da Silva, Cleiziane Bispo;
dos Santos, Hellen Ribeiro Martins;
Marbach, Phellippe Arthur Santos;
de Souza, Jorge Teodoro;
Cruz-Magalhães, Valter;
Argôlo-Filho, Ronaldo Costa;
Loguercio, Leandro Lopes
First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds
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Media type:
E-Article
Title:
First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds
Contributor:
da Silva, Cleiziane Bispo;
dos Santos, Hellen Ribeiro Martins;
Marbach, Phellippe Arthur Santos;
de Souza, Jorge Teodoro;
Cruz-Magalhães, Valter;
Argôlo-Filho, Ronaldo Costa;
Loguercio, Leandro Lopes
Published:
PeerJ, 2019
Published in:
PeerJ, 7 (2019), Seite e7452
Language:
English
DOI:
10.7717/peerj.7452
ISSN:
2167-8359
Origination:
Footnote:
Description:
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 correspondingAluI-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 ofAluI 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 theAluI-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.