prokaryotic species definition, counting on a set of specific genes with sufficient evolutionary signals to allow fine discrimination between genetically close strains (Gevers et al., 2005). Polyphasic approaches (Vandamme et al., 1996) including all these methods have been regarded as the most suitable manner for taxonomic characterization of bacteria (Das et al., 2014; Sarethy, Pan & Danquah, 2014).

An often overlooked aspect to consider in the analyses of newly isolated strains from environmental samples (including endophytes) is how they are preliminarily assigned to a taxon (Woo et al., 2011). Studies have shown that horizontal gene transfer and recombination within the 16S rDNA do occur in bacteria (Schouls, Schot & Jacobs, 2003; Zhaxybayeva et al., 2006; Kitahara & Miyazaki, 2013; Tian et al., 2015). Experimental transfer of complete rRNA operons within and between species have resulted in viable organisms with heterologous 16S rRNA genes from two other species (Asai et al., 1999). Since some strains can carry up to 15 copies of the 16S rRNA gene (Klappenbach et al., 2001; López-López et al., 2007; Engene & Gerwick, 2011; Sun et al., 2013), transfer and recombination of segments can lead to a mosaic-like structure, i.e., different sequences of the 16S rDNA within the same cell (Eardly, Wang & Van Berkum, 1996; Schouls, Schot & Jacobs, 2003; Tian et al., 2015). The presence of multiple 16S rDNA copies and possible intragenomic heterogeneity can be, therefore, a limiting factor for both correct identification and counting of operational taxonomic units (OTUs) in biodiversity studies of both culturable and unculturable bacteria (Coenye & Vandamme, 2003; Pei et al., 2010; Chen et al., 2015). Although culturable microbes represent only 0.1–5% of the estimated total microbial diversity of any given environment (Bull, 2004), the levels of 16S rDNA intragenomic variation in a culturable population may provide a glance of what can occur in the whole community. Knowing such variation is useful in studies on microbial ecology and diversity, to prevent overestimation of this latter parameter (Sun et al., 2013) and to provide correction factors to compensate it.

Due to an intimate interaction with the plants, endophytic microbial symbionts can play important roles in host adaptation and evolution (e.g., Bulgarelli et al., 2013; Turner, James & Poole, 2013; Agler et al., 2016), performing relevant biological functions, such as increased photosynthetic efficiency, growth promotion, tolerance to abiotic and resistance to biotic stresses, including antagonism towards phytopathogens (Barrow et al., 2008; Hanada et al., 2009; Andreote, Gumiere & Durrer, 2014; Hardoim et al., 2015; Berg et al., 2017). Endophytes also represent a potential source of bioactive compounds with a variety of applications in agriculture and industry (Schulz et al., 2002; Schulz & Boyle, 2005; Leite et al., 2013; Gouda et al., 2016). The great genetic, molecular and biochemical diversification in microorganisms is the basis of these processes, with their greatest biodiversity being long acknowledged in tropical latitudes (Strobel & Daisy, 2003; Arnold & Lutzoni, 2007; Duarte et al., 2013). The cacao tree is an interesting experimental model for these regions, due to a globally recognized importance for its economic, social, and environmental characteristics (Donald, 2004; Schroth et al., 2011; Beg et al., 2017; Wickramasuriya & Dunwell, 2018). Moreover, exploratory studies on tropical cacao communities of endophytic fungi and bacteria in different plant tissues have been conducted, with emphasis on improvement of the plant productivity based on biological control and growth promotion (Rubini et al., 2005; Crozier et al., 2006; Mejía et al., 2008; Hanada et al., 2009; Hanada et al., 2010; Melnick et al., 2011; Leite et al., 2013; Tchinda et al., 2016).

