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Res Microbiol ; 167(3): 190-201, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26708984

RESUMO

In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway.


Assuntos
Azospirillum brasilense/enzimologia , Azospirillum brasilense/fisiologia , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Azospirillum brasilense/genética , Técnicas Bacteriológicas , Coenzimas/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Flavina-Adenina Dinucleotídeo/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/metabolismo , Microscopia Confocal , Fósforo-Oxigênio Liases/isolamento & purificação , Coloração e Rotulagem
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