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

Cleiziane Bispo Da Silva,Ronaldo Costa Argôlo-Filho,Phellippe Arthur Santos Marbach,Valter Cruz-Magalhães,Hellen Ribeiro Martins Dos Santos,Jorge Teodoro De Souza,Leandro Lopes Loguercio

doi:10.7717/peerj.7452

Cleiziane Bispo Da Silva, Ronaldo Costa Argôlo-Filho + Show 5 more

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https://doi.org/10.7717/peerj.7452

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Abstract

BackgroundIntragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner.MethodsTotal genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5–V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the ‘number of AluI bands’ and ‘type of eletropherogram’.ResultsTwo types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity.ConclusionThe approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

Highlights

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
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)
Knowing the levels of intragenomic variation (i) in individual bacterial strains, (ii) in a genus or species, or (iii) in more diverse ecosystems 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)

Summary

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. 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). The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed

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