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Bacterial photobiology: Why do so many bacteria use photoreceptors?

The challenge

A quarter of all bacterial genomes encode a photoreceptor protein, but in most cases their biological function is entirely unknown.


Questions and hypotheses to address

  • We want to find out what all these bacteria are looking at.
  • In addition to phototaxis, we found evidence that light is an important signal regulating bacterial biofilm formation. We are exploring this phenomenon.
  • We want to identify the signal transduction chains involved in bacterial photosensing.

 

Who cares and so what?

Light is a key source of both energy and information that pervades the biosphere. The unexpected discovery of genes encoding photosensory proteins in many bacteria has revealed a largely unexplored area of bacterial photobiology. The photosensory proteins involved often are excellent model systems for protein biophysics and are finding many applications in the novel field of optogenetics.

 

Key methods we use

  • Bacterial physiology experiments.
  • Bioinformatics.
  • Biochemistry approaches.

 

Selected contributions that we made to the field

  • We discovered that a deep sea bacterium (where no sunlight penetrates) encodes a functional photoreceptor that regulates biofilm formation.
  • We reported evidence indicating that many bacteria contain photoreceptors involved in light-regulation of biofilm formation.

 

Selected publications from our lab on this topic

  • Riggs J, Hoff WD. 2019. Phototaxis in Archaea and Bacteria. In: Schmidt, Thomas M. (ed.) Encyclopedia of Microbiology, 4th Edition. vol. 3, pp. 520-526. UK: Elsevier.
  • Gomelsky M, Hoff WD. 2011. Light helps bacteria make important lifestyle decisions. Trends Microbiol. 19, 441-448.
  • van der Horst MA, Stalcup TP, Kaledhondar S, Kumauchi M, Hara M, Xie A, Hellingwerf KJ, Hoff, WD. 2009. Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis. J. Am. Chem. Soc. 131, 17443-17451.
  • Kumauchi M, Hara M, Stalcup P, Xie A, Hoff WD. 2008. Identification of six new photoactive yellow proteins: diversity and structure-function relationships in a bacterial blue light photoreceptor. Photochem. Photobiol. 84, 956-969.

Antibiotics resistance: can evolutionary strategies be used to address this challenge?

The challenge

As bacterial pathogens increasingly evolve resistance to antibiotics, humanity is faced with the risk of losing one of the pillars of modern medicine. Since this challenge is evolutionary in nature, we have started to explore possible evolutionary strategies to addressing this problem.

 

Questions and hypotheses to address

  • We are examining evolutionary strategies to combat antibiotics resistance. Currently we are exploring the rate with which unused antibiotics resistance genes are lost in the absence of the antibiotics.
  • This work also has implications for a broad question in molecular evolution: how quickly are (temporarily) unused genes lost?

 

Who cares and so what?

The emergence of widespread antibiotics resistance in infectious agents is widely identified as a major and growing medical problem, and addressing this challenge likely will require novel strategies.

 

Key methods we use

In-the-lab evolution experiments to measure the rate of loss of unselected genes, including genes encoding various types of antibiotics resistance.


tRNA sets: how and why do tRNA sets stay constant or evolve across the tree of life?

The challenge

While tRNAs are central to the functioning of all cells, key questions remain about their evolutionary origin and diversification. Can we understand the factors driving the evolution of tRNA sets?

 

Questions and hypotheses to address

  • Is there a universal standard tRNA set and how are these sets different tRNA sets in Bacteria, Archaea, and Eukaryotes?
  • What evolutionary pressures cause organisms to diverge from the domain-specific tRNA sets?
  • How did tRNA sets evolve to become different in the different domains of life?
  • What insights can be obtained on the emergence of translation during the origin of life?

 

Who cares and so what?

tRNAs are ancient molecules at the core of protein synthesis in all cellular life. While the genetic code implemented by cellular tRNA sets is remarkably conserved, tRNA sets exhibit considerable variability that has implications for molecular biology, the evolution of translation, and the use of tRNAs as potential drug targets.

