Single Molecule Microbiology

A paper co-authored with Crits-Cristoph and colleagues on environmental sampling at the epicenter of the COVID-19 pandemic has been published: Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic. We describe how metatranscriptomics data can be used to identify animals and animal viruses in Huanan Seafood Wholesale Market. Zach helped analyze statistical correlations in the data and commented on the manuscript for New Scientist:

Ruilan and Zach presented posters at the Single-Molecule Localization Microscopy Symposium in Lisbon. Ruilan presented her work building on our recently updated preprint on studying an M. tuberculosis protein-protein interaction in the context of E. coli cells in order to observe the two proteins without other Mtb cell division proteins. Zach presented a summary of our progress towards efficiently counting single mRNAs as they are transcribed and quantifying transcription elongation rates for single mRNAs. This included Margarida Perreira’s work in her undergraduate research project in the lab testing whether newly reported fluorescent proteins mStayGold and mBaoJin improved upon mNeonGreen for mRNA imaging.

We’ve updated the research page to include updates on new preprints frkom our lab and in collaboration with other labs. Congrats to João and Ruilan for their work on the new preprint: Mycobacterium tuberculosis FtsB and PerM interact via a C-terminal helix in FtsB to modulate cell division. Congrats to lab alumnus Sara Costa as well for her paper from Mariana Pinho’s lab published last month: The role of GpsB in Staphylococcus aureus cell morphogenesis

Work led by Liv Zimmermann and Petr Chlanda was recently published in Nature Communications: SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle. The work used cryo-electron tomography to find that cells expressing only SARS2 non-structural proteins nsp3 and nsp4 produced hexameric pores decorating double-membrane vesicles (DMV). Previously, expression of IBV nsp4 alone was found to induce membrane pairing and nsp6 was considered to be a likely possible constituent of DMV pores in MHV- and SARS2-infected cells.

Congratulations to Sara Costa, who defended her thesis and was awarded her PhD today. In Sara’s thesis, New approaches to study Staphlococcus aureus elongation and division, she discusses work implementing fluorescence microscopy techniques for studying protein-protein interaction in S. aureus and reports identifying GpsB as a non-essential protein that impacts cell elongation in S. aureus – this work is preprinted at biorxiv and was overseen by Sara’s co-supervisor, Mariana Pinho.

A new preprint on environmental sampling has been published. Crits-Cristoph and colleagues describe how metatranscriptomics data can be used to identify animals and animal viruses in Huanan Seafood Wholesale Market at the beginning of the COVID-19 pandemic. Zach helped analyze statistical correlations in the data, demonstrating Carr et al’s claim that it can be difficult, if not impossible, to infer direct association of abudandance from statistical association. The supplemental figure here shows that combining samples from two different days, with two different sampling strategies, can transform a lack of correlation into an apparent, unrealistically strong correlation. Co-corresponding author Florence Débarre noted that this is a demonstration of Simpson’s Paradox.

We aspire to keep this updated from this point forward and are looking ahead to sharing our ongoing work soon.

It’s that time of year, so the Single Molecule Microbiology lab (Zach Hensel, Sara Costa and Cleusia Manuel) got together and designed and 3d-printed holiday lights based on the x-ray structure of the GFP variant mTurquoise2.

Congrats and good luck to Teresa Carvalho and Ricardo Quiteres who submitted their Masters theses in the last two months and will be defending in the next month! Teresa studied how bacterial effectors influence actin filamentation with a combination of in vitro and in vivo approaches (pictured left) and Ricardo studied intermediate filament defects associated with a neurodegenerative disorder by superresolution microscopy (pictured right).

João Silva’s Masters research culimated in a paper published in mSphere today: Plasmids for Independently Tunable, Low-Noise Expression of Two Genes. The paper reports the development and characterization of two compatible plasmids that we co-transformed into E. coli to independently induce GFP (GFPmut2) and RFP (mScarlet-I) expression. Together with Soraia Lopes and Diogo Grilo in the lab, we also showed how the new IPTG-inducible plasmid that João created can be used for imaging single mRNA molecules.

After almost a year and a half, Soraia’s time in our lab came to an end this week. She was instrumental in getting the lab off the ground and contributed to many on-going projects in RNA detection. She’s off to work in regenerative medicine approaches to ALS treatment!

Congratulations to João Silva and everyone else in the lab who contributed to the first manuscript arising from his masters work. The manuscript, “Plasmids for independently tunable, low-noise expression of two genes,” is now available on bioRxiv. We characterized new plasmids that can be combined to tune the expression of two different genes in E. coli, and we discuss how these plasmids can be used to improve a variety of microbiology experiments. We also showed some data for single-molecule mRNA detection using the bacteriophage PP7 coat protein. The figure below shows how we can use this system to change the expression levels of GFP and mScarlet-I independently.

New plasmids from the lab are now available at AddGene. First, a collection of plasmids for RNA imaging in E. coli using the coat protein from the bacteriophage PP7 fused to different fluorescent proteins. These are based on work from a few papers from Robert Singer’s lab, and make it possible to observe single RNAs containing tandem repeats of a specific sequence with either a cyan or red fluorescent protein. Second, plasmids from João Silva’s Masters project are available for low-noise gene expression. See more information on those in this Twitter thread:

Congrats to João Silva submitted his thesis today on “Engineering low-noise gene expression systems for single-molecule experiments”. João developed new gene-expression plasmids that are compatible with ones that we previously reported. He showed that LacI/IPTG can work just as well as TetR/tetracycline, and that the systems can be moved to a compatible plasmid backbone with lower copy number. The plasmids are on their way to AddGene. The image shows how expression can be tuned to have low noise with only a few hundred molecules per cell in order to see spots that are labeled with a couple dozen molecules above background.

In the first selected talk for the group, Zach presented recent work by the Single Molecule Microbiology lab on new techniques for single mRNA imaging at the 84th Harden Conference on Single Molecule Bacteriology at Oxford University. His talk was titled “Improved tools for single-molecule mRNA and protein detection in living E. coli.”

Welcome to Sara Costa who recently joined the lab and presented her project today for the MolBioS PhD Program! Sara is co-supervised by Mariana Pinho. Her project is “Understanding the role of gene expression regulation in controlling cell division in Staphylococcus aureus.”

Thanks to the Science Sketcher Cirenia Baldrich for illustrating a lecture on fluorescence microscopy techniques for the Gene Expression Mechanisms course!

This is a simple lab website that Zach put together using GitHub Pages with a lightly-customized Cayman theme. The repo is public if you want to clone it for your own lab. Photo credit for the banner goes to Zach and the students at the 2016 Okinawa Children’s School of Science who helped frame the shot.