# Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator

> **NIH NIH R01** · NORTH DAKOTA STATE UNIVERSITY · 2021 · $290,000

## Abstract

PROJECT SUMMARY/ABSTRACT
 The CDC recently released a report detailing antibiotic resistant threats in the US. Of particular
emphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR-
GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteria
rely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) to
import critical nutrients from their environment, especially metals like iron, which are bound by high-affinity,
metal chelating compounds called siderophores. Recent antibiotic developments have shown that
siderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this delivery
mechanism overcomes several key antibiotic resistance mechanisms. A significant limitation of this delivery
system is the low expression levels of the TBDTs. However, a subset of these TBDTs controls their own
expression through a cell-surface signaling (CSS) process that up-regulates their own expression. The long-
term objective of this research is to understand the CSS regulatory process and manipulate TBDT expression
to enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. Research outlined
in this proposal will help elucidate the structural basis for CSS by a sigma-regulator. As a model system, the
pseudobactin BN7/8 transport system of Psuedomonas putida, which consists of the TBDT, PupB, the inner
membrane σ-regulator, PupR, and the cytoplasmic σ-factor, PupI, is being used. To accomplish this
proposal's objective the following three specific aims will be pursued: 1) establish that PupR anti-σ-factor
domain dimerization influences transcriptional activation by PupI, 2) identify the structural determinants and
delineate the role of the PupR:PupB periplasmic interactions on the stability of the PupR periplasmic C-
terminal CSS domain (CCSSD), and 3) determine changes in the full-length PupB:PupR CCSSD complex in
the presence and absence of its cognate siderophore, pseudobactin BN7/8. These aims will be accomplished
using a multidisciplinary approach; including X-ray crystallography, small-angle X-ray scattering, molecular
biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry and circular
dichroism spectroscopy. This research will provide critical structural information about a σ-regulator; explain
how it interacts with a σ-factor at the inner membrane, and the extent to which periplasmic conformational
changes between the TBDT and σ-regulator lead to proteolytic degradation that is important for controlling
transcriptional activation.

## Key facts

- **NIH application ID:** 10240569
- **Project number:** 5R01GM126207-04
- **Recipient organization:** NORTH DAKOTA STATE UNIVERSITY
- **Principal Investigator:** Christopher L Colbert
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $290,000
- **Award type:** 5
- **Project period:** 2018-09-20 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10240569

## Citation

> US National Institutes of Health, RePORTER application 10240569, Structural basis for cell surface siganling by a Gram-negative bacteria sigma-regulator (5R01GM126207-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10240569. Licensed CC0.

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