 

Key methods we use

  • Bioinformatics.
  • Theoretical considerations from the field of RNA structural biology.

 

Selected contributions that we made to the field

  • We reconstructed the tRNA set of LUCA, the ancestor of all known cellular organisms.
  • We refined approaches to identify the tRNA sets encoded in archaeal genomes.

 

Selected publications from our lab on this topic

  • van der Gulik PTS, Egas M, Kraaijeveld K, Dombrowski N, Groot AT, Spang A, Hoff WD, Gallie J. On distinguishing between canonical tRNA genes and tRNA gene fragments in prokaryotes. RNA Biology 20:48-58.
  • van der Gulik PTS, Hoff WD. 2016. Anticodon modifications in the tRNA set of LUCA and the fundamental regularity in the Standard Genetic Code. PLoS ONE 11(7): e0158342.
  • van der Gulik TS, Hoff WD. 2011. Unassigned codons, nonsense suppression, and anticodon modifications in the evolution of the genetic code. J. Mol. Evol. 73, 59-69.

The tree of life: what are the large-scale trends in the molecular evolution of life?

The challenge

How to incorporate the massive amount of novel insights about biological diversity and evolution, particularly the discovery of the Archaea and many bacterial phyla, the immense diversity of viruses, horizontal gene transfer, and eukaryogenesis, into an updated view of the tree of life and the evolutionary processes that generated it?


Questions and hypotheses to address

  • Since Eukaryotes originated from a merger between a Asgard archaeon and a Proteobacterium, are there two or three domains of life?
  • Given the massive amount of horizontal gene transfer, should the tree of life concept be replaced by a web of life concept?
  • Now that it has become clear that most diversity of life is microbial/microscopic, how should Linnaean taxonomy by updated?

 

Who cares and so what?

Linnaean taxonomy has served as a key organizing principle for biological diversity. During the past four decades it has become increasingly clear that most of this diversity is microbial. The ongoing avalanche of novel microorganisms being discovered has led to intense debates regarding the most productive view of evolutions on the planet and has been yielding unexpected novel evolutionary insights, particularly regarding eukaryogenesis.

 

Key methods we use

Wide reading of relevant literature and developing a description that most accurately reflects the emerging biological realities.

 

Selected contributions that we made to the field

  • We made specific proposals to achieve a much-needed update to the Linnaean taxonomy of life.
  • We reported a coherent view on combining the three Domain view of life with the novel model for eukaryogenesis.

 

Selected publications from our lab on this topic

  • van der Gulik PTS, Hoff WD, Speijer D. 2024. The contours of evolution: In defence of the Tree of Life paradigm, BioEssays, in press.
  • van der Gulik PTS, Hoff WD, Speijer D. Renewing Linnaean taxonomy: A proposal to restructure the highest levels of the Natural System. Biological Reviews 98: 584–602.
  • van der Gulik, PTS, Hoff WD, Speijer D. 2017. In defense of the Three-Domains of life paradigm. BMC Evolutionary Biology 17: 218.

Extreme halophiles: how do organisms adapt their proteome to thrive high salt conditions?

The challenge

Can we understand the strategies that microorganisms use to survive even in saturated salt environments?

 

Questions and hypotheses to address

  • What is the diversity in halophilic strategies?
  • Why do some extreme halophiles evolve a highly acidic proteome?

 

Who cares and so what?

A striking aspect of Bacteria and Archaea is their ability to thrive in seemingly hostile environments based on biochemical innovations. We are examining how microorganisms are able to grow in saturated salt solutions and what information these adaptations hold for a fundamental understanding of the interactions between proteins, water, and salt.

 

Key methods we use

  • Genomics and bioinformatics.
  • Experimental measurements of salts and osmoprotectants in the cytoplasm of extreme halophiles.

 

Selected contributions that we made to the field

  • We demonstrated that an extremely halophilic bacterium with a highly acidic proteome can thrive without high cytoplasmic potassium concentrations.
  • We identified an osmoprotectant switch in a extremely halophilic bacterium, which can switch between the two main osmoprotection strategies (KCl accumulation and compatible solute accumulation) depending on growth conditions.

