Origin of the Chordate Notochord

The