Mixed electropherograms obtained from Sanger sequencing can be an interesting first-tier approach to assess polymicrobial samples composition or intragenomic heterogeneity of rRNA genes/operons. Previous studies have demonstrated the feasibility of employing direct sequencing of PCR-amplified 16S rDNA stretches to detect the presence of multiple microbes (Kommedal, Karlsen & Sæbø, 2008; Hartmeier & Justesen, 2010) or intragenomic rDNA variability in clinical samples of culturable isolates (Chen et al., 2015) by visually inspecting the corresponding mixed chromatograms. In the present work, a similar approach of Sanger-type chromatogram-based assessment, but complemented with frequent-cutter (AluI) restriction analyses was applied to study intragenomic variability in 16S rDNA in a set of culturable endophytic bacteria from cacao seeds. The results confirmed detection of intragenomic variation in individual isolates, suggesting this feature as being present in natural/environmental populations at high frequencies.

Materials & Methods

Results

A total of 65 culturable endophytic isolates obtained from cacao pulp adhered to seeds (Da Silva, 2013) were considered in this study. Their DNA was individually extracted and subjected to PCR amplification with specific primers targeting the V5–V9 hypervariable region of the 16S rDNA, 799F and U1492R (Chelius & Triplett, 2001). Each isolate generated a single PCR amplicon (electrophoretic band) that were individually sequenced in a direct manner (Sanger method), after purification from the gel. Under these conditions, no apparent unspecific amplification was observed in the gel for all samples. The preliminary identification of isolates, patterns of chromatogram obtained, and levels of sequence identity revealed by the BlastN are summarized in Table 1. The electropherograms from the 16S rDNA sequences presented two major patterns (Fig. 1): 15 isolates showed clear, single-peaks electropherograms of high quality sequences (‘clean’), whereas 50 presented mixed eletropherograms, with variable number and intensity of peaks underneath the main base-called sequences subjected to BlastN (Table 1). When the AluI restriction analysis was performed on the same PCR-amplified and gel-purified amplicons from the endophytic isolates, all but one of the clean sequences (93.3%, Table 1) presented the expected electrophoretic pattern of fragments, according to the number of AluI sites identified in silico for those sequences (Fig. 1).

In silico analysis for the AluI sites of the mixed electropherograms could not be done safely, due to the uncertainty of their base-called sequences (Table 1); hence, the number of restriction fragments generated was assessed by electrophoresis only. For these 50 mixed sequences, the majority of AluI restriction patterns (63.3%) showed ≥6 bands (Fig. 1). A chi-square analysis (2 × 2 contingency table) was performed to test the null hypothesis (H_o) of independence between the ‘number of AluI fragments’ (≤5 or ≥6 bands) and the ‘type of sequence’ (single or mixed). The results (χ² = 12.968; P = 0.0003) allowed to reject H_o, thereby indicating that the number of AluI restriction fragments was essentially dependent upon the type of chromatogram obtained. Interestingly, in certain cases of mixed sequences, possible indels in the 16S rRNA genes have apparently generated mixed templates detectable from specific points in the electropherogram (Fig. 2) (Chen et al., 2015). From a visual inspection and alignment, indels could be identified between the main and the underneath sequences, although the nucleotide signals for the latter were of such a lower level that further fluorescence-background interference prevented their safe detection. In the example shown (Fig. 2), the main base-called sequence was 99% identical to a Bacillus strain, whereas the underneath sequence dropped the identity to 96% closest to a Paenibacillus strain. These results strongly indicated the presence of intracell heterogeneity for the 16S rDNA for that particular isolate. Obviously, the observation of clean or mixed chromatograms (which pointed to unique or multiple templates, respectively) did not allow any inference about the number of copies of the 16S rRNA gene present in the organism.

