The contours of evolution: In defence of Darwin's tree of life paradigm.

Both the concept of a Darwinian tree of life (TOL) and the possibility of its accurate reconstruction have been much criticized. Criticisms mostly revolve around the extensive occurrence of lateral gene transfer (LGT), instances of uptake of complete organisms to become organelles (with the associated subsequent gene transfer to the nucleus), as well as the implications of more subtle aspects of the biological species concept. Here we argue that none of these criticisms are sufficient to abandon the valuable TOL concept and the biological realities it captures. Especially important is the need to conceptually distinguish between organismal trees and gene trees, which necessitates incorporating insights of widely occurring LGT into modern evolutionary theory. We demonstrate that all criticisms, while based on important new findings, do not invalidate the TOL. After considering the implications of these new insights, we find that the contours of evolution are best represented by a TOL.

Spectroscopic Validation of Crystallographic Structures of a Protein Active Site by Chiroptical Spectroscopy.

Out-of-plane distortions of a cofactor molecule in a protein active site are functionally important, and in photoreceptors, it has been proposed that they are crucial for spectral tuning and energy storage in photocycle intermediates. However, these subtle structural features are often beyond the grasp of structural biology. This issue is strikingly exemplified by photoactive yellow protein: its 14 independently determined crystal structures exhibit considerable differences in the dihedral angles defining the chromophore geometry, even though most of these are at excellent resolution. Here we developed a strategy to verify cofactor distortions in crystal structures by using quantum chemical calculations and chiroptical spectroscopy, particularly Raman optical activity and electronic circular dichroism spectroscopies. Based on this approach, we identify seven crystal structures with the chromophore geometries inconsistent with the experimentally observed data. The strategy implemented here promises to be widely applicable to uncovering cofactor distortions at active sites and to studies of reaction intermediates.

On distinguishing between canonical tRNA genes and tRNA gene fragments in prokaryotes.

Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.

Renewing Linnaean taxonomy: a proposal to restructure the highest levels of the Natural System.

During the last century enormous progress has been made in the understanding of biological diversity, involving a dramatic shift from macroscopic to microscopic organisms. The question now arises as to whether the Natural System introduced by Carl Linnaeus, which has served as the central system for organizing biological diversity, can accommodate the great expansion of diversity that has been discovered. Important discoveries regarding biological diversity have not been fully integrated into a formal, coherent taxonomic system. In addition, because of taxonomic challenges and conflicts, various proposals have been made to abandon key aspects of the Linnaean system. We review the current status of taxonomy of the living world, focussing on groups at the taxonomic level of phylum and above. We summarize the main arguments against and in favour of abandoning aspects of the Linnaean system. Based on these considerations, we conclude that retaining the Linnaean Natural System provides important advantages. We propose a relatively small number of amendments for extending this system, particularly to include the named rank of world (Latin alternative mundis) formally to include non-cellular entities (viruses), and the named rank of empire (Latin alternative imperium) to accommodate the depth of diversity in (unicellular) eukaryotes that has been uncovered. We argue that in the case of both the eukaryotic domain and the viruses the cladistic approach intrinsically fails. However, the resulting semi-cladistic system provides a productive way forward that can help resolve taxonomic challenges. The amendments proposed allow us to: (i) retain named taxonomic levels and the three-domain system, (ii) improve understanding of the main eukaryotic lineages, and (iii) incorporate viruses into the Natural System. Of note, the proposal described herein is intended to serve as the starting point for a broad scientific discussion regarding the modernization of the Linnaean system.

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.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

Health Functionalities of Betaine in Patients With Homocystinuria.

Homocystinuria is a medical condition that can have widespread and harmful effects on multiple organ systems within the body. This disease is caused by a deficiency in one of the enzymes involved in the methionine metabolism pathway. One example would be a deficiency in cystathionine-ß-synthase (CBS), which is seen in classical homocystinuria. A deficiency in CBS can lead to elevated levels of homocysteine (HCY) and possible depletion of methionine and/or cysteine. There are several different treatment options for patients with this condition, one of which is the administration of the drug betaine. Here we review the use of betaine to decrease these elevated levels of homocysteine back to within normal ranges. Published literature indicates that the use of this choline derivative is most beneficial to patients who are either not compliant with the recommended low methionine and low protein diet or wish to consume a less restricted diet.

"Watching" a Molecular Twist in a Protein by Raman Optical Activity.

Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.

A potassium chloride to glycine betaine osmoprotectant switch in the extreme halophile Halorhodospira halophila.