 

Selected publications from our lab on this topic

  • Deole R, Hoff WD. 2020. A potassium chloride to glycine betaine osmoprotectant switch in the purple photosynthetic extreme halophile Halorhodospira halophila. Sci. Rep. 10, 3383.
  • Youssef NH, Savage-Ashlock KN, McCully AM, Ludetke B, Shaw EI, Hoff WD, Elshahed MS. 2013. Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales. The ISME Journal 8: 636–649.
  • Deole R, Challacombe J, Raiford DW, Hoff WD. 2013. An extremely halophilic proteobacterium combines a highly acidic proteome with a low cytoplasmic potassium content. J. Biol. Chem. 288, 581-588.
  • Challacome JF, Majid S, Deole R, Brettin TS, Bruce D, Delano SF, Detter JC, Gleasner CD, Han CS, Misra M, Reitenga KG, Saunders E, Tapia R, Lapidus A, Ivanova N, Hoff WD. 2013. Complete genome sequence of Halorhodospira halophila SL1. Standards in Genome Science 8, 206-214.

Structure-function relationships: how to predict the properties of entire protein families?

The challenge

Protein (super)families often exhibit high levels of both functional diversity and sequence diversity. Is it possible to unravel the combinations of substitutions that are responsible for functional diversity and the effects that govern the observed patterns of residue conservation and variance at specific positions in the protein?

 

Questions and hypotheses to address

  • How to identity the combination of substitutions that cause functional variation in protein families?
  • How does protein function map in amino acid sequence space to allow robustness and evolvability?
  • How to obtain predictive structure-function rules that apply to an entire protein family?
  • What is the functional role of residues that are conserved in protein (super)families?

 

Who cares and so what?

While major progress has been made in predicting the structure of proteins based on their amino acid sequence, predicting the biological function of proteins remains highly challenging. This major gap in knowledge limits both biotechnology and functionally informative genome annotation.

 

Key methods we use

  • Biophysics and spectroscopy of purified proteins, their mutant variants, and homologs from other organisms.
  • Gene synthesis and protein biochemistry of resurrected ancestral proteins.
  • Bioinformatics.

 

Selected contributions that we made to the field

  • We identified that some superfamily-conserved residues in PAS domains form functionally important and highly conserved patterns in residue-residue or residue-backbone interactions.
  • For the PYP model system we found that almost all point mutants remain functional, but that about half exhibit altered functional properties: a striking and likely universal combination of robustness to mutational disruption with a high degree of functional evolvability.

 

Selected publications from our lab on this topic

  • Philip AF, Kumauchi M, Hoff WD. 2010. Robustness and evolvability in the functional anatomy of a PAS domain. Proc. Natl. Acad. Sci. USA 107, 17986-17991.
  • Kumauchi M, Kaledhonkar S, Philip AF, Wycoff J, Hara M, Li Y, Xie A, Hoff WD. 2010. A conserved helical capping hydrogen bond in PAS domains controls signaling kinetics in the superfamily prototype photoactive yellow protein. J. Am. Chem. Soc. 132, 15820-15830.
  • Philip AF, Eisenman KT, Papadantonakis GA, Hoff WD. 2008. Functional tuning of photoactive yellow protein by active site residue 46. Biochemistry 47, 13800-13810.

Fundamental and universal processes in proteins: understanding protein dynamics, intramolecular proton transfer, and receptor activation using photoreceptor model systems

The challenge

Great progress has been made in measuring and predicting the static structures of proteins The next frontier is understanding structural changes that enable protein function.

 

Questions and hypotheses to address

  • How can we identify functionally important motions (FIMs) during protein function?
  • Can we uncover the forces that drive these motions and how they contribute to functional processes such as receptor activation?
  • What drives directional proton transfer in many of these transitions?

 

Who cares and so what?

How the dynamics of proteins contributes to their function is a matter of intense debate. On the one hand the study of protein dynamics is emerging as the key frontier in structural biology, but on other hand there is little understanding of the underling mechanisms of how protein dynamics contributes to function. We are studying these questions with emphasis on experimentally identifying functionally important motions and on understanding the widely occurring process of proton transfer in proteins.

