Nucleotides as Bacterial Second Messengers.

In addition to comprising monomers of nucleic acids, nucleotides have signaling functions and act as second messengers in both prokaryotic and eukaryotic cells. The most common example is cyclic AMP (cAMP). Nucleotide signaling is a focus of great interest in bacteria. Cyclic di-AMP (c-di-AMP), cAMP, and cyclic di-GMP (c-di-GMP) participate in biological events such as bacterial growth, biofilm formation, sporulation, cell differentiation, motility, and virulence. Moreover, the cyclic-di-nucleotides (c-di-nucleotides) produced in pathogenic intracellular bacteria can affect eukaryotic host cells to allow for infection. On the other hand, non-cyclic nucleotide molecules pppGpp and ppGpp are alarmones involved in regulating the bacterial response to nutritional stress; they are also considered second messengers. These second messengers can potentially be used as therapeutic agents because of their immunological functions on eukaryotic cells. In this review, the role of c-di-nucleotides and cAMP as second messengers in different bacterial processes is addressed.

Genomic insights into the c-di-GMP signaling and biofilm development in the saprophytic spirochete Leptospira biflexa.

C-di-GMP is a bacterial second messenger with central role in biofilm formation. Spirochete bacteria from Leptospira genus present a wide diversity, with species of medical importance and environmental species, named as saprophytic. Leptospira form biofilms in the rat's reservoir kidneys and in the environment. Here, we performed genomic analyses to identify enzymatic and effector c-di-GMP proteins in the saprophytic biofilm-forming species Leptospira biflexa serovar Patoc. We identified 40 proteins through local alignments. Amongst them, 16 proteins are potentially functional diguanylate cyclases, phosphodiesterases, or hybrid proteins. We also identified nine effectors, including PilZ proteins. Enrichment analyses suggested that c-di-GMP interacts with cAMP signaling system, CsrA system, and flagella assembly regulation during biofilm development of L. biflexa. Finally, we identified eight proteins in the pathogen Leptospira interrogans serovar Copenhageni that share high similarity with L. biflexa c-di-GMP-related proteins. This work revealed proteins related to c-di-GMP turnover and cellular response in Leptospira and their potential roles during biofilm development.

The Sinorhizobium meliloti NspS-MbaA system affects biofilm formation, exopolysaccharide production and motility in response to specific polyamines.

We previously showed that specific polyamines (PAs) present in the extracellular environment markedly affect extracellular polysaccharide (EPS) production, biofilm formation and motility in Sinorhizobium meliloti Rm8530. We hypothesized that extracellular PA signals were sensed and transduced by the NspS and MbaA proteins, respectively, which are homologs of the PA-sensing, c-di-GMP modulating NspS-MbaA proteins described in Vibrio cholerae. Here we show that the decrease in biofilm formation and EPS production in the quorum-sensing (QS)-deficient S. meliloti wild-type strain 1021 in cultures containing putrescine or spermine did not occur in a 1021 nspS mutant (1021 nspS). The transcriptional expression of nspS in strain 1021 was significantly increased in cultures containing either of these polyamines, but not by exogenous cadaverine, 1,3-diaminopropane (DAP), spermidine (Spd) or norspermidine (NSpd). Cell aggregation in liquid cultures did not differ markedly between strain 1021 and 1021 nspS in the presence or absence of PAs. The S. meliloti QS-proficient Rm8530 wild-type and nspS mutant (Rm8530 nspS) produced similar levels of biofilm under control conditions and 3.2- and 2.2-fold more biofilm, respectively, in cultures with NSpd, but these changes did not correlate with EPS production. Cells of Rm8530 nspS aggregated from two- to several-fold more than the wild-type in cultures without PAs or in those containing Spm. NSpd, Spd and DAP differently affected swimming and swarming motility in strains 1021 and Rm8530 and their respective nspS mutants. nspS transcription in strain Rm8530 was greatly reduced by exogenous Spm. Bioinformatic analysis revealed similar secondary structures and functional domains in the MbaA proteins of S. meliloti and V. cholerae, while their NspS proteins differed in some residues implicated in polyamine recognition in the latter species. NspS-MbaA homologs occur in a small subset of soil and aquatic bacterial species that commonly interact with eukaryotes. We speculate that the S. meliloti NspS-MbaA system modulates biofilm formation, EPS production and motility in response to environmental or host plant-produced PAs.

CdgC, a Cyclic-di-GMP Diguanylate Cyclase of Azospirillum baldaniorum Is Involved in Internalization to Wheat Roots.

