An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in Bradyrhizobium diazoefficiens.

Noemí Fernández,Eulogio J Bedmar,Hans-Martin Fischer,Juan J Cabrera,Christian H Ahrens,Stefanie Lutz,Socorro Mesa,Adithi R Varadarajan,Gabriella Pessi,Bernd Roschitzki,Leo Eberl,Raphael Ledermann

doi:10.3389/fmicb.2019.00924

Abstract

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont Bradyrhizobium diazoefficiens USDA 110 (formerly B. japonicum USDA 110). As a first step, the complete genome of B. diazoefficiens 110spc4, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal cbb3 oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK2: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from fixK2, additional B. diazoefficiens genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis.

Highlights

Rhizobia are primary contributors to biological nitrogen fixation, a process that is highly relevant both for agronomy and, by reducing the need of chemical fertilizers, for the environment
A genome comparison uncovered several differences compared to the USDA 110 reference genome (Table 1), most notably a 202 kb deletion of a genomic region corresponding to nucleotides 1–70,634 and 8,974,768–9,105,828 of the USDA 110 reference genome sequence (Figure 1)
With the de novo assembled and annotated genome of our model strain B. diazoefficiens 110spc4 at hand, we aimed to identify the set of proteins differentially expressed in microoxia, which has been recognized as a key signal for the induction of symbiosis-related genes during different steps of bacteriahost plant interaction (Terpolilli et al, 2012; Poole et al, 2018)

Summary

Introduction

Rhizobia are primary contributors to biological nitrogen fixation, a process that is highly relevant both for agronomy and, by reducing the need of chemical fertilizers, for the environment. Rhizobia comprise a large group of both alpha- and betaproteobacteria (reviewed in Sprent et al, 2017) which can establish symbiotic relationships with legumes They encode the enzyme nitrogenase, which catalyzes the conversion of atmospheric nitrogen gas to ammonium inside plant root (and occasionally stem) nodules (reviewed in Dixon and Kahn, 2004; Terpolilli et al, 2012). During the establishment of a rhizobialegume symbiosis, bacteria need to adapt their physiology from the free-living state in soil to the highly specialized environment of a plant cell, the so called bacteroid state This multi-step process is tightly controlled at different stages of the symbiotic interaction and includes various signals released and sensed by both the bacteria and the host plant. Microoxia represents a critical signal both for the expression and activity of nitrogenase and the cbb3-type highaffinity terminal oxidase required for bacterial respiration inside nodules (reviewed in Fischer, 1994, 1996; Dixon and Kahn, 2004; Terpolilli et al, 2012; Poole et al, 2018)

Methods

Results

Conclusion