 

Key methods we use

  • Static and time-resolved Fourier transform infrared (FTIR) spectroscopy, with the long-term goal of developing infrared structural biology of proteins (in collaboration with Prof. Aihua Xie).
  • Ultrafast laser spectroscopy to unravel the first steps in photoreceptor activation as a model system for the fastest steps in protein dynamics (with collaboration with Prof. Delmar Larsen).
  • Site-directed mutagenesis and site-specific isotope editing of proteins.

 

Selected contributions that we made to the field

  • We used the PYP system to show that light-triggered intra-molecular proton transfer generates a buried charged residue that acts as the “electrostatic epicenter” for the “protein quake” that activates signaling by PYP.
  • We found that the sequence of events of ultrafast (picosecond) photochemistry, followed by slower (~100 microseconds) proton transfer, followed by large protein conformational changes (~millisecond) occurs in a wide variety of different types of photoreceptor proteins.

 

Selected publications from our lab on this topic

  • Mix T, Hara M, Fuzell J, Kumauchi M, Kaledhonkar S, Xie A; Hoff WD, Larsen D. 2021. Not all Photoactive Yellow Proteins are built alike: surprises and insights into chromophore photoisomerization, protonation, and thermal reisomerization of the Photoactive Yellow Protein isolated from Salinibacter ruber. J. Am. Chem. Soc. 143: 19614-19628.
  • Kottke T, Xie A, Larsen DS, Hoff WD. 2018. Photoreceptors take charge: emerging principles for light sensing. Annu Rev. Biophys. 47, 291-313.
  • Mix LT, Hara M, Rathod R, Kumauchi M, Hoff WD, Larsen DS. 2016. Non-canonical photocycle initiation dynamics of the photoactive yellow protein domain of the PYP-phytochrome-related (Ppr) photoreceptor. J. Phys. Chem. Lett. 7: 5212−5218.
  • Creelman M, Kumauchi M, Hoff WD, Mathies RA. 2014. Chromophore dynamics in the PYP photocycle from femtosecond stimulated Raman spectroscopy. J. Phys. Chem. B 118: 659–667.
  • Xie A, Kelemen L, Hendriks J, White BJ, Hellingwerf KJ, Hoff WD. 2001. Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation. Biochemistry 40, 1510-1517.

Active site strain and protein-ligand interactions using photoreceptor model systems

The challenge

Proteins create intricate active sites to achieve the myriad functional properties they perform. Residues and cofactors at active sites often acquire physical and chemical properties quite distinct from the ones they have in solution. What protein ligand interactions cause these effects and what is the contribution of active site strain, an effect that until recently was very difficult to measure?

 

Questions and hypotheses to address

  • What causes the spectral tuning effects in light-absorbing proteins that occur throughout photobiology and are also at the basis of human color vision?
  • Can the novel technique of Raman Optical Activity be developed to quantify molecular strain and distortions at the active site of chromophoric proteins?
  • What resolution in determining the atomic coordinates of atoms in a protein would be needed to deeply understand protein function? We hypothesize that vibrational spectroscopies offer an approach to obtain structural information on active sites at ultra-high resolution, opening up novel frontiers in understanding protein function.

 

Who cares and so what?

Biochemical textbooks often depict active site strain as one of the key mechanisms that enzymes use to accelerate chemical reactions. However, measuring such strain has proven highly challenging. We are using vibrational spectroscopies to address this gap in knowledge.

Key methods we use

  • Cloning, mutating, and purifying various photoreceptor protein variants.
  • Pre-resonance Raman and Raman Optical Activity (ROA) spectroscopy (in collaboration with Prof. Masashi Unno and Prof. Tomotsumi Fujisawa).
  • Static and time-resolved Fourier Transform Infrared (FTIR) spectroscopy (in collaboration with Prof. Aihua Xie).