Azospirillum baldaniorum is a plant growth-promoting rhizobacterium (PGPR) capable of fixing nitrogen, the synthesis of several phytohormones including indole-acetic acid, and induction of plant defenses against phytopathogens. To establish a successful and prolonged bacteria-plant interaction, A. baldaniorum can form biofilms, bacterial communities embedded in a self-made matrix formed by extracellular polymeric substances which provide favorable conditions for survival. A key modulator of biofilm formation is the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), which is synthesized by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases. In this study, we analyzed the contribution of a previously uncharacterized diguanylate cyclase designated CdgC, to biofilm formation and bacterial-plant interaction dynamics. We showed that CdgC is capable of altering c-di-GMP levels in a heterologous host, strongly supporting its function as a DGC. The deletion of cdgC resulted in alterations in the three-dimensional structure of biofilms in a nitrogen-source dependent manner. CdgC was required for optimal colonization of wheat roots. Since we also observed that CdgC played an important role in exopolysaccharide production, we propose that this signaling protein activates a physiological response that results in the strong attachment of bacteria to the roots, ultimately contributing to an optimal bacterium-plant interaction. Our results demonstrate that the ubiquitous second messenger c-di-GMP is a key factor in promoting plant colonization by the PGPR A. baldaniorum by allowing proficient internalization in wheat roots. Understanding the molecular basis of PGPR-plant interactions will enable the design of better biotechnological strategies of agro-industrial interest.

In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes.

BACKGROUND: The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences. RESULTS: The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5T, A. lipoferum 4B, Azospirillum sp. B510, A. thiophilum BV-ST, A. halopraeferens DSM3675, A. oryzae A2P, and A. brasilense Sp7, Sp245 and Az39 encode for 29 to 41 of these predicted proteins. Notably, only 15 proteins were conserved in all nine genomes: eight GGDEF, three EAL and four GGDEF-EAL hybrid domain proteins, all of which corresponded to core genes in the genomes. The predicted proteins exhibited variable lengths, architectures and sensor domains. In addition, the predicted cellular localizations showed that some of the proteins to contain transmembrane domains, suggesting that these proteins are anchored to the membrane. Therefore, as reported in other soil bacteria, the Azospirillum genomes encode a large number of proteins that are likely involved in c-di-GMP metabolism. In addition, the data obtained here strongly suggest host specificity and environment specific adaptation. CONCLUSIONS: Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.

The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.

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.

[Networks involving quorum sensing, cyclic-di-GMP and nitric oxide on biofilm production in bacteria]. / Redes de señalización en la producción de biopelículas en bacterias: quorum sensing, di-GMPc y óxido nítrico.

Bacterial biofilms are ubiquitous in nature, and their flexibility is derived in part from a complex extracellular matrix that can be made-to-order to cope with environmental demand. Although common developmental stages leading to biofilm formation have been described, an in-depth knowledge of genetic and signaling is required to understand biofilm formation. Bacteria detect changes in population density by quorum sensing and particular environmental conditions, using signals such as cyclic di-GMP or nitric oxide. The significance of understanding these signaling pathways lies in that they control a broad variety of functions such as biofilm formation, and motility, providing benefits to bacteria as regards host colonization, defense against competitors, and adaptation to changing environments. Due to the importance of these features, we here review the signaling network and regulatory connections among quorum sensing, c-di-GMP and nitric oxide involving biofilm formation.

Redes de señalización en la producción de biopelículas en bacterias: quorum sensing, di-GMPc y óxido nítrico / Networks involving quorum sensing, cyclic-di-GMP and nitric oxide on biofilm production in bacteria

Las bacterias forman biopelículas de manera ubicua, y esta característica les otorga una flexibilidad que es resultado, en parte, de una matriz compleja construida según las exigencias de las condiciones ambientales. Aunque los estadios de la formación de las biopelículas bacterianas se conocen con detalle, para entender con profundidad la formación de las biopelículas es deseable un conocimiento mayor de los mecanismos de señalización. Las bacterias detectan cambios en la densidad de población por regulación del quórum y condiciones específicas, empleando señales como el di-GMPc y el óxido nítrico. La importancia del conocimiento de estas vías de señalización radica en que controlan una variedad de funciones, como la formación de biopelículas y la movilidad, y proporcionan a las bacterias beneficios en la colonización del hospedador, la defensa contra competidores y los cambios adversos del entorno. Por la trascendencia que revisten estos aspectos, revisamos aquí las redes de regulación y la conexión de la señalización entre quorum sensing, di-GMPc y óxido nítrico

Bacterial biofilms are ubiquitous in nature, and their flexibility is derived in part from a complex extracellular matrix that can be made-to-order to cope with environmental demand. Although common developmental stages leading to biofilm formation have been described, an in-depth knowledge of genetic and signaling is required to understand biofilm formation. Bacteria detect changes in population density by quorum sensing and particular environmental conditions, using signals such as cyclic di-GMP or nitric oxide. The significance of understanding these signaling pathways lies in that they control a broad variety of functions such as biofilm formation, and motility, providing benefits to bacteria as regards host colonization, defense against competitors, and adaptation to changing environments. Due to the importance of these features, we here review the signaling network and regulatory connections among quorum sensing, c-di-GMP and nitric oxide involving biofilm formation