To verify whether the strains retrieved by the BlastN search also show evidence of different 16S rRNA genes in fully sequenced genomes of the same species, an in silico analysis was performed with the 15 clean and the two mixed sequences showing 99% identity to database entries (see Table 1). These sequences were chosen due to their safer taxonomic identification, resulting from clearer and undoubtful base-called sequences that provided the highest cover and identity percentages when aligned with GenBank entries (Table 1, Fig. 1). From those 17 safe sequences, we were able to identify 11 deposited strains with fully sequenced genomes from four bacterial species: Bacillus thuringiensis, B. pumilus, Gluconobacter oxydans and Staphylococcus epidermidis (Fig. 3). The results showed a range of one to seven aligned copies of 16S rRNA gene that were different from each other and from our isolates. Most of the variations were base substitutions, but deletions and duplications were also identified (Fig. 3). Interestingly, a copy of 16S rRNA gene within the genome of the B. pumilus strain CP009108.1 showed an insertion of 14 bp not shared by the other seven copies, nor by our isolate ‘23’, which made it only 97% identical to them (Fig. 3B). It is also noteworthy that, for most of the clean-chromatogram’s isolates subjected to BlastN, the corresponding list of hits depicted from 7 to 51 different species with nucleotide identities ≥98%, thus providing a series of distinct genera/spp as possible candidates for taxonomic definition of an isolate, based on this 16S rDNA region.

Discussion

Sequences of 16S rRNA gene have long been used for identification of prokaryotic organisms (Srinivasan et al., 2015), at least in a preliminary manner. However, the intragenomic variation naturally present for this gene, both quantitatively (number of copies) and qualitatively (different DNA sequences), has been adding complexity to this matter (Coenye & Vandamme, 2003; Sun et al., 2013; Chen et al., 2015; Valdivia-Anistro et al., 2016). Due to the extensive use of 16S rRNA gene sequences for taxonomy, phylogenetics and metagenomics, for both culture-dependent and -independent methods, the assessment of the extent of these heterogeneities in natural populations appears as an important issue (Pei et al., 2010; Michon et al., 2010). Knowing the levels of intragenomic variation (i) in individual bacterial strains, (ii) in a genus or species, or (iii) in more diverse ecosystems (e.g., water, soils, plants, animals and humans’ microbiomes) would help researchers to adjust biodiversity estimates of populations/communities based on 16S rDNA sequences, as well as to choose which rDNA region is more appropriate to use for taxonomic identification purposes (Sun et al., 2013; Chen et al., 2015). In the present study, previously isolated endophytic bacteria from cacao seeds (Da Silva, 2013; Dos Santos, 2017) were used as a model system to address levels of intragenomic variation of this proxy gene in a natural population from a tropical environment. The assessment of an electropherogram profile from direct Sanger sequencing of rDNA-amplified fragment from a single culturable bacterial isolate, coupled with a frequent-cutter (AluI) restriction analysis of the amplicon, could serve properly as a first-tier indication of intragenomic heterogeneity in the 16S rRNA gene. In this case, the frequent-cutter restriction analysis was able to properly replace the need of cloning and re-sequencing the PCR amplicons (e.g., Chen et al., 2015) to confirm the multiple template condition.

The results obtained from the combined analyses of electropherograms and AluI restriction patterns (Fig. 1, Table 1) showed that clean chromatograms tend to be indicative of a unique template (with the number of restriction fragments in electrophoresis agreeing with the number of AluI sites seen in silico), whereas the mixed ones indicate more than a single template (frequently with ≥ 6 AluI bands) present in the sequenced amplicon. These results, which were fully supported by the chi-square analysis, is in close agreement with previous work with polymicrobial (Kommedal, Karlsen & Sæbø, 2008) and with clinical strictly anaerobical samples (Chen et al., 2015). The number of underneath peaks varied among mixed sequences, which may correspond to the number of templates available (only one underneath peak suggests two distinct templates; two underneath peaks, three templates, and so on; Kommedal, Karlsen & Sæbø, 2008).

The generally much lower identities (≤96%) obtained from 47 mixed sequences (Table 1) were likely due to the overlapping electropherograms that generated a lot of confounding signals for base calling, and so, likely formed “chimeric sequences” (in addition to those potentially formed as a PCR-sequencing artifacts; (Haas et al., 2011) that far departed from any previously described organism. Contrariwise, the ≥98% identity with database entries found for three mixed sequences (Table 1) can be explained by a lower-intensity signal of the secondary (underneath) peaks, which did not significantly disturb the base calling process. Under these circumstances, such mixed electropherograms in several microbial studies (that have likely not been reported) may have been mistakenly overlooked or discarded as either poor sequencing or isolate contamination (Chen et al., 2015), when, in fact, they might have been indicative of intragenomic heterogeneity in 16S rDNAs.

