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Most Free-Living Bacteria Likely Make All 20 Amino Acids

ENIGMA researchers at Lawrence Berkeley Lab and the University of Missouri systematically used high-throughput genetics to fill gaps in amino acid biosynthesis pathways. This explains how bacteria can grow on their own, in contrast to widespread speculation that bacteria cross-feed amino acids among community members.

According to Morgan Price, for many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio Vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

Price, M.N.; G. M. Zane, J. V. Kuehl, R. A. Melnyk, J. D. Wall, A. M. Deutschbauer, A. P. Arkin (2018) Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High‐throughput Genetics. PLOS Genetics.

Results can be viewed

Methionine synthesis in Phaeobacter inhibens by a three-part methionine synthase and two vitamin B12 reactivation proteins



Rapidly Moving New Bacteria to Model Organism Status

ENIGMA researchers describe our proposed workflow as a general guideline for studying new environmental microbes in a fast and comprehensive way.

Liu, H.; A.M. Deutschbauer (2018) Rapidly moving new bacteria to model‐organism status. Current Opinion in Biotechnology. DOI: 10.1016/j.copbio.2017.12.006

Currently, there is a need to rapidly move new bacteria to “model organism” status, given the importance of a diverse range of these microorganisms to human health, the environment, and biotechnology. Hualan Liu proposes at a minimum, a new model bacterium should have a complete and accurately annotated genome, tools for genetic manipulation, and a computational framework for data analysis. ENIGMA  now has a number of tools available to accelerate the development of a new model bacterium, aided by advances in next-generation sequencing, functional genomics, and shared computational platforms for systems biology




Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq) wins R&D100 Award

Mutalik_VivekENIGMA’s Vivek Mutalik, Adam Deutschbauer, Pavel Novichkov, and Adam Arkin win R&D100 Award for a tool developed under our “Discovery Program”; where high-risk projects are funded for a short duration to encourage high impact changes in science or technological capability that extend and enhance our ongoing research

Dub-seq technology is based on creating a genomic fragment library in association with dual barcodes on broad-host vectors.  DNA (from any source) is sheared and cloned between dual barcodes. Then, the cloned genomic fragment is identified and associated with unique DNA barcode upfront and is done only once for each library. Once this step of associating barcodes to genomic fragment is performed, only one of the barcodes is used as a proxy for clonal isolate in the following experiments. This standardization enables researchers to assay the same library across diverse conditions with minimal cost per genome-wide assay. A simple analytic approach aids in connecting fragment score and gene score to gene function.

This technology fills a critical gap in elucidating gene-function in a very high-throughput assay set up and saves time, labor, and money as compared to state-of-the-art methods. Dub-seq technology is applicable in the discovery of novel enzymes/biocatalysts for biofuel production, improving and tolerance traits for toxic biochemicals, ensemble functional assessment of microbial communities, plant growth-promoting factors, bioremediation routes, novel green chemistries and biotechnologies in improving energy and environment missions. The technology is also extendable in diverse health and agriculture associated biotechnologies.

For more information associated with the award:

2017 R&D 100 Winner


Key Cellular Components Persist in Bacteria Although Response to Salt Stress Changes

ENIGMA researchers at University of Oklahoma, Lawrence Berkeley Lab, University of Washington, and University of Missouri reveal that the accumulation and induction of key cellular components persist in bacteria for greater than 5000 generations. However, the physiological and transcriptional responses to high salinity are altered. The mechanistic changes in evolved genotypes suggested that growth energy efficiency might be a key factor for selection.  Aifen Zhou is interested in the interaction between bacteria and the environment; here, the co-existence of sulfate-reducing bacterium (SRB) and high salinity in natural habitats and heavy metal contaminated field sites laid the foundation for the study of the salt adaptation of DvH, a model SRB, with experimental evolution.

Questions addressed

  • What is the major driving force for salt adaptation in DvH over a long-term experimental evolution?
  • How did the cellular components such as metabolites and phospholipid fatty acids (PLFAs) change over long term evolution?


Major findings

Comparison between the best-adapted clones ES10-5 and ES9-11, which had evolved under salt stress for 5,000 and 1,200 generations, respectively, demonstrated the continuous improvement of salt tolerance; glutamate as a key osmolyte and i17:1ω9c as the major PLFA  for salt tolerance in DvH; growth energy efficiency might be a key factor for selection; there is a restorative trend of metabolic, physiological, and transcriptional changes in the long-term evolution of DvH; the relationship between a genotype and a phenotype is very complex.