 

Selected contributions that we made to the field

  • We showed that the pre-resonance regime is ideal for driving structurally informative ROA data on structural distortions in the chromophore at the active site of photoreceptor proteins.
  • We have shown how ROA data can be used to obtain quantitative information on the degree of functionally relevant distortion of specific dihedral angles in such active site chromophores.
  • We complemented the well-studied kinetic isotope effect in enzymes by demonstrating and explaining a spectral isotope effect in photoreceptor proteins.
  • We developed a novel approach to understanding the mechanism of spectral tuning by mapping the energy surfaces involved using a combined analysis of absorbance and fluorescence emission spectra.

 

Selected publications from our lab on this topic

  • Fujisawa T, Shingae T, Ren J, Haraguchi S, Hanamoto T, Hoff WD, Unno M. 2023. spectroscopic validation of crystallographic structures of a protein active site by chiroptical spectroscopy. J. Phys. Chem. Lett. 14: 9304-9309.
  • Matsuo J, Kikukawa T, Fujisawa T, Hoff WD, Unno M. 2020. “Watching” a molecular twist in a protein by Raman optical activity. J. Phys. Chem. Lett. 11: 579–8584.
  • Haraguchi S, Shingae T, Fujisawa T, Kasai N, Kumauchi M, Hanamoto T, Hoff WD, Unno M. 2018. A spectroscopic ruler for measuring active site distortions based on Raman optical activity of a hydrogen out-of-plane vibration. Proc. Natl. Acad. Sci. USA 115: 8671-8675.
  • Haraguchi S, Hara M, Shingae T, Kumauchi M, Hoff WD, Unno M. 2015. Experimental detection of the intrinsic difference in Raman optical activity of a photoreceptor protein under pre-resonance and resonance conditions. Angew. Chem. Intl. Ed. 127: 11717 –11720.
  • Kaledhonkar S, Hara M, Stalcup* TP, Xie A, Hoff WD. 2013. Strong hydrogen bonding causes a spectral isotope effect in the p-coumaric chromophore of the photoactive yellow protein from Salinibacter ruber. Biophys. J. 105: 2577–2585.
  • Philip AF, Nome RA, Papadantonakis GA, Scherer NF, Hoff WD. 2010. Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface. Proc. Natl. Acad. Sci. USA 107, 5821-5826.

Course-embedded research on bacterial diversity and genomics

The challenge

Research on STEM education has shown that learning new material during the process of doing authentic research leads to much better learning outcomes. How to make such authentic research more widely available by incorporating it into undergraduate courses?

 

Questions and hypotheses to address

  • Can undergraduate students generate publishable scientific results while performing course-embedded research?
  • Can undergraduate students function as authors and peer reviewers of original scientific manuscripts?

 

Who cares and so what?

Successful implementation of course-embedded research promises to offer the dual benefits of providing students with an approach that helps them achieve deeper understanding and better learning outcomes, while at the same time yielding novel scientific insights. Bacterial genomics offers an exciting area for such approaches.

 

Key methods we use

  • Whole genome sequencing of genomic DNA extracted from isolated bacterial strains.
  • Assembly and bioinformatics analysis of the resulting sequence information (in collaboration with Prof. Noha Youssef).

 

Selected contributions that we made to the field

Undergraduate student researchers at OSU isolated novel bacterial strains and extracted genomic DNA from these organisms for whole genome sequencing. Prof. Youssef used these genomes in her genomics course, and a sequence of homework assignments resulted in the data resulting in a series of genome announcement and analysis publications.

 