A relevant result from our sampling of culturable endophytes from cacao was the high percentage of intragenomic variation for the 16S rRNA gene in this type of environmental community of culturable bacteria (Table 1). Out of the 65 isolates under study, at least 32 (49.2%) showed a mixed chromatogram coupled with an AluI restriction pattern of ≥ 6 fragments, which safely detected more than a single template in the corresponding 799F/U1492R PCR amplicon. Considering the tropical origin of these isolates, this result suggests that part of the high levels of microbial diversity in these regions on Earth (Arnold et al., 2002; Strobel & Daisy, 2003; Duarte et al., 2013) is likely due to higher levels of genetic variability and gene transfer among bacteria (Schouls, Schot & Jacobs, 2003; Zhaxybayeva et al., 2006; Jensen, Frost & Torsvik, 2009; Kitahara & Miyazaki, 2013). An alternative possibility, though, that the culture medium used for isolation might have favored those bacteria with intrinsically higher rates of mutation/gene transfer cannot be ruled out. A common technical, though simplistic view for mixed chromatograms/sequences is that they result from contamination, due to certain associations of bacteria that are more difficult to separate by standard microbiological techniques, i.e., the case of syntrophs (Caldwell, 1995; Sanders, 2012). Nevertheless, such possibility is not expected to account for so many cases, mainly when very careful procedures for single-colonies isolation were employed throughout (see methods), and considering that endophytes do not tend to form syntrophic associations, due to the mostly aerobic growth conditions in planta (Morris et al., 2013). Therefore, it is fair to assume that contamination/syntrophy are likely not contributing to the results in a decisive manner (Chen et al., 2015). In addition, completely sequenced genomes in databases show that intragenomic 16S rDNA heterogeneity is not rare in Bacteria, with different levels and types of variation being taxon-dependent (Sun et al., 2013; Chen et al., 2015; Valdivia-Anistro et al., 2016); the same bacterial cell may have up to 15 copies of the 16S rRNA gene, and more than 2–3 types of sequences (Fig. 3) (Klappenbach et al., 2001; Jensen, Frost & Torsvik, 2009; Liu et al., 2015). Hence, such a condition of intragenomic 16s rDNA variation ought to be more careful addressed and considered, as it has direct consequences for species identification and analyses of richness, abundance and composition of bacterial communities in environmental samples (Coenye & Vandamme, 2003; Pei et al., 2010; Tian et al., 2015). Improved fitness to environmental stresses has been suggested as a driver for higher number and types of 16s rDNA in bacterial genomes (López-López et al., 2007; Jensen, Frost & Torsvik, 2009; Chen et al., 2015), although these relationships are the likely result of a complex multi-factorial interaction (Valdivia-Anistro et al., 2016).

The approach here presented did show some limitations in identifying the 16S rDNA intragenomic heterogeneity. The fact that 36% of the mixed-sequences isolates showed a number of restriction fragments compatible to clean ones (Table 1) suggests that other interfering factors leading to mixed-type chromatograms have also occurred. Several technical aspects that can add experimental variability to chromatograms/restriction analyses are, for instance, (i) quality of the gel-purified PCR amplicon sent to sequencing; (ii) potential presence of non-specific priming sites; (iii) quality/consistency of the sequencing procedure, including signal strength, base-calling sensitivity and PCR-derived chimeric sequence formation (Haas et al., 2011); (iv) possible mutation(s) during PCR amplification or bacterial culture (Martinez & Baquero, 2000); (v) occasional V5–V9 regions with a single or none AluI site that can lead to mixed chromatograms with ≤ 5 bands (Ashby et al., 2007); and (vi) occasional incomplete restriction digestion for certain sequences, as the conditions for full digestion (DNA and enzyme amounts, digestion time and temperature) vary among DNA structures/sequences and extracted biological samples. It is noteworthy that the efficiency/completeness of a type IIP enzyme digestion (such as AluI) depends on specific sequences around the restriction sites and/or proximity between sites in a single DNA molecule (Armstrong & Bauer, 1982; Alves et al., 1984; Pingoud & Jeltsch, 2001). The latter condition is a possible explanation for the pattern of ≥ 6 bands obtained for one clean-electropherogram isolate (Table 1), since analysis of its V5–V9 sequence revealed two of the AluI sites located less than 17 bp apart. Alternatively, other restriction enzymes can be used in the attempt to solve issues of this nature (e.g., Stakenborg et al., 2005; Jensen, Frost & Torsvik, 2009; Dos Santos, 2017). Once the technical aspects are addressed properly, the approach here employed can provide useful insights concerning the presence of 16S rDNA variation in natural populations at a first-tier level.

