# Molecular Genetics of Rhizobium Nodulation Plasmids

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $406,623

## Abstract

Project Summary/Abstract
For more than three decades, research supported by this grant has had a major impact on our understanding
of how Sinorhizobium meliloti invades nodules and establishes the chronic intracellular infection that underlies
the symbiosis with its legume host. Our work has also identified common bacterial functions that are important
for both symbiotic and pathogenic bacteria to interact with their respective eukaryotic hosts. In addition, it has
also led to unanticipated fundamental discoveries, including the missing enzyme in vitamin B12 biosynthesis,
and YbeY, a previously unrecognized, extremely highly conserved endoribonuclease that plays key roles in
bacterial RNA metabolism and has human and plant homologs. The proposed research builds on our past
progress to address critical issues, including how plant-encoded defensin-like NCR (Nodule Cysteine Rich)
peptides modulate S. meliloti's cell cycle and physiology during symbiosis, and how the RNase YbeY exerts its
multiple biological roles. NCR peptides play key roles in the striking process in which the bacteria undergo
rounds of endoreduplication and terminally differentiate into bacteroids. We will continue to evaluate the
symbiotic and antimicrobial activities of chemically synthesized NCR peptides and their variants and also
representative antimicrobial peptides; continue to develop and exploit a novel strategy to observe the
physiological consequences of expressing NCR peptides, antimicrobial peptides, or variants in different
subcellular locations; and continue to use crosslinking to identify direct targets of NCR peptides. Our prior
research has identified cellular functions that enable S. meliloti to respond appropriately to NCR peptides while
resisting their antimicrobial action. We will build on our prior discovery and analysis of the symbiotically critical
BacA protein and numerous other functions identified in our recent Tn-seq study. We will analyze structure-
function relationships of the symbiotically important S. meliloti BacA protein; establish the membrane topology
of BacA and collaborate to obtain a crystal structure; and characterize other bacterial proteins that affect the
antimicrobial activity of NCR247 and assess their possible importance for the S. meliloti-legume symbiosis. We
initially identified YbeY because of its critical role in symbiosis and have subsequently shown that it is a single-
strand endoribonuclease that plays key roles in 16S rRNA 3'-processing, 70S ribosome quality control, and
small RNA regulation. We will continue our investigations of the mechanism of action and physiological
importance of YbeY. We will complete our in vitro study of the role of YbeY in the maturation of the 3' terminus
of 16S rRNA; determine the nature of the ribosomal defects in a ΔybeY strain; identify global RNA processing
targets of YbeY; and test whether any known inhibitors of Mycobacterium tuberculosis act by targeting YbeY.

## Key facts

- **NIH application ID:** 9959416
- **Project number:** 5R01GM031030-38
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** GRAHAM C WALKER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $406,623
- **Award type:** 5
- **Project period:** 1982-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9959416

## Citation

> US National Institutes of Health, RePORTER application 9959416, Molecular Genetics of Rhizobium Nodulation Plasmids (5R01GM031030-38). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9959416. Licensed CC0.

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