Natural product discovery in actinomycetes and investigation into the regulation of SPI-1 in salmonella typhimurium

Linaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated, alkene containing amino acid derived from threonine. This work describes the development of a linaridin-specific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome mining tool tailored toward RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 nonredundant) linaridin biosynthetic gene clusters. Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in data set validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening and isolated from Streptomyces noursei and Streptomyces auratus, respectively. Reactivity-based screening involves the use of a probe that chemoselectively modifies an organic functional group present in the natural product. The dehydrated amino acids present in linaridins as α/β-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4-addition using a thiol-functionalized probe. The data presented within significantly expand the number of predicted linaridin biosynthetic gene clusters and serve as a roadmap for future work in the area. The combination of bioinformatics and reactivity-based screening is a powerful approach to accelerate natural product discovery. Salmonella enterica serovar Typhimurium relies on a type three secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI-1) to invade epithelial cells in the small intestine. This invasion leads to inflammatory diarrhea in the host, a typical symptom of Salmonella infection. Induction of expression of SPI-1 is tightly regulated by the master regulator hilA. In turn, hilA expression is controlled by three AraC-like transcription factors HilC, HilD, and RtsA. The three transcription factors HilC, HilD, and RtsA form a complex feed-forward regulatory loop. These proteins contain a C-terminal DNA-binding domain and an N-terminal dimerization domain. These transcription factors can form homo- or heterodimers which bind the same portion of DNA resulting in induction of their own, and each other’s, expression. This positive regulation results in robust expression of SPI-1. Despite the apparent redundancy of these three proteins most external regulatory inputs act on HilD. HilD is tightly regulated at low levels of expression by the repressor HilE. HilE specifically represses HilD but not HilC or RtsA, despite a high level of homology between all three proteins, preventing expression of hilA. HilE is understood to repress HilD through a protein-protein interaction which prevents HilD from binding DNA but leaves dimerization intact. To better understand the interaction between HilE and HilD our work attempts to determine the specific residues on HilD which HilE recognizes and binds. Through the creation of HilD/HilC hybrid proteins we show that the sequence of the DNA-binding domain is not important in HilE repression. Instead, these studies suggest that a 137 amino acid region within the dimerization domain contains the residues required for repression. Through targeted mutations and alanine substitutions within this region of HilD we have further narrowed the potentially important residues to 49 amino acids. Finally, our work has also investigated the structure of HilE and what amino acid residues within its sequence may be important for HilD recognition and binding. Computational structural predictions indicate that HilE may function as a hexamer. This aligns with the way its closest evolutionary homolog, Hcp (T6SS sheath protein), functions. The structural prediction indicated that the 28 N-terminal amino acids of HilE may be expendable, which was confirmed through deletion studies. This indicates that this 28 amino acid region does not affect the multimeric structure of HilE, nor does it affect repression of HilE.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Georgiou, Matthew Alex

Contributors dc:contributor

• Slauch, James M
• Imlay, James A
• Cronan, John E
• Hatoum-Aslan, Asma

• Streptomyces
• Linaridin
• Ripps
• Reactivity-based Screening
• Salmonella
• Hild
• Hile
• Regulation

Statement dc:rights

• Copywrite 2024 Matthew Georgiou

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/125763

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F125763