Biology subjects

Getz, L.

Publications and source records attributed to Getz, L..

3 recordsLinked to original sources

bioRxiv2024

Functional genomics of chitin degradation by Vibrio parahaemolyticus reveals finely integrated metabolic contributions to support environmental fitness

Vibrio species are marine prokaryotes that inhabit diverse ecological niches, colonizing abiotic and biotic surfaces. These bacteria are vital players in the global carbon cycle, assimilating billions of tonnes of chitin for carbon (and nitrogen) metabolites. Many bacterial proteins involved in the process-including chitinases, sugar transporters, and modifying enzymes-have been well studied. However, the genetic functional interplay and key drivers of Vibrio competitive survival in the presence of chitin as the dominant carbon source is not understood. To address this question, we carried out transposon sequencing (Tn-seq) to determine the genetic fitness of Vibrio parahaemolyticus mutants grown on chitin as a sole carbon source. Along with validating known Vibrio genes associated with chitin metabolism, our data newly identified vital roles for an unclassified OprD-like import porin and a HexR family transcriptional regulator. Furthermore, we functionally implicated HexR in regulating multiple physiological processes involved in V. parahaemolyticus environmental survival including carbon assimilation and cell growth, biofilm formation, and cell motility. Notably, our data revealed a strict requirement for HexR in filamentous cell morphology, a critical trait for V. parahaemolyticus environmental fitness. Therefore, a vital import porin and genomic regulation mediated by HexR supports multiple physiological processes for Vibrio chitinolytic growth and environmental fitness.

microbiology↗

bioRxiv2018

Discovery and Characterization of Novel Lignocellulose-Degrading Enzymes from the Porcupine Microbiome

Plant cell walls are comprised of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade these components to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with novel lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four genes from the porcupine microbiome encoding putative novel lignocellulose-degrading enzymes, including a {beta}-xylanase, endoxylanase, {beta}-glucosidase, and an -L-arabinofuranosidase. These genes were identified via conserved catalytic domains associated with cellulose and hemicellulose degradation. We cloned the putative {beta}-xylanase into the pET26b(+) plasmid, enabling inducible gene expression in Escherichia coli (E. coli) and periplasmic localization. We demonstrated IPTG-inducible accumulation of {beta}-xylanase protein but failed to detect xylobiose degrading activity in a reporter assay. Alternative assays may be required to measure activity of this putative {beta}-xylanase. In this report, we describe how a synthetic metagenomic pipeline can be used to identify novel microbial lignocellulose-degrading enzymes and take initial steps to introduce a hemicellulose-degradation pathway into E. coli to enable biofuel production from wood pulp feedstock.

synthetic biology↗

bioRxiv2018

Tandem tyrosine residues in the EPEC multicargo chaperone CesT support differential type III effector translocation and early host colonization

Enteropathogenic Escherichia coli (EPEC) are worldwide human enteric pathogens inflicting significant morbidity and causing large economic losses. A type 3 secretion system (T3SS) is critical for EPEC intestinal colonization, and injection of effectors into host cells contributes to cellular subversion and innate immune evasion. Here, we demonstrate that two strictly conserved C-terminal tyrosine residues, Y152 and Y153, within the multicargo T3SS chaperone CesT serve differential roles in regulating effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine attenuated EPEC type 3 effector injection into host cells, and significantly limited Tir effector mediated intimate adherence, a key feature of attaching and effacing pathogenesis. Whereas CesT Y153 supported normal levels of Tir translocation, CesT Y152 was strictly required for the effector NleA to be expressed and subsequently translocated into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y152, Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating Y152 in CesT functionality. A mouse infection model of EPEC intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for strictly conserved tandem tyrosine residues within CesT. Tyrosine 152 of CesT is implicated in NleA expression, providing functional relevance for localized amino acid conservation. Therefore, CesT via its novel C-terminal domain, has relevant roles beyond typical T3SC that interact and stabilize effector proteins.

microbiology↗