Selected publications from our lab on this topic

  • Youssef N, Farag I, Rudy S, Mulliner A, Walker K, Caldwell F, Miller M, Hoff WD, and Elshahed M. 2019.  The Wood-Ljungdahl pathway as a key driver of metabolic versatility in Candidate phylum Bipolaricaulota (Acetothermia, OP1), Environmental Microbiology Reports 11: 538–547.
  • Youssef N, Farag I, Hahn CR, Jarrett J, Becraft E, Eloe-Fadrosh E, Lightfoot J, Bourgeois A, Cole T, Ferrante S, Truelock M, Marsh W, Jamaleddine M, Ricketts S, Simpson R, McFadden A, Hoff WD, Ravin N, Sievert S, Stepanauskas R, Woyke T, Elshahed M. 2019. Analysis of the Predicted metabolic abilities, structure features, and ecological preferences of candidate division LCP-89, a member of the FCB superphylum, Appl. Env, Microbiol. 85: e00110-19.
  • Calkins SS, Couger MB, Jackson C, Zandler J, Hudgins GC, Hanafy RA, Budd C, French DP, Hoff WD, Youssef N. 2016. Draft genome sequence of Staphylococcus hominis strain Hudgins isolated from human skin implicates metabolic versatility and several virulence determinants. Genomics Data 10: 91-96.
  • Hanafy RA, Couger MB, Baker K, Murphy C, O’Kane SD, Budd C, French DP, Hoff WD, Youssef N. 2016. Draft genome sequence of Micrococcus luteus strain O’Kane implicates metabolic versatility and the potential to degrade polyhydroxybutyrates. Genomics Data 9: 148–153.

Peer reviewed science writing by undergraduate students

The challenge

Scientific and technical writing is an important skill at risk of not being explicitly taught because of the time investments needed for detailed feedback on scientific texts written by undergraduate students.

 

Questions and hypotheses to address

Peer-reviewed science writing by undergraduate students is an effective and practically feasible approach for students to improve their scientific and technical writing skills.

 

 

Who cares and so what?

Scientific writing is widely considered to be a key skill, but it is rarely taught in science classes. We use peer-reviewed science writing as an exciting and scalable approach to provide students with the opportunity to improve their technical writing abilities.

 

Key methods we use

We are using the Open Journal System (OJS) to allow students to write, submit, peer-review, revise, and publish on scientific topics of their own choice.

 

Selected contributions that we made to the field

As part of their course work, undergraduate students at OSU select topics have been successfully publishing original scientific manuscripts on topics of their choice in the area of the molecular life sciences and cancer.

 

Peer-reviewed online undergraduate science journals that we created for this purpose


Key collaborators (alphabetically)

  • Dr. Sabrina Beckman, MMG OSU: extreme halophiles.
  • Dr. Rob Burnap, MMG OSU: bioinformatics.
  • Dr. Ratnakar Deole, Boston University: extreme halophiles.
  • Dr. Marijn Egas, Rijksuniversiteit Groningen, the Netherlands: tRNA sets.
  • Dr. Mostafa Elshahed, MMG OSU: bioinformatics.
  • Dr. Donald French, Integrative Biology OSU: course-embedded authentic research.
  • Dr. Tomotsumi Fujisawa, Saga University, Japan: protein biophysics and ROA spectroscopy.
  • Dr. Jenna Gallie, Max Planck Institute for Evolutionary Biology, Germany: tRNA sets.
  • Dr. Astrid Groot, University of Amsterdam, the Netherlands: tRNA sets.
  • Dr. Peter van der Gulik, Centrum Wiskunde & Informatica, the Netherlands: tRNA sets; evolution in the tree of life.
  • Dr. Ken Kraaijeveld, University of Applied Sciences Leiden, The Netherlands: tRNA sets.
  • Dr. Delmar Larsen, University of California – Davis: protein biophysics and ultrafast spectroscopy.
  • Dr. Josiah Meints (and Dr. Anna Sicari and Dr. Rebecca Damron), OSU Writing Center: peer-reviewed science writing by undergraduate students.
  • Dr. Akhilesh Ramachandran (Vet Med OSU): evolutionary solutions to antibiotics resistance.
  • Dr. Dave Speijer, University of Amsterdam, the Netherlands: evolution in the tree of life.
  • Dr. Masashi Unno, Saga University, Japan: protein biophysics and ROA spectroscopy.
  • Dr. Aihua Xie, Physics OSU: protein biophysics and FTIR spectroscopy; teaching Foundations of Cancer; co-organizing sessions at the March Meeting of the APS.
  • Dr. Noha Youssef, MMG OSU: bacterial genomics; bioinformatics.
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