Production of 3',3'-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme, Bd0367, regulates exit from prey by gliding motility.

Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3',3'-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.

Evaluation of California-Grown Blood and Cara Cara Oranges Through Consumer Testing, Descriptive Analysis, and Targeted Chemical Profiling.

The acceptability of Moro, Tarocco, Cara Cara, Shahani, Bream Tarocco, Boukhobza, and Sanguinelli oranges from both commercial and research orchards was tested with adult (n = 152) and child (n = 72) consumers. Qualitative focus groups were also conducted to understand consumer familiarity and thoughts about the fruit. Sensory descriptive and chemical analyses were carried out to identify drivers of liking. Overall, consumers preferred the lighter colored varieties consisting of Tarocco, Cara Cara, and Boukhobza. One cluster of adults (n = 80) showed preferences towards sweet and fruity flavors and away from sourness and citric acid. The second adult cluster (n = 72) was tolerant of the sour fruit but did not like fruit high in bitterness and flavonoid content. The largest child cluster (n = 42) showed preferences for samples higher in orange and tropical flavors (Cara Cara, Tarocco, and Boukhobza varieties). The appearance of the Cara Cara was strongly liked by the consumer population in both quantitative and qualitative settings. Hunter scale a color values strongly correlated to the higher berry/dried fruit flavors, and concentrations of naringenin. Focus group participants noted that they were relatively unfamiliar with blood oranges. Growers and producers may want to invest in the lighter colored varieties, such as Cara Cara, Tarocco, Boukhobza and Shahani, as these were liked by a majority of consumers and were low in less desirable sensory characteristics, such as bitterness and sourness. PRACTICAL APPLICATION: Through consumer tests, sensory evaluation, and chemical analyses, this research uncovered which sensory properties may drive consumer acceptance of blood and Cara Cara oranges, and informed potential production and marketing strategies for increasing their consumption. This information should benefit the citrus industry as a whole and may enhance the use of specialty oranges by the food, beverage, and food service industries.