Phenotypic, genetic, and regulatory analyses of carbohydrate metabolism systems in Vibrio parahaemolyticus
Date
2019
Authors
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Publisher
University of Delaware
Abstract
Utilizing unique carbohydrates or utilizing them more efficiently help bacteria expand and colonize new niches. Horizontal gene transfer (HGT) of catabolic systems is a powerful mechanism by which bacteria can acquire new metabolic traits that can increase survival and fitness in different niches. In this dissertation work, we investigated horizontal acquisition of catabolism systems and two major global regulators of metabolism in the survival and fitness of Vibrio parahaemolyticus, a marine species that is also an important human and fish pathogen. In humans, V. parahaemolyticus is the leading cause of bacterial seafood-related gastroenteritis worldwide. ☐ In chapter two, we examined carbon catabolism diversity within Vibrio parahaemolyticus and among members of the family Vibrionaceae in general. Bioinformatics analysis showed that the ability to utilize D-galactose was present in all V. parahaemolyticus but at least two distinct transporters were present; a major facilitator superfamily (MFS) transporter and a sodium/galactose transporter (SGLT). Growth and genetic analyses demonstrated that SGLT was a more efficient transporter of D-galactose and was the predominant type among strains. The ability to utilize D- gluconate was universal within the species, however deletion of eda (VP0065), which encodes aldolase, a key enzyme in the Entner-Doudoroff (ED) pathway, reached a similar biomass to wild type when grown on D-gluconate as a sole carbon source. Two additional eda genes were identified, VPA1708 (eda2) associated with a D- glucuronate cluster and VPA0083 (eda3) that clustered with an oligogalacturonide (OGA) metabolism cluster that were both induced by D-gluconate and both of these regions were variably present among strains. A metabolism island was identified that contained citrate fermentation, L-rhamnose and OGA metabolism gene clusters as well as a CRISPR-Cas system. Phylogenetic analysis showed that CitF (a key enzyme in the citrate fermentation pathway) and RhaA (a key enzyme in the L-rhamnose pathway) had a limited distribution among V. parahaemolyticus. Our analysis showed that the L-rhamnose pathway was acquired at least three times in this species. Our data suggest that horizontal transfer of metabolic systems among Vibrionaceae is an important source of metabolic diversity. This work describes previously uncharacterized metabolic islands that were hotspots for the gain and loss of functional modules likely mediated by transposons. ☐ In chapter 3, we investigated the role of an important global regulator of metabolism, cAMP receptor protein (CRP) in V. parahaemolyticus for the first time. In bacteria CRP is activated by cAMP, a secondary messenger synthesized from ATP by adenylate cyclase (AC). We constructed Dcrp and DcyaA mutant strains by deleting the crp and cyaA genes to inactivate CRP. The Dcrp mutant was unable to grow in minimal media supplemented with non-glucose carbon sources demonstrating a requirement for CRP. Unexpectedly, the cyaA mutant behaved similar to wild-type, which suggested the presence of an uncharacterized adenylate cyclase. Bioinformatics analysis identified CyaB (VP1760), a class IV adenylate cyclase present in all strains, which complemented an Escherichia coli DcyaA mutant. The DcyaB mutant reached a similar biomass when grown on alternative carbon sources, which suggested that CyaB and CyaA can complement each other. A DcyaB/DcyaA double mutant confirmed this, as this mutant behaved identical to the Dcrp strain in all growth pattern analyses. Capsule polysaccharide (CPS) production and biofilm formation showed a defect in the Dcrp and DcyaB mutants, whereas the DcyaA behaved like wild type. This indicated a specific requirement of CRP activation by CyaB. In an Artemia franciscana (brine shrimp) model of virulence, the Dcrp and DcyaB/DcyaA strains showed attenuated killing demonstrating that cAMP-CRP is required for virulence. Phylogenetic analysis of cyaB from V. parahaemolyticus showed that it is highly divergent from other members of the Campbellii, which contained this gene and not phylogenetically widespread. This is the first study to determine the physiological role of a class IV adenylate cyclase. ☐ In chapter four, we examined the role of FIS (factor for inversion stimulation) in V. parahaemolyticus biology for the first time. FIS has been demonstrated to be a global regulator controlling genes belonging to multiple functional categories in several species. We constructed a deletion of fis (VP2885) in V. parahaemolyticus and demonstrated a motility defect in both swimming and swarming assays suggesting FIS is an important positive regulator of these phenotypes. Bioinformatics analysis identified putative FIS binding sites in the regulatory region of both polar and lateral flagellum biosynthesis gene clusters which was confirmed by electrophoretic mobility shift assays (EMSAs). In in vitro growth competition assays, the Dfis mutant was outcompeted by wild type in minimal media supplemented with L-arabinose, D- glucosamine, or D-gluconate. The effects of FIS were direct as purified FIS protein bound to the regulatory regions of these catabolism gene clusters. The Dfis mutant was more sensitive to antimicrobial peptides such as polymyxin B. In in vivo competition assays in a streptomycin pretreated adult mouse colonization model the fis deletion mutant had a colonization defect compared to wild type. Thus, similar to other enteric pathogens, FIS plays a central role as a regulator of global gene regulation in this species.