Poole, P. S. | Search bioRxiv

bioRxiv2021

Metabolic constraints on nitrogen fixation by rhizobia in legume nodules

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which use plant-derived dicarboxylates as energy and electron sources for reduction of atmospheric N2 into ammonia for secretion to plants. Using heterogeneous genome-scale datasets, we reconstructed a model of bacteroid metabolism to investigate the effects of varying dicarboxylate and oxygen supply on carbon and nitrogen allocation. Modelling and 13C metabolic flux analysis in bacteroids indicate that microaerobiosis restricts the decarboxylating arm of the TCA cycle and limits ammonia assimilation into glutamate. Catabolism of dicarboxylates induces a higher oxygen demand but also a higher NADH/NAD+ ratio compared to sugars. Carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules with alanine secretion increasing as oxygen tension decreases. Our results provide a framework for understanding fundamental constraints on rhizobial metabolism during symbiotic nitrogen fixation.

microbiology↗

bioRxiv2020

A multi-sensor system provides spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis

Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include fixNOQP, encoding the high-affinity cbb3-type terminal oxidase used in symbiosis. In vitro, the Rlv3841 hFixL-FxkR-FixK cascade was active at 1% O2, and confocal microscopy showed the cascade is active in the earliest stages of Rlv3841 differentiation in nodules (zones I-II). In vitro and in vivo work showed that the hFixL-FxkR-FixK cascade also induces transcription of fnrN at 1% O2 and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in fixNOQP expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III-IV of pea nodules. Quantification of fixNOQP expression in nodules showed this was driven primarily by FnrN, with minimal direct hFixL-FxkR-FixK induction. Thus, FnrN is key for full symbiotic expression of fixNOQP. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK system effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In Zone III of mature nodules, the near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required to achieve wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O2 sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O2 concentration. Multi-sensor O2 regulation systems are prevalent in rhizobia, suggesting the fine-tuned control they enable is common and maximizes the effectiveness of the symbioses. Author SummaryRhizobia are soil bacteria that form a symbiosis with legume plants. In exchange for shelter from the plant, rhizobia provide nitrogen fertilizer, produced by nitrogen fixation. Fixation is catalysed by the nitrogenase enzyme, which is inactivated by oxygen. To prevent this, plants house rhizobia in root nodules, which create a low oxygen environment. However, rhizobia need oxygen, and must adapt to survive low oxygen in the nodule. Key to this is regulating their genes based on oxygen concentration. We studied one Rhizobium species which uses three different protein sensors of oxygen, each turning on at a different oxygen concentration. As the bacteria get deeper inside the plant nodule and the oxygen concentration drops, each sensor switches on in turn. Our results also show that the first sensor to turn on, hFixL, primes the second sensor, FnrN. This prepares the rhizobia for the core region of the nodule where oxygen concentration is lowest and most nitrogen fixation takes place. If both sensors are removed, the bacteria cannot fix nitrogen. Many rhizobia have several oxygen sensing proteins, so using multiple sensors is likely a common strategy that makes it possible for rhizobia to adapt to low oxygen gradually in stages during symbiosis.

microbiology↗

bioRxiv2020

Robust gene coexpression networks using signed distance correlation

MotivationEven within well studied organisms, many genes lack useful functional annotations. One way to generate such functional information is to infer biological relationships between genes/proteins, using a network of gene coexpression data that includes functional annotations. However, the lack of trustworthy functional annotations can impede the validation of such networks. Hence, there is a need for a principled method to construct gene coexpression networks that capture biological information and are structurally stable even in the absence of functional information. ResultsWe introduce the concept of signed distance correlation as a measure of dependency between two variables, and apply it to generate gene coexpression networks. Distance correlation offers a more intuitive approach to network construction than commonly used methods such as Pearson correlation. We propose a framework to generate self-consistent networks using signed distance correlation purely from gene expression data, with no additional information. We analyse data from three different organisms to illustrate how networks generated with our method are more stable and capture more biological information compared to networks obtained from Pearson or Spearman correlations. Code availabilityhttps://github.com/javier-pardodiaz/sdcorGCN.

bioinformatics↗