title: The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.
abstract: 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.
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif. 103J, Av. San Claudio S/N, Col. San Manuel, Puebla Pue CP 72570, Mexico. Electronic address: aramirezmata@gmail.com.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ramírez-Mata
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif. 103J, Av. San Claudio S/N, Col. San Manuel, Puebla Pue CP 72570, Mexico. Electronic address: 13_isela13@hotmail.com.
authors.forename: Lilia I
authors.initials: LI
authors.lastname: López-Lara
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif. 103J, Av. San Claudio S/N, Col. San Manuel, Puebla Pue CP 72570, Mexico. Electronic address: luisa.xiqui@hotmail.com.
authors.forename: Ma Luisa
authors.initials: ML
authors.lastname: Xiqui-Vázquez
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif. 103J, Av. San Claudio S/N, Col. San Manuel, Puebla Pue CP 72570, Mexico. Electronic address: jims_pcm@hotmmail.com.
authors.forename: Saúl
authors.initials: S
authors.lastname: Jijón-Moreno
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif. 103J, Av. San Claudio S/N, Col. San Manuel, Puebla Pue CP 72570, Mexico. Electronic address: angy.4behappy@gmail.com.
authors.forename: Angelica
authors.initials: A
authors.lastname: Romero-Osorio
authors.affiliation: Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edif. 103J, Av. San Claudio S/N, Col. San Manuel, Puebla Pue CP 72570, Mexico. Electronic address: beatriz.baca@correo.buap.mx.
authors.forename: Beatriz E
authors.initials: BE
authors.lastname: Baca
mesh_terms: D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D001431:Bacteriological Techniques; D018441:Biofilms / Q000254:growth & development*; D003067:Coenzymes / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D018613:Microscopy, Confocal; D019761:Phosphorus-Oxygen Lyases / Q000302:isolation & purification / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D013194:Staining and Labeling
chemical_list: D003067:Coenzymes; D029968:Escherichia coli Proteins; D011135:Polysaccharides, Bacterial; D005182:Flavin-Adenine Dinucleotide; C062025:bis(3',5')-cyclic diguanylic acid; D006160:Guanosine Triphosphate; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP; D008274:Magnesium
keywords: Azospirillum brasilense; Biofilm production; Cyclic di-GMP; Diguanylate cyclase activity