Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Rivier, Alex J.; Myers, Kevin S.; Garcia, Amanda K.; Sobol, Morgan S.; Kaçar, Betül

doi:10.1128/spectrum.02815-23

Title: Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Journal Article · 12 September 2023 · Microbiology Spectrum

DOI: https://doi.org/10.1128/spectrum.02815-23 · OSTI ID:2202765

Rivier, Alex J. ^[1]; Myers, Kevin S. ^[2];

^[2];

^[2]

University of Wisconsin-Madison, WI (United States); University of Wisconsin-Madison Department of Bacteriology
University of Wisconsin-Madison, WI (United States)

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

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Research Organization:: Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018409

OSTI ID:: 2202765

Journal Information:: Microbiology Spectrum, Journal Name: Microbiology Spectrum Journal Issue: 5 Vol. 11; ISSN 2165-0497

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (59)

Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii Carruthers, Brooke M.; Garcia, Amanda K.; Rivier, Alex Current Protocols, Vol. 1, Issue 3 https://doi.org/10.1002/cpz1.57	journal	March 2021
UCSF ChimeraX : Structure visualization for researchers, educators, and developers Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Protein Science, Vol. 30, Issue 1 https://doi.org/10.1002/pro.3943	journal	October 2020
ggplot2 Wickham, Hadley Springer New York, NY https://doi.org/10.1007/978-0-387-98141-3	book	January 2009
Expression of the N2 fixation gene operon of Paenibacillus sp. WLY78 under the control of the T7 promoter in Escherichia coli BL21 Zhang, Lihong; Liu, Xiaomeng; Li, Xinxin Biotechnology Letters, Vol. 37, Issue 10 https://doi.org/10.1007/s10529-015-1874-5	journal	June 2015
Reactive nitrogen in the environment and its effect on climate change Erisman, Jan Willem; Galloway, Jim; Seitzinger, Sybil Current Opinion in Environmental Sustainability, Vol. 3, Issue 5 https://doi.org/10.1016/j.cosust.2011.08.012	journal	October 2011
Enigmatic evolution of microbial nitrogen fixation: insights from Earth’s past Rucker, Holly R.; Kaçar, Betül Trends in Microbiology https://doi.org/10.1016/j.tim.2023.03.011	journal	April 2023
The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii : Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis Ledbetter, Rhesa N.; Garcia Costas, Amaya M.; Lubner, Carolyn E. Biochemistry, Vol. 56, Issue 32 https://doi.org/10.1021/acs.biochem.7b00389	journal	August 2017
Structural Enzymology of Nitrogenase Enzymes Einsle, Oliver; Rees, Douglas C. Chemical Reviews, Vol. 120, Issue 12 https://doi.org/10.1021/acs.chemrev.0c00067	journal	June 2020
Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase Stripp, Sven T.; Duffus, Benjamin R.; Fourmond, Vincent Chemical Reviews, Vol. 122, Issue 14 https://doi.org/10.1021/acs.chemrev.1c00914	journal	July 2022
Biosynthesis of Nitrogenase Cofactors Burén, Stefan; Jiménez-Vicente, Emilio; Echavarri-Erasun, Carlos Chemical Reviews, Vol. 120, Issue 12 https://doi.org/10.1021/acs.chemrev.9b00489	journal	January 2020
Reduction of Substrates by Nitrogenases Seefeldt, Lance C.; Yang, Zhi-Yong; Lukoyanov, Dmitriy A. Chemical Reviews, Vol. 120, Issue 12 https://doi.org/10.1021/acs.chemrev.9b00556	journal	March 2020
Survival During Long-Term Starvation: Global Proteomics Analysis of Geobacter sulfurreducens under Prolonged Electron-Acceptor Limitation Bansal, Reema; Helmus, Ruth A.; Stanley, Bruce A. Journal of Proteome Research, Vol. 12, Issue 10 https://doi.org/10.1021/pr400266m	journal	September 2013
Functional optimization of gene clusters by combinatorial design and assembly Smanski, Michael J.; Bhatia, Swapnil; Zhao, Dehua Nature Biotechnology, Vol. 32, Issue 12, p. 1241-1249 https://doi.org/10.1038/nbt.3063	journal	November 2014
MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets Steinegger, Martin; Söding, Johannes Nature Biotechnology, Vol. 35, Issue 11 https://doi.org/10.1038/nbt.3988	journal	October 2017
Genetic regulation of biological nitrogen fixation Dixon, Ray; Kahn, Daniel Nature Reviews Microbiology, Vol. 2, Issue 8 https://doi.org/10.1038/nrmicro954	journal	August 2004
Type IV pili: dynamics, biophysics and functional consequences Craig, Lisa; Forest, Katrina T.; Maier, Berenike Nature Reviews Microbiology, Vol. 17, Issue 7 https://doi.org/10.1038/s41579-019-0195-4	journal	April 2019
Improved protein structure prediction using potentials from deep learning Senior, Andrew W.; Evans, Richard; Jumper, John Nature, Vol. 577, Issue 7792 https://doi.org/10.1038/s41586-019-1923-7	journal	January 2020
