Identification and characterization of the TetR family transcriptional regulator NffT in
Rhizobium johnstonii
Authors: Li Xiaofang , Li Zhangqun , Wei Yajuan , Chen Zirui , Xie Shijie 
Published: March 2024

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF,
nifA-fixA
signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased
fixA
expression, was discovered through a
fixA
-promoter-β-glucuronidase fusion assay performed with
Rhizobium johnstonii
. Real-time quantitative PCR analysis showed that
nffT
deletion increased the expression of symbiotic genes including
nifA
and
fixA
in
nifA-fixA
signaling, and
fixL
,
fixK, fnrN,
and

fixN
9

in
fixL-fixN
signaling.
nffT
overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091–93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located −32 to −99 bp upstream of RL0091–93 and NffT deletion significantly increased the expression of RL0091–93.
nffT-
promoter-β-glucuronidase fusion assay indicated that
nffT
expression was regulated by the
cobNTS
genes and cobalamin. Mutations in
cobNTS
significantly decreased the expression of
nffT
, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of
R. johnstonii-Pisum sativum
.

IMPORTANCE

Symbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of
fixA
in
Rhizobium johnstonii
. Furthermore, NffT was confirmed to play pleiotropic roles in
R. johnstonii-Pisum sativum
symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected
R. johnstonii-P. sativum
symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.