Halophiles utilize two distinct osmoprotection strategies. The accumulation of organic compatible solutes such as glycine betaine does not perturb the functioning of cytoplasmic components, but represents a large investment of energy and carbon. KCl is an energetically attractive alternative osmoprotectant, but requires genome-wide modifications to establish a highly acidic proteome. Most extreme halophiles are optimized for the use of one of these two strategies. Here we examine the extremely halophilic Proteobacterium Halorhodospira halophila and report that medium K+ concentration dramatically alters its osmoprotectant use. When grown in hypersaline media containing substantial K+ concentrations, H. halophila accumulates molar concentrations of KCl. However, at limiting K+ concentrations the organism switches to glycine betaine as its major osmoprotectant. In contrast, the closely related organism Halorhodospira halochloris is limited to using compatible solutes. H. halophila performs both de novo synthesis and uptake of glycine betaine, matching the biosynthesis and transport systems encoded in its genome. The medium K+ concentration (~10 mM) at which the KCl to glycine betaine osmoprotectant switch in H. halophila occurs is near the K+ content of the lake from which it was isolated, supporting an ecological relevance of this osmoprotectant strategy.

Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.

We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.

Candidatus Krumholzibacterium zodletonense gen. nov., sp nov, the first representative of the candidate phylum Krumholzibacteriota phyl. nov. recovered from an anoxic sulfidic spring using genome resolved metagenomics.

The accumulation of genomes of uncultured organisms has highlighted the need for devising a taxonomic and nomenclature scheme to validate names and prevent redundancies. We here report on the recovery and analysis of four phylogenetically related genomes recovered from an anoxic sulfide and sulfur-rich spring (Zodletone spring) in southwestern Oklahoma. Phylogenetic analysis based on 120 single copy markers attested to their position as a novel distinct bacterial phylum. Genomic analysis suggests Gram-negative flagellated organisms that possess type IV pili. The organisms are predicted to be rod-shaped, slow-growers, with an anoxic, heterotrophic, and fermentative lifestyle. Predicted substrate utilization pattern includes multiple amino acids, dipeptides, tripeptides, and oligpopeptides; as well as few sugars. Predicted auxotrophies include proline, vitamin B6, lipoic acid, biotin, and vitamin B12. Assessment of the putative global distribution pattern of this novel lineage suggests its preference to anoxic marine, terrestrial, hydrocarbon-impacted, and freshwater habitats. We propose the candidatus name Krumholzibacterium zodletonense gen. nov, sp. nov. for Zgenome0171T, with the genome serving as the type material for the novel family Krumholzibacteriaceae fam. nov., order Krumholzibacteriales ord. nov., class Krumholzibacteria class nov., and phylum Krumholzibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number QTKG01000000.

Spectroscopic ruler for measuring active-site distortions based on Raman optical activity of a hydrogen out-of-plane vibration.

Photoactive yellow protein (PYP), from the phototrophic bacterium Halorhodospira halophila, is a small water-soluble photoreceptor protein and contains p-coumaric acid (pCA) as a chromophore. PYP has been an attractive model for studying the physical chemistry of protein active sites. Here, we explore how Raman optical activity (ROA) can be used to extract quantitative information on distortions of the pCA chromophore at the active site in PYP. We use 13C8-pCA to assign an intense signal at 826 cm-1 in the ROA spectrum of PYP to a hydrogen out-of-plane vibration of the ethylenic moiety of the chromophore. Quantum-chemical calculations based on density functional theory demonstrate that the sign of this ROA band reports the direction of the distortion in the dihedral angle about the ethylenic C=C bond, while its amplitude is proportional to the dihedral angle. These results document the ability of ROA to quantify structural deformations of a cofactor molecule embedded in a protein moiety.

Photoreceptors Take Charge: Emerging Principles for Light Sensing.

The first stage in biological signaling is based on changes in the functional state of a receptor protein triggered by interaction of the receptor with its ligand(s). The light-triggered nature of photoreceptors allows studies on the mechanism of such changes in receptor proteins using a wide range of biophysical methods and with superb time resolution. Here, we critically evaluate current understanding of proton and electron transfer in photosensory proteins and their involvement both in primary photochemistry and subsequent processes that lead to the formation of the signaling state. An insight emerging from multiple families of photoreceptors is that ultrafast primary photochemistry is followed by slower proton transfer steps that contribute to triggering large protein conformational changes during signaling state formation. We discuss themes and principles for light sensing shared by the six photoreceptor families: rhodopsins, phytochromes, photoactive yellow proteins, light-oxygen-voltage proteins, blue-light sensors using flavin, and cryptochromes.

Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from Halorhodospira halophila.

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.

Noncanonical Photocycle Initiation Dynamics of the Photoactive Yellow Protein (PYP) Domain of the PYP-Phytochrome-Related (Ppr) Photoreceptor.

The photoactive yellow protein (PYP) from Halorhodospira halophila (Hhal) is a bacterial photoreceptor and model system for exploring functional protein dynamics. We report ultrafast spectroscopy experiments that probe photocycle initiation dynamics in the PYP domain from the multidomain PYP-phytochrome-related photoreceptor from Rhodospirillum centenum (Rcen). As with Hhal PYP, Rcen PYP exhibits similar excited-state dynamics; in contrast, Rcen PYP exhibits altered photoproduct ground-state dynamics in which the primary I0 intermediate as observed in Hhal PYP is absent. This property is attributed to a tighter, more sterically constrained binding pocket around the p-coumaric acid chromophore due to a change in the Rcen PYP protein structure that places Phe98 instead of Met100 in contact with the chromophore. Hence, the I0 state is not a necessary step for the initiation of productive PYP photocycles and the ubiquitously studied Hhal PYP may not be representative of the broader PYP family of photodynamics.

Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira halophila.

We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PYP, but this residue is Ile in wild-type Lbif PYP. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PYP family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PYP photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PYP confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.

Draft genome sequence of Microbacterium oleivorans strain Wellendorf implicates heterotrophic versatility and bioremediation potential.

Microbacterium oleivorans is a predominant member of hydrocarbon-contaminated environments. We here report on the genomic analysis of M. oleivorans strain Wellendorf that was isolated from an indoor door handle. The partial genome of M. oleivorans strain Wellendorf consists of 2,916,870 bp of DNA with 2831 protein-coding genes and 49 RNA genes. The organism appears to be a versatile mesophilic heterotroph potentially capable of hydrolysis a suite of carbohydrates and amino acids. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, fructose, rhamnose, galactose, xylose, arabinose, alanine, aspartate, asparagine, glutamate, serine, glycine, threonine and cysteine. This is the first detailed analysis of a Microbacterium oleivorans genome.

Draft genome sequence and detailed analysis of Pantoea eucrina strain Russ and implication for opportunistic pathogenesis.

The genus Pantoea is a predominant member of host-associated microbiome. We here report on the genomic analysis of Pantoea eucrina strain Russ that was isolated from a trashcan at Oklahoma State University, Stillwater, OK. The draft genome of Pantoea eucrina strain Russ consists of 3,939,877 bp of DNA with 3704 protein-coding genes and 134 RNA genes. This is the first report of a genome sequence of a member of Pantoea eucrina. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of all amino acids as well as glucose, fructose, mannose, xylose, arabinose and galactose, suggesting the organism is a versatile heterotroph. The genome also encodes an extensive secretory machinery including types I, II, III, IV, and Vb secretion systems, and several genes for pili production including the new usher/chaperone system (pfam 05,229). The implications of these systems for opportunistic pathogenesis are discussed.

Draft genome sequence of Staphylococcus hominis strain Hudgins isolated from human skin implicates metabolic versatility and several virulence determinants.

Staphylococcus hominis is a predominant member of the human skin microbiome. We here report on the genomic analysis of Staphylococcus hominis strain Hudgins that was isolated from the wrist area of human skin. The partial genome assembly of S. hominis Hudgins consists of 2,211,863 bp of DNA with 2174 protein-coding genes and 90 RNA genes. Based on the genomic analysis of KEGG pathways, the organism is expected to be a versatile heterotroph potentially capable of hydrolyzing the sugars glucose, fructose, mannose, and the amino acids alanine, aspartate, glutamate, glycine, threonine, cysteine, methionine, valine, isoleucine, leucine, lysine, arginine, phenylalanine, tyrosine, and tryptophan for energy production through aerobic respiration, with occasional lactate and acetate fermentation. Evidence for poly-gamma glutamate capsule and type IV Com system pili were identified in the genome. Based on COG analysis, the genome of S. hominis Hudgins clusters away from the previously published S. hominis genome ZBW5.

Draft genome sequence of Micrococcus luteus strain O'Kane implicates metabolic versatility and the potential to degrade polyhydroxybutyrates.

Micrococcus luteus is a predominant member of skin microbiome. We here report on the genomic analysis of Micrococcus luteus strain O'Kane that was isolated from an elevator. The partial genome assembly of Micrococcus luteus strain O'Kane is 2.5 Mb with 2256 protein-coding genes and 62 RNA genes. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, galactose, fructose, mannose, alanine, aspartate, asparagine, glutamate, glutamine, glycine, serine, cysteine, methionine, arginine, proline, histidine, phenylalanine, and fatty acids. Genomic comparison to other M. luteus representatives identified the potential to degrade polyhydroxybutyrates, as well as several antibiotic resistance genes absent from other genomes.

Draft genome sequence of Pseudomonas moraviensis strain Devor implicates metabolic versatility and bioremediation potential.

Pseudomonas moraviensis is a predominant member of soil environments. We here report on the genomic analysis of Pseudomonas moraviensis strain Devor that was isolated from a gate at Oklahoma State University, Stillwater, OK, USA. The partial genome of Pseudomonas moraviensis strain Devor consists of 6016489 bp of DNA with 5290 protein-coding genes and 66 RNA genes. This is the first detailed analysis of a P. moraviensis genome. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of fructose, xylose, mannose and all amino acids with the exception of tryptophan and valine, implying that the organism is a versatile heterotroph. The genome of P. moraviensis strain Devor was rich in transporters and, based on COG analysis, did not cluster closely with P. moraviensis R28-S genome, the only previous report of a P. moraviensis genome with a native mercury resistance plasmid.