ColabFold: making protein folding accessible to all Mirdita, Milot; Schütze, Konstantin; Moriwaki, Yoshitaka Nature Methods, Vol. 19, Issue 6 https://doi.org/10.1038/s41592-022-01488-1	journal	May 2022
Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion Wu, Chao; Herold, Ryan A.; Knoshaug, Eric P. Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-49717-6	journal	September 2019
Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants Jiang, Xi; Payá-Tormo, Lucía; Coroian, Diana Communications Biology, Vol. 4, Issue 1 https://doi.org/10.1038/s42003-020-01536-6	journal	January 2021
Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening Barahona, Emma; Jiménez-Vicente, Emilio; Rubio, Luis M. Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep38291	journal	December 2016
Exploring the alternatives of biological nitrogen fixation Mus, Florence; Alleman, Alexander B.; Pence, Natasha Metallomics, Vol. 10, Issue 4 https://doi.org/10.1039/c8mt00038g	journal	January 2018
Keeping the nitrogen-fixation dream alive Vicente, Emilio Jimenez; Dean, Dennis R. Proceedings of the National Academy of Sciences, Vol. 114, Issue 12 https://doi.org/10.1073/pnas.1701560114	journal	March 2017
Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in Azotobacter vinelandii Martin del Campo, Julia S.; Rigsbee, Jack; Bueno Batista, Marcelo Critical Reviews in Biochemistry and Molecular Biology, Vol. 57, Issue 5-6 https://doi.org/10.1080/10409238.2023.2181309	journal	November 2022
clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters Yu, Guangchuang; Wang, Li-Gen; Han, Yanyan OMICS: A Journal of Integrative Biology, Vol. 16, Issue 5 https://doi.org/10.1089/omi.2011.0118	journal	May 2012
Fast and accurate short read alignment with Burrows-Wheeler transform Li, H.; Durbin, R. Bioinformatics, Vol. 25, Issue 14 https://doi.org/10.1093/bioinformatics/btp324	journal	May 2009
The Sequence Alignment/Map format and SAMtools Li, H.; Handsaker, B.; Wysoker, A. Bioinformatics, Vol. 25, Issue 16 https://doi.org/10.1093/bioinformatics/btp352	journal	June 2009
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data Robinson, M. D.; McCarthy, D. J.; Smyth, G. K. Bioinformatics, Vol. 26, Issue 1 https://doi.org/10.1093/bioinformatics/btp616	journal	November 2009
Pathview: an R/Bioconductor package for pathway-based data integration and visualization Luo, Weijun; Brouwer, Cory Bioinformatics, Vol. 29, Issue 14 https://doi.org/10.1093/bioinformatics/btt285	journal	June 2013
RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies Stamatakis, Alexandros Bioinformatics, Vol. 30, Issue 9 https://doi.org/10.1093/bioinformatics/btu033	journal	January 2014
Trimmomatic: a flexible trimmer for Illumina sequence data Bolger, Anthony M.; Lohse, Marc; Usadel, Bjoern Bioinformatics, Vol. 30, Issue 15 https://doi.org/10.1093/bioinformatics/btu170	journal	April 2014
HTSeq--a Python framework to work with high-throughput sequencing data Anders, S.; Pyl, P. T.; Huber, W. Bioinformatics, Vol. 31, Issue 2 https://doi.org/10.1093/bioinformatics/btu638	journal	September 2014
Operon-mapper: a web server for precise operon identification in bacterial and archaeal genomes Taboada, Blanca; Estrada, Karel; Ciria, Ricardo Bioinformatics, Vol. 34, Issue 23 https://doi.org/10.1093/bioinformatics/bty496	journal	June 2018
Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins Garcia, Amanda K.; Kolaczkowski, Bryan; Kaçar, Betül Genome Biology and Evolution, Vol. 14, Issue 3 https://doi.org/10.1093/gbe/evac031	journal	February 2022
MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability Katoh, K.; Standley, D. M. Molecular Biology and Evolution, Vol. 30, Issue 4 https://doi.org/10.1093/molbev/mst010	journal	January 2013
KEGG: Kyoto Encyclopedia of Genes and Genomes Kanehisa, Minoru; Goto, Susumu Nucleic Acids Research, Vol. 28, Issue 1, p. 27-30 https://doi.org/10.1093/nar/28.1.27	journal	January 2000
The global nitrogen cycle in the twenty-first century Fowler, David; Coyle, Mhairi; Skiba, Ute Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 368, Issue 1621 https://doi.org/10.1098/rstb.2013.0164	journal	July 2013
Azotobacter vinelandii: the source of 100 years of discoveries and many more to come Noar, Jesse D.; Bruno-Bárcena, Jose M. Microbiology, Vol. 164, Issue 4 https://doi.org/10.1099/mic.0.000643	journal	April 2018
Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization Garcia, Amanda K.; McShea, Hanon; Kolaczkowski, Bryan Geobiology, Vol. 18, Issue 3 https://doi.org/10.1111/gbi.12381	journal	February 2020
Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing Benjamini, Yoav; Hochberg, Yosef Journal of the Royal Statistical Society: Series B (Methodological), Vol. 57, Issue 1 https://doi.org/10.1111/j.2517-6161.1995.tb02031.x	journal	January 1995
Coordinated regulation of nitrogen fixation and molybdate transport by molybdenum: Molybdenum regulation of nitrogen fixation Demtröder, Lisa; Narberhaus, Franz; Masepohl, Bernd Molecular Microbiology, Vol. 111, Issue 1 https://doi.org/10.1111/mmi.14152	journal	November 2018
Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa Wang, Congcong; Chen, Wenhui; Xia, Aiguo Applied and Environmental Microbiology, Vol. 85, Issue 22 https://doi.org/10.1128/AEM.01705-19	journal	November 2019
Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes Setubal, J. C.; dos Santos, P.; Goldman, B. S. Journal of Bacteriology, Vol. 191, Issue 14 https://doi.org/10.1128/JB.00504-09	journal	May 2009
Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii Hamilton, T. L.; Ludwig, M.; Dixon, R. Journal of Bacteriology, Vol. 193, Issue 17 https://doi.org/10.1128/JB.05099-11	journal	July 2011
Rnf and Fix Have Specific Roles during Aerobic Nitrogen Fixation in Azotobacter vinelandii Alleman, Alexander B.; Garcia Costas, Amaya; Mus, Florence Applied and Environmental Microbiology, Vol. 88, Issue 17 https://doi.org/10.1128/aem.01049-22	journal	September 2022
Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations Dingler, C.; Kuhla, J.; Wassink, H. Journal of Bacteriology, Vol. 170, Issue 5 https://doi.org/10.1128/jb.170.5.2148-2152.1988	journal	May 1988
Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. Jacobson, M. R.; Brigle, K. E.; Bennett, L. T. Journal of Bacteriology, Vol. 171, Issue 2 https://doi.org/10.1128/jb.171.2.1017-1027.1989	journal	February 1989
Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. Mouncey, N. J.; Mitchenall, L. A.; Pau, R. N. Journal of Bacteriology, Vol. 177, Issue 18 https://doi.org/10.1128/jb.177.18.5294-5302.1995	journal	September 1995
Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii. Premakumar, R.; Jacobitz, S.; Ricke, S. C. Journal of Bacteriology, Vol. 178, Issue 3 https://doi.org/10.1128/jb.178.3.691-696.1996	journal	February 1996
Quantitative Control for Stoichiometric Protein Synthesis Taggart, James C.; Lalanne, Jean-Benoît; Li, Gene-Wei Annual Review of Microbiology, Vol. 75, Issue 1 https://doi.org/10.1146/annurev-micro-041921-012646	journal	October 2021
The Glyoxylate Shunt, 60 Years On Dolan, Stephen K.; Welch, Martin Annual Review of Microbiology, Vol. 72, Issue 1 https://doi.org/10.1146/annurev-micro-090817-062257	journal	September 2018
Oxygen and Hydrogen in Biological Nitrogen Fixation Robson, R. L.; Postgate, J. R. Annual Review of Microbiology, Vol. 34, Issue 1 https://doi.org/10.1146/annurev.mi.34.100180.001151	journal	October 1980
BLAST+: architecture and applications Camacho, Christiam; Coulouris, George; Avagyan, Vahram BMC Bioinformatics, Vol. 10, Issue 1 https://doi.org/10.1186/1471-2105-10-421	journal	January 2009
Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves Sprouffske, Kathleen; Wagner, Andreas BMC Bioinformatics, Vol. 17, Issue 1 https://doi.org/10.1186/s12859-016-1016-7	journal	April 2016
Clustering gene expression time series data using an infinite Gaussian process mixture model McDowell, Ian C.; Manandhar, Dinesh; Vockley, Christopher M. PLOS Computational Biology, Vol. 14, Issue 1 https://doi.org/10.1371/journal.pcbi.1005896	journal	January 2018
Energy Starved Candidatus Pelagibacter Ubique Substitutes Light-Mediated ATP Production for Endogenous Carbon Respiration Steindler, Laura; Schwalbach, Michael S.; Smith, Daniel P. PLoS ONE, Vol. 6, Issue 5 https://doi.org/10.1371/journal.pone.0019725	journal	May 2011
Genetic and Biochemical Analysis of the Azotobacter vinelandii Molybdenum Storage Protein Navarro-Rodríguez, Mónica; Buesa, José María; Rubio, Luis M. Frontiers in Microbiology, Vol. 10 https://doi.org/10.3389/fmicb.2019.00579	journal	March 2019
Engineering Nitrogenases for Synthetic Nitrogen Fixation: From Pathway Engineering to Directed Evolution Bennett, Emily M.; Murray, James W.; Isalan, Mark BioDesign Research, Vol. 5 https://doi.org/10.34133/bdr.0005	journal	January 2023
Nitrogenase resurrection and the evolution of a singular enzymatic mechanism Garcia, Amanda K.; Harris, Derek F.; Rivier, Alex J. eLife, Vol. 12 https://doi.org/10.7554/eLife.85003	journal	February 2023

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59 BASIC BIOLOGICAL SCIENCES
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Title: Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

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