# Structure analysis of F pilus biogenesis

> **NIH NIH R21** · UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON · 2020 · $192,916

## Abstract

The transmission of DNA polymers across biological membranes is a fundamental process in all cells. Early
studies in bacteria demonstrated the remarkable capacity of a mobile genetic element (MGE), the conjugative
F plasmid, to integrate into the genome and promote single-stranded transfer of the entire chromosome to
recipient bacteria in uninterrupted matings. In the ensuing ~75 years, studies established the broad importance
of MGEs in shaping of bacterial genomes over evolutionary time as well as their capacity to rapidly
disseminate antibiotic resistance and other fitness traits under selective pressure. F plasmids are particularly
problematic from a medical perspective because they are distributed among pathogenic members of the
Enterobacteriaceae and they often harbor resistance to multiple antibiotics including last-resort β-lactam
antibiotics, e.g., carbapenems. F plasmids also have the capacity to transfer at very high frequencies and
elaborate conjugative pili in large numbers around the cell surface; these attachment organelles play important
roles in establishment of robust biofilms, which are typically refractory to antibiotic treatment. Bacterial MGEs,
including the F plasmids, are transmitted intercellularly through nanomachines termed type IV secretion
systems (T4SSs). F-encoded and phylogenetically-related conjugation systems have served as important
models for deciphering the mechanism of action of T4SSs, but there remains a fundamental lack of structural
detail for these machines. We have solved, for the first time, structures of F pilus-associated T4SS
nanomachines in the native environment of the bacterial cell envelope by cryoelectron tomography
(cryoET). Strikingly, we discovered that the F system elaborates four distinct F-substructures, not just one or
two as previous models have predicted. These include a presumptive F channel complex and three distinct
substructures associated with F pili. Our overarching hypothesis is that the F channel complex transitions to an
active translocation channel or a pilus assembly factory (PAF) in response to distinct signals; the PAF in turn
has the potential to produce many pili for deposition on other F-encoded platforms. This exploratory study will
test this new model by solving WT and mutant T4SSs at the highest resolutions achievable using in situ
cryoET. In Aim 1, we will generate 3D maps of WT and mutant substructures at resolutions sufficient to define
the structural contributions of the F pilus and TraA pilin, as well as the TraC and TraD T4SS ATPases, to the
visualized substructures. Aim 2 initiates a long-term goal of defining compositions and assembly pathways for
the F-encoded substructures through analyses of tra gene deletion mutations and detection of novel densities
contributed by traceable tags fused to Tra proteins for spatial assignments. Our studies will generate important
new insights into the composition, architecture, and biogenesis of F T4SSs, and answer long-stan...

## Key facts

- **NIH application ID:** 9828070
- **Project number:** 5R21AI142378-02
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
- **Principal Investigator:** Bo Hu
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $192,916
- **Award type:** 5
- **Project period:** 2018-11-21 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9828070

## Citation

> US National Institutes of Health, RePORTER application 9828070, Structure analysis of F pilus biogenesis (5R21AI142378-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9828070. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*