Compatible solutes are accumulated in response to osmotic stress and are used as an abundant nutrient source in marine bacteria
Date
2024
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University of Delaware
Abstract
Marine bacteria thrive in environments with variable levels of salinity and have adapted strategies to combat the osmotic stress they encounter. Bacteria in the genus Vibrio are Gram-negative marine bacteria that belong to the family Vibrionaceae and are distributed throughout aquatic environments. Vibrio species have been isolated from salt marshes, deep sea sediment, the water column, and are known to colonize marine flora and fauna. Many Vibrio species are significant pathogens of humans and cause infectious disease in shellfish and fish. This diversity makes Vibrio an ideal model to investigate osmotic stress response mechanisms in marine bacteria. When bacteria encounter an environment with high salinity, they respond by accumulating small low molecular weight compounds called compatible solutes that can be accumulated to molar concentrations within the cell without interfering with essential cellular processes. Intracellular accumulation of compatible solutes is completed through uptake from their surroundings or biosynthesis. Transporters are used to scavenge free compatible solutes from the environment, and these include betaine-carnitine-choline-transporter (BCCT) family transporters, ATP-binding cassette (ABC) family transporters, Major facilitator superfamily (MFS) transporters, and tripartite ATP-independent periplasmic (TRAP) family transporters. Bacteria also biosynthesize compatible solutes such as glycine betaine (GB) and ectoine, but this is energetically more costly than uptake from the environment. In Chapter 2, to better understand how compatible solute biosynthesis is regulated at the transcriptional level, we examined the de novo biosynthesis of ectoine in Vibrio parahaemolyticus, the number one cause of bacterial seafood-borne gastroenteritis in humans. In Chapter 3, we investigated the catabolism of ectoine by Vibrio species, specifically in V. diabolicus, a species related to V. parahaemolyticus, but not known to cause human disease. In Chapter 4, the discovery of the catabolism of myo-inositol a compatible solute and signaling molecule is described in V. coralliilyticus, a coral pathogen. ☐ In Chapter 2, we uncovered novel regulators of the ectoine biosynthesis ectABC-asp_ect operon. The de novo biosynthesis of ectoine is carried out by approximately 70% of members of Vibrionaceae family, many of which are marine halophiles. Using a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region, we identified, amongst others, three regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. This study identified LeuO as a positive regulator of ectABC-asp_ect and showed that LeuO is an anti-silencer of H-NS, which is a global gene silencer. In addition, NhaR was shown to be a negative regulator of the ectoine biosynthesis operon. Growth analysis showed that the ∆leuO, ∆nhaR and ∆hns mutants all had significant growth defects in high salinity, indicating that all three regulators play a wider role in salinity tolerance. ☐ Chapter 3 described for the first-time the genes for the catabolism of the compatible solute ectoine in Vibrionaceae. The genes for ectoine degradation were present in a 13 gene cluster comprised of catabolism, transporter, and regulatory genes. The work showed that Vibrio diabolicus, a species originally recovered from hydrothermal deep-sea vents, was able to both biosynthesize and catabolize ectoine as a sole carbon source. Interestingly, V. diabolicus catabolized ectoine as efficiently as glucose, producing similar growth yields. Within Vibrionaceae, the ectoine catabolism gene cluster was found in species of Vibrio, Salinivibrio, Enterovibrio, Grimontia, and one species of Photobacterium and was widespread among marine species. Ectoine is a well-established compatible solute used to relieve osmotic stress, but its function as a nutrient presents bacteria with a versatile source of energy. ☐ In Chapter 4, we further investigated the ability of Vibrio species to take advantage of compatible solutes in their environment as diverse nutrient sources. Myo-inositol is a stress protectant that is biosynthesized and used by algae for protection from osmotic stress among other functions. The genes necessary for myo-inositol catabolism (iol) were found in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species within the Vibrionaceae family. The work in this chapter showed that V. coralliilyticus is able to use myo-inositol as a sole carbon source and verified an essential gene (iolG) in this pathway through the construction of a deletion mutant. We also demonstrated that a major facilitator superfamily (MFS) transporter (iolT1) and an ABC-family (iolXYZ) transporter uptake myo-inositol for use as a carbon source. Our phylogenetic analysis of myo-inositol catabolism genes showed that the evolutionary history is complex and multiple acquisition events were identified. Similar to other Vibrio species, V. coralliilyticus is a host-associated bacteria and encounters competition for nutrients. Therefore, V. coralliilyticus has evolved metabolic strategies to better compete for food.
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Keywords
Compatible solutes, Ectoine, Myo-inositol catabolism, Osmotic stress, Vibrio