The GGDEF-EAL protein CdgB from Azospirillum baldaniorum Sp245, is a dual function enzyme with potential polar localization.

Azospirillum baldaniorum Sp245, a plant growth-promoting rhizobacterium, can form biofilms through a process controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP). A. baldaniorum has a variety of proteins potentially involved in controlling the turnover of c-di-GMP many of which are coupled to sensory domains that could be involved in establishing a mutualistic relationship with the host. Here, we present in silico analysis and experimental characterization of the function of CdgB (AZOBR_p410089), a predicted MHYT-PAS-GGDEF-EAL multidomain protein from A. baldaniorum Sp245. When overproduced, CdgB behaves predominantly as a c-di-GMP phosphodiesterase (PDE) in A. baldaniorum Sp245. It inhibits biofilm formation and extracellular polymeric substances production and promotes swimming motility. However, a CdgB variant with a degenerate PDE domain behaves as diguanylate cyclase (DGC). This strongly suggest that CdgB is capable of dual activity. Variants with alterations in the DGC domain and the MHYT domain negatively affects extracellular polymeric substances production and induction of swimming motility. Surprisingly, we observed that overproduction of CdgB results in increased c-di-GMP accumulation in the heterologous host Escherichia coli, suggesting under certain conditions, the WT CdgB variant can behave predominantly as a DGC. Furthermore, we also demonstrated that CdgB is anchored to the cell membrane and localizes potentially to the cell poles. This localization is dependent on the presence of the MHYT domain. In summary, our results suggest that CdgB can provide versatility to signaling modules that control motile and sessile lifestyles in response to key environmental signals in A. baldaniorum.

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.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense.

Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

A Review of Eight High-Priority, Economically Important Viral Pathogens of Poultry within the Caribbean Region.

Viral pathogens cause devastating economic losses in poultry industries worldwide. The Caribbean region, which boasts some of the highest rates of poultry consumption in the world, is no exception. This review summarizes evidence for the circulation and spread of eight high-priority, economically important poultry viruses across the Caribbean region. Avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), fowl adenovirus group 1 (FADV Gp1), and egg drop syndrome virus (EDSV) were selected for review. This review of serological, molecular, and phylogenetic studies across Caribbean countries reveals evidence for sporadic outbreaks of respiratory disease caused by notifiable viral pathogens (AIV, IBV, NDV, and ILTV), as well as outbreaks of diseases caused by immunosuppressive viral pathogens (IBDV and FADV Gp1). This review highlights the need to strengthen current levels of surveillance and reporting for poultry diseases in domestic and wild bird populations across the Caribbean, as well as the need to strengthen the diagnostic capacity and capability of Caribbean national veterinary diagnostic laboratories.

Separate evolution of virulent newcastle disease viruses from Mexico and Central America.

An outbreak of Newcastle disease (ND) in poultry was reported in Belize in 2008. The characteristics of three virulent Newcastle disease virus (NDV) isolates from this outbreak (NDV-Belize-3/08, NDV-Belize-4/08, and NDV-Belize-12/08) were assessed by genomic analysis and by clinicopathological characterization in specific-pathogen-free (SPF) chickens. The results showed that all three strains belong to NDV genotype V and are virulent, as assessed by the intracerebral pathogenicity index and the polybasic amino acid sequence at the fusion protein cleavage site. In 4-week-old SPF chickens, NDV-Belize-3/08 behaved as a typical velogenic viscerotropic NDV strain, causing severe necrohemorrhagic lesions in the lymphoid organs, with systemic virus distribution. Phylogenetic analysis of multiple NDV genotype V representatives revealed that genotype V can be divided into three subgenotypes, namely, Va, Vb, and Vc, and that all tested Belizean isolates belong to subgenotype Vb. Furthermore, these isolates are nearly identical to a 2007 isolate from Honduras and appear to have evolved separately from other contemporary viruses circulating in Mexico, clustering into a new clade within NDV subgenotype Vb.

The Caribbean animal health network: new tools for harmonization and reinforcement of animal disease surveillance.

The Caribbean Animal Health Network (CaribVET) is a collaboration of veterinary services, diagnostic laboratories, research institutes, universities, and regional/international organizations to improve animal health in the Caribbean. New tools were used by the network to develop regional animal health activities: (1) A steering committee, a coordination unit, and working groups on specific diseases or activities were established. The working group on avian influenza used a collaborative Web site to develop a regionally harmonized avian influenza surveillance protocol and performance indicators. (2) A specific network was implemented on West Nile virus (WNV) to describe the WNV status of the Caribbean countries, to perform a technology transfer of WNV diagnostics, and to establish a surveillance system. (3) The CaribVET Web site (http://www.caribvet.net) encompasses information on surveillance systems, diagnostic laboratories, conferences, bibliography, and diseases of major concern in the region. It is a participatory Web site allowing registered users to add or edit information, pages, or data. An online notification system of sanitary information was set up for Guadeloupe to improve knowledge on animal diseases and facilitate early alert.