Related Experiment Video
Updated: Jun 9, 2026
Microtiter Dish Biofilm Formation Assay
Published on: January 30, 2011
Analysis of HmsH and its role in plague biofilm formation
Arwa Abu Khweek1, Jacqueline D Fetherston1, Robert D Perry1
1Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, 800 Rose St., Lexington, KY, USA.
Abstract:
The Yersinia pestis Hms(+) phenotype is a manifestation of biofilm formation that causes adsorption of Congo red and haemin at 26 degrees C but not at 37 degrees C. This phenotype is required for blockage of the proventricular valve of the oriental rat flea and plays a role in transmission of bubonic plague from fleas to mammals. Genes responsible for this phenotype are located in three separate operons, hmsHFRS, hmsT and hmsP. HmsH and HmsF are outer membrane (OM) proteins, while the other four Hms proteins are located in the inner membrane. According to the Hidden Markov Method-based predictor, HmsH has a large N terminus in the periplasm, a beta-barrel structure with 16 beta-strands that traverse the OM, eight surface-exposed loops, and seven short turns connecting the beta-strands on the periplasmic side. Here, we demonstrate that HmsH is a heat-modifiable protein, a characteristic of other beta-barrel proteins, thereby supporting the bioinformatics analysis. Alanine scanning mutagenesis was used to identify conserved amino acids in the HmsH-like family that are critical for the function of HmsH in biofilm formation. Of 23 conserved amino acids mutated, four residues affected HmsH function and three likely caused protein instability. We used formaldehyde cross-linking to demonstrate that HmsH interacts with HmsF but not with HmsR, HmsS, HmsT or HmsP. Loss-of-function HmsH variants with single alanine substitutions retained their beta-structure and interaction with HmsF. Finally, using a polar hmsH : : mini-kan mutant, we demonstrated that biofilm development is not important for the pathogenesis of bubonic or pneumonic plague in mice.
Insights
The Yersinia pestis Hms(+) biofilm phenotype is crucial for plague transmission via fleas. However, HmsH protein
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- The Yersinia pestis Hms(+) phenotype is essential for biofilm formation, mediating Congo red and haemin adsorption at 26°C.
- This biofilm is critical for blocking the oriental rat flea's proventricular valve, facilitating bubonic plague transmission.
- The Hms(+) phenotype is governed by genes in three operons: hmsHFRS, hmsT, and hmsP.
Purpose of the Study:
- To investigate the structural and functional characteristics of the HmsH outer membrane protein in Yersinia pestis biofilm formation.
- To identify key amino acid residues within HmsH essential for its role in biofilm development.
- To elucidate the interaction of HmsH with other Hms proteins.
Main Methods:
- Bioinformatic analysis using the Hidden Markov Method to predict HmsH structure.
- Heat-modification assays to confirm HmsH's structural properties.
- Alanine scanning mutagenesis to probe critical residues in HmsH function.
- Formaldehyde cross-linking to assess HmsH protein interactions.
Main Results:
- HmsH exhibits heat-modifiable properties, consistent with its predicted beta-barrel structure.
- Mutagenesis identified four critical residues for HmsH function in biofilm formation.
- HmsH directly interacts with HmsF, but not with HmsR, HmsS, HmsT, or HmsP.
- Loss-of-function HmsH variants maintained their beta-structure and HmsF interaction.
Conclusions:
- HmsH is a heat-modifiable outer membrane protein crucial for Yersinia pestis biofilm formation.
- Specific residues in HmsH are vital for its function and interaction with HmsF.
- Despite its role in flea transmission, HmsH-mediated biofilm formation is not essential for bubonic or pneumonic plague pathogenesis in mice.