Rapid genetic and phenotypic adaptation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough to salt stress was observed during experimental evolution. In order to identify key metabolites important for salt tolerance, a clone, ES10-5, isolated from population ES10 and allowed to experimentally evolve under salt stress for 5,000 generations, was analyzed and compared to clone ES9-11, which was isolated from population ES9 and had evolved under the same conditions for 1,200 generations. These two clones were chosen because they represented the best-adapted clones among six independently evolved populations. ES10-5 acquired new mutations in genes potentially involved in salt tolerance, in addition to the preexisting mutations and different mutations in the same genes as in ES9-11. Most basal abundance changes of metabolites and phospholipid fatty acids (PLFAs) were lower in ES10-5 than ES9-11, but an increase of glutamate and branched PLFA i17:1ω9c under high salinity was persistent. ES9-11 had decreased cell motility compared to the ancestor; in contrast, ES10-5 showed higher cell motility under both nonstress and high salinity conditions. Both genotypes displayed better growth energy efficiencies than the ancestor under nonstress or high salinity. Consistently, ES10-5 did not display most of the basal transcriptional changes observed in ES9-11. Still, it showed increased expression of genes involved in glutamate biosynthesis, cation efflux, and energy metabolism under high salinity. These results demonstrated the role of glutamate as a key osmolyte and i17:1ω9c as the major PLFA for salt tolerance in D. vulgaris.

DOI: 10.1128/mBio.01780-1714 November 2017 mBio vol. 8no. 6 e01780-17

Key Metabolites and Mechanistic Changes for Salt Tolerance in an Experimentally Evolved Sulfate-Reducing Bacterium, Desulfovibrio vulgaris. 

Aifen ZhouaRebecca LaubRichard BaranbJincai MaaFrederick von Netzerc, Weiling ShiaDrew Gorman-LewisdMegan L. KempheraZhili HeaYujia QinaZhou ShiaGrant M. ZaneeLiyou WuaBenjamin P. BowenbTrent R. NorthenbKristina L. HilleslandfDavid A. StahlcJudy D. WalleAdam P. Arkinb,gJizhong Zhoua,h,i

New ENIGMA Study Finds Anaerobic Nitrate-Reducing Conditions Influence Metal Toxicity

Prior observations of chromium (Cr[VI]) and uranium (U[VI]) toxicity under aerobic conditions, where metal toxicity was caused not only directly by the metal, but also indirectly due to redox reactions of the metal with oxygen and the resulting reactive oxygen species.  ENIGMA researchers from University of Georgia, University of Missouri, and Lawrence Berkeley Lab report the results of random barcode transposon site sequencing experiments performed on the chromium-contaminated environmental isolate, Pseudomonas stutzeri RCH2, grown under anaerobic denitrifying conditions. The mechanisms by which uranium and chromium cause toxicity under anaerobic denitrifying conditions and the defense mechanisms Pseudomonas stutzeri RCH2 uses to defend against these metals were examined using RB-TnSeq technology combined with physiology, biochemistry, and genetics.  Novel insights include characterization of a gene of previously unknown function (Psest_2088), involved in sulfite reduction and a key to Cr resistance.

ENIGMA Researcher at University of Georgia

This comprehensive analysis of (U[VI]) and (Cr[VI]) toxicity under anaerobic denitrifying conditions was possible only by leveraging all of the resources available in ENIGMA; which allowed Michael Thorgersen et al to elucidate on a genome wide scale the anaerobic toxicity targets of Cr[VI] and U[VI] and the mechanisms used by RCH2 to defend against these metals.  For Cr[VI], DNA is a toxicity target even under anaerobic conditions.  Cr[VI]-dependent fitness defects were seen under anaerobic conditions for strains lacking proteins involved in homologous recombination and nucleotide excision DNA repair.  Fitness data together with physiological growth studies on wild-type RCH2 and the Δ2088 mutant strain were used to develop a model in which the reduced thiol pool is an additional target of Cr[VI] toxicity.  In this model, Psest_2088, a protein of previously unknown function, is a key protein involved in sulfur assimilation at the step of sulfite reduction.  Both Cr[VI] and U[VI] toxicity have large fitness effects on RCH2 strains with defects in nitrate reduction. We propose that both metals interfere with cytochrome components of the remainder of the denitrification pathway, which is critical to respiration and survival when nitrate reduction is hindered.  This could hinder the remediation of sites contaminated with both nitrate and heavy metals such as Cr[VI] and U[VI].  Finally, exopolysaccharide biosynthesis and the universal stress protein UspA were identified as possible defenses mechanisms against U[VI] toxicity.  Cr[VI] and U[VI] damage living organisms in diverse ways, and RB-TnSeq technology is a powerful tool that can be used to study these processes, and identify the metabolic pathways involved.

RCH2 data can be found in the Fitness Browser:

Thorgersen MP, Lancaster WA, Ge X, Zane GM, Wetmore KM, Vaccaro BJ, Poole FL 2nd, Younkin AD, Deutschbauer AM, Arkin AP, Wall JD, Adams MWW. (2017) Mechanisms of Chromium and Uranium Toxicity in Pseudomonas stutzeri RCH2 Grown under Anaerobic Nitrate-Reducing Conditions. Front Microbiol. 8:1529  10.3389/fmicb.2017.01529 

Andrea Rocha is featured in DOE’s online Women @ Energy Series



Physical Biosciences ENIGMA Annual Retreat at the UC EBB – group photo, discussions, a poster session – August 11, 2015.

Andrea Rocha is featured in DOE’s online Women @ Energy Series in a formal announcement from Minorities in Energy Kickoff Year 2 event in Washington, D.C.