Increased c-di-GMP Levels Lead to the Production of Alginates of High Molecular Mass in Azotobacter vinelandii.

Azotobacter vinelandii produces the linear exopolysaccharide alginate, a compound of significant biotechnological importance. The biosynthesis of alginate in A. vinelandii and Pseudomonas aeruginosa has several similarities but is regulated somewhat differently in the two microbes. Here, we show that the second messenger cyclic dimeric GMP (c-di-GMP) regulates the production and the molecular mass of alginate in A. vinelandii The hybrid protein MucG, containing conserved GGDEF and EAL domains and N-terminal HAMP and PAS domains, behaved as a c-di-GMP phosphodiesterase (PDE). This activity was found to negatively affect the amount and molecular mass of the polysaccharide formed. On the other hand, among the diguanylate cyclases (DGCs) present in A. vinelandii, AvGReg, a globin-coupled sensor (GCS) DGC that directly binds to oxygen, was identified as the main c-di-GMP-synthesizing contributor to alginate production. Overproduction of AvGReg in the parental strain phenocopied a ΔmucG strain with regard to alginate production and the molecular mass of the polymer. MucG was previously shown to prevent the synthesis of high-molecular-mass alginates in response to reduced oxygen transfer rates (OTRs). In this work, we show that cultures exposed to reduced OTRs accumulated higher levels of c-di-GMP; this finding strongly suggests that at least one of the molecular mechanisms involved in modulation of alginate production and molecular mass by oxygen depends on a c-di-GMP signaling module that includes the PAS domain-containing PDE MucG and the GCS DGC AvGReg.IMPORTANCE c-di-GMP has been widely recognized for its essential role in the production of exopolysaccharides in bacteria, such as alginate produced by Pseudomonas and Azotobacter spp. This study reveals that the levels of c-di-GMP also affect the physical properties of alginate, favoring the production of high-molecular-mass alginates in response to lower OTRs. This finding opens up new alternatives for the design of tailor-made alginates for biotechnological applications.

Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii.

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Prevalencia de parásitos intestinales en niños del Área Metropolitana de Barranquilla, Colombia / Intestinal parasites prevalence in children from Barranquilla (Colombia) Metropolitan Area

Introducción: la parasitosis intestinal constituye un importante problema de salud pública, la Organización Mundial de la Salud, considera que la infección por geohelmintos afecta un aproximado de 1500 millones de personas en el mundo y se calcula que 46 millones de niños están en riesgo de contraer infecciones por geohelmintos en América Latina. Objetivos: determinar la prevalencia de parasitismo intestinal en niños menores de 10 años de tres poblaciones pertenecientes al Área Metropolitana de Barranquilla, Colombia. Métodos: estudio descriptivo de corte transversal, en el que se analizaron 411 muestras fecales de niños entre 1 mes y 10 años de edad; recolectadas durante el año 2014 en tres diferentes poblaciones del Área Metropolitana de Barranquilla (Distrito de Barranquilla, Corregimiento de la Playa y Municipio de Galapa). El análisis parasitológico se realizó mediante examen directo de las heces en solución salina, lugol, y concentración con el método formol-éter. Se estableció la frecuencia absoluta y relativa de los parásitos presentes y se compararon los resultados entre los tres lugares de muestreo. Resultados: se observó una prevalencia de parasitismo intestinal del 45,3 por ciento en todo el AMB, la cual fue mayor en Galapa y La Playa; presentándose además, en La Playa una alta prevalencia de helmintos 19,2 por ciento. El protozoario de mayor prevalencia fue Blastocystis sp 22,1 por ciento y el patógeno más frecuente encontrado fue Giardia intestinalis, presente en el 9,7 por ciento de las muestras analizadas. Conclusiones: la alta prevalencia de parásitos en los niños plantea la necesidad de realizar programas de vigilancia y control a toda la población a nivel local. La presencia de protozoarios como Blastocystis sp., parásito relacionado con precarias condiciones higiénicas del agua de consumo, hace evidente la urgencia de crear estrategias para mejorar el saneamiento básico y la educación sanitaria como ejes fundamentales en el control de las parasitosis(AU)

The Na+-translocating NADH : ubiquinone oxidoreductase of Azotobacter vinelandii negatively regulates alginate synthesis.

Azotobacter vinelandii is a nitrogen-fixing soil bacterium that produces the exopolysaccharide alginate. In this report we describe the isolation and characterization of A. vinelandii strain GG4, which carries an nqrE : : Tn5 mutation resulting in alginate overproduction. The nqrE gene encodes a subunit of the Na+-translocating NADH : ubiquinone oxidoreductase (Na+-NQR). As expected, Na+-NQR activity was abolished in mutant GG4. When this strain was complemented with the nqrEF genes this activity was restored and alginate production was reduced to wild-type levels. Na+-NQR may be the main sodium pump of A. vinelandii under the conditions tested ( approximately 2 mM Na+) since no Na+/H+-antiporter activity was detected. Collectively our results indicate that in A. vinelandii the lack of Na+-NQR activity caused the absence of a transmembrane Na+ gradient and an increase in alginate production.

Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production.

Azotobacter vinelandii is a soil gamma-proteobacteria that fixes nitrogen and forms desiccation-resistant cysts. The exopolysaccharide alginate is an integral part of the layers surrounding the cysts. Here, we reported the cloning of A. vinelandii algC, encoding the enzyme catalyzing the second step of alginate pathway. We showed that AlgC is involved not only in alginate production, but also in lipopolysaccharide (LPS) synthesis and that it seems to have both phosphomannomutase and phosphoglucomutase activities. The transcriptional analysis of the A. vinelandii algC gene showed that it contained two start sites, one of which was dependent on the alternative sigma factor AlgU/AlgT. This finding explains why alginate biosynthesis is dependent on AlgU activity, since all other alginate biosynthetic genes have been characterized previously and algC is the only alginate structural gene that is directly transcribed by this sigma factor.

Expression of the Azotobacter vinelandii poly-beta-hydroxybutyrate biosynthetic phbBAC operon is driven by two overlapping promoters and is dependent on the transcriptional activator PhbR.

The Azotobacter vinelandii phbBAC genes encode the enzymes for poly-beta-hydroxybutyrate (PHB) synthesis. The phbR gene, which is located upstream of and in the opposite direction of phbBAC, encodes PhbR, a transcriptional activator which is a member of the AraC family of activators. Here we report that a mutation in phbR reduced PHB accumulation and transcription of a phbB-lacZ fusion. We also report that phbB is transcribed from two overlapping promoters, p(B)1 and p(B)2. The region corresponding to the -35 region of p(B)1 overlaps the p(B)2 -10 region. In the phbR mutant, expression of phbB from the p(B)1 promoter is significantly reduced, whereas expression from the p(B)2 promoter is slightly increased. Two phbR promoters, p(R)1 and p(R)2, were also identified. Transcription from p(R)2 was shown to be dependent on sigma(S). Six conserved 18-bp sites, designated R1 to R6, are present within the phbR-phbB intergenic region and are proposed to be putative binding targets for PhbR. R1 overlaps the -35 region of the p(B)1 promoter. A model for the regulation of phbB transcription by PhbR is proposed.