Biology subjects

Boas Lichty, K.

Publications and source records attributed to Boas Lichty, K..

4 recordsLinked to original sources

bioRxiv2025

Dual roles of glycine betaine (GB), dimethylglycine, and sarcosine as osmoprotectants and nutrient sources for Vibrio natriegens

Bacteria respond to osmotic stress by intracellularly accumulating low molecular weight compounds called compatible solutes (CS), also known as osmolytes. Glycine betaine (N,N,N-trimethylglycine, GB) is a highly effective and widely available osmolyte used by bacteria, algae, and plants for abiotic stress protection. Here, we highlight the dual roles of GB, dimethyl glycine (DMG), and sarcosine for both osmoprotection and a less known role as sole carbon sources. First, we showed that the marine halophile Vibrio natriegens can grow in 1% to 7% NaCl and biosynthesize GB, ectoine, and glutamate, and imported GB, DMG, and sarcosine in response to osmotic stress. Betaine-carnitine-choline transporters (BCCTs) for the uptake of GB and DMG, but not sarcosine, were identified. Bioinformatics analyses uncovered homologs of GB, DMG, and sarcosine catabolism genes (dgcAB_fixAB, gbcA, gbcB, purU, soxBDAG, glyA, glxA) clustered in the V. natriegens genome and these genes had a limited distribution among vibrios. We showed V. natriegens ATCC 14048 grew on GB, DMG, and sarcosine as sole carbon sources and gbcA and dgcA were required for growth. A contiguous catabolism cluster was present in a subset of V. fluvialis strains, and we demonstrated growth of V. fluvialis 2013V-1197 in DMG and sarcosine as sole carbon sources. Phylogenetic analysis revealed the catabolism cluster did not share a common ancestor among members of the family Vibrionaceae. IMPORTANCECompatible solutes are frequently the most concentrated organic components in marine organisms allowing them to adapt to high saline environments as well as affording protection to other abiotic stresses. These organic compounds are significant energy stores that have been overlooked for their potential as abundant nutrient sources for bacteria. Our study characterized GB, DMG, and sarcosine catabolism genes and showed their efficient use as carbon and energy sources by marine halophilic vibrios.

microbiology↗

bioRxiv2024

The coral pathogen Vibrio coralliilyticus utilizes the compatible solute and signaling molecule myo-inositol as a sole carbon source

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna. IMPORTANCEHost associated bacteria such as V. coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

microbiology↗

bioRxiv2024

Regulators H-NS and LeuO inversely control swarming motility and biofilm formation in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a halophile present in marine environments worldwide and is a leading cause of bacterial seafood-borne gastroenteritis. Free living Vibrio parahaemolyticus planktonic cells can either attach to surfaces to form swarming cells or develop into a sessile three-dimensional biofilm structure. Swarming motility requires lateral flagella (laf operon) and the expression of the surface sensing operon scrABC to produce a spreading cauliflower colony morphology. Biofilms are formed from capsule polysaccharide (CPS) encoded by the cpsA-K operon that is positively regulated by CpsR and CpsQ. In enteric bacteria, H-NS is a global gene silencer and LeuO is an antagonist of H-NS. In this work, we examined the role of these regulators in the decision between swarming and biofilm behaviors using deletion mutants of leuO, hns, and a double deletion leuO/hns. The wild type and {Delta}leuO strains produced swarming colonies whereas {Delta}hns produced a hyper swarming whereas in {Delta}leuO/{Delta}hns, the phenotype reverted to wild type. Transcriptional reporter assays of PlafB-gfp and PscrA-gfp showed significantly increased fluorescence in {Delta}hns compared to wild type. In the {Delta}leuO/{Delta}hns mutant, PlafB-gfp fluorescence reverted to wild type levels and PscrA-gfp showed increased fluorescence compared to wild type. In CPS assays, {Delta}leuO had a less dense rugose morphology compared to wild type and {Delta}hns produced a smooth colony, which also produced significantly less biofilm. {Delta}leuO/{Delta}hns had an opaque morphology and produced significantly more biofilm. Reporter expression assays of PcpsA-gfp and PcpsR-gfp confirmed the roles of both H-NS and LeuO in CPS and biofilm formation. ImportanceThis study determined the role of LeuO and H-NS in controlling the decision between two surface based behaviors, swarming motility and sessile biofilm formation in V. parahaemolyticus. The effects of deletions of leuO, hns, and a double {Delta}leuO/{Delta}hns mutant showed that H-NS was a negative regulator of swarming, but a positive regulator of biofilm formation. The mechanism of this control was in part due to H-NS inhibition of LeuO at loci required for swarming and biofilm formation.

microbiology↗

bioRxiv2022

LeuO and H-NS are part of an expanded regulatory network for ectoine biosynthesis expression

Bacteria accumulate compatible solutes, to maintain cellular turgor pressure when exposed to high salinity. In the marine halophile Vibrio parahaemolyticus, the compatible solute ectoine is biosynthesized de novo, which is energetically more costly than uptake; therefore, tight regulation is required. To uncover novel regulators of the ectoine biosynthesis ectABC-asp_ect operon, a DNA affinity pulldown of proteins interacting with the ectABC-asp_ect regulatory region was performed. Mass spectrometry analysis identified, amongst others, three regulators: LeuO, NhaR, and the nucleoid associated protein H-NS. In-frame non-polar deletions were made for each gene and PectA-gfp promoter reporter assays were performed in exponential and stationary phase cells. PectA-gfp expression was significantly repressed in the {Delta}leuO mutant and significantly induced in the {Delta}nhaR mutant compared to wild type, suggesting positive and negative regulation, respectively. In the {Delta}hns mutant, PectA-gfp showed increased expression in exponential phase cells, but no change compared to wild type in stationary phase cells. To examine whether H-NS interacts with LeuO or NhaR at the ectoine regulatory region, double deletion mutants were created. In a {Delta}leuO/{Delta}hns mutant, PectA-gfp showed reduced expression, but significantly more than {Delta}leuO suggesting H-NS and LeuO interact to regulate ectoine expression. Whereas {Delta}nhaR/{Delta}hns had no additional effect as compared to {Delta}nhaR suggesting NhaR regulation is independent of H-NS. To examine leuO regulation further, a PleuO-gfp reporter analysis was examined that showed significantly increased expression in the {Delta}leuO, {Delta}hns and {Delta}leuO/{Delta}hns mutants as compared to wild type, indicating both are repressors. Growth pattern analysis of the mutants in M9G 6%NaCl, showed growth defects compared to wild type, indicating that these regulators play an important physiological role in salinity stress tolerance. ImportanceEctoine is a commercially used compatible solute that acts as a biomolecule stabilizer because of its additional role as a chemical chaperone. A better understanding of how the ectoine biosynthetic pathway is regulated in natural bacterial producers can be used to increase efficient industrial production. The de novo biosynthesis of ectoine is essential for bacteria to survive osmotic stress when exogenous compatible solutes are absent. This study identified LeuO as a positive regulator and NhaR as a negative regulator of ectoine biosynthesis and also showed that similar to enteric species, LeuO is an anti-silencer of H-NS. In addition, defects in growth in high salinity among all the mutants suggest that these regulators play a broader role in the osmotic stress response beyond ectoine biosynthesis regulation.

microbiology↗