In the scope of ecology, diversity and evolution studies, certain aspects concerning intragenomic variability of 16S rDNA are important to highlight. It has been indicated that <97% identity between two 16S rRNA gene sequences is an indicative of different bacterial species (Gevers et al., 2005; Janda & Abbott, 2007). Interestingly, from our results, at least two different 16S rRNA gene sequences present in a single genome can account for differences higher than 3% identity (Figs. 2 and 3). Indeed, other authors have reported variations as high as 9.7% among the copies of 16S rDNA (e.g., Pei et al., 2010; Sun et al., 2013). This situation of cells containing heterologous 16S rRNA genes (Asai et al., 1999) is not so unexpected, as it can occur by horizontal transfer of complete operons between bacteria of the same or different species through transformation, conjugation and transduction (Armougom & Raoult, 2009; Smillie et al., 2010; Arber, 2014); studies have shown that Bacillus is a genus specially rich in terms of strains/cells harboring different types and copy number of rRNA operons (Liu et al., 2015), with such a variability being likely relevant for its wide niche occupancy (Valdivia-Anistro et al., 2016) and references therein).

Therefore, analysis of unculturable populations from environmental samples through 16S rDNA sequencing may lead to a relevant overestimation of the community diversity and composition (Pei et al., 2010; Haas et al., 2011; Sun et al., 2013; Chen et al., 2015), as different copies of this gene, although originating from the same cell, can match to different species in the database (Fig. 2). Our results suggest that assessments of microbial diversity from any environment, even if based upon high-throughput molecular techniques (e.g., Sinclair et al., 2015), may benefit from also obtaining culturable isolates to investigate the overall levels of their 16S rDNAs intragenomic heterogeneity. The concomitant use of a variety of different media (e.g., those focusing on culturing of specific bacterial subsets of interest; (López-López et al., 2007; Jensen, Frost & Torsvik, 2009; Michon et al., 2010; Tchinda et al., 2016) could help minimizing possible culturing biases of using a single-type medium, thereby providing more representative samplings. In this context, diversity overestimations of culture-independent methods can be somehow more precisely compensated. For instance, finding a given proportion of culturable isolates displaying such intragenomic variability (from a set of properly chosen media) could be applied to correct the OTUs diversity estimation found by a high-throughput molecular approach in the same sample. Depending on the previous knowledge existing in a study system, the information on OTUs overestimations provided by Sun et al. (2013) can be used with the overall approach presented here, which may further help in devising diversity-compensation strategies.

Conclusions

In conclusion, the coupling of direct Sanger sequencing with restriction enzyme analysis on PCR-purified DNA from culturable isolates was capable to consistently reveal intragenomic 16S rDNA heterogeneity in endophytic culturable populations of bacteria. To our knowledge, this is a pioneer report assessing this issue in bacteria inhabiting cacao fruits/seeds, which can be viewed as a relevant representative of tropical environments for endophytes. Despite that full-genome sequencing is the most accurate way to verify such a intragenomic variability, this strategy depends on specific conditions of logistics or facilities for the laboratories worldwide. The approach presented here combined simple and classical procedures that can fast detect intragenomic variation in 16S rDNAs at a first-tier level in any bacterial population. This appears as an alternative not only for initial fingerprinting of isolates in culturable collections, but also to investigate microbial ecology and biodiversity in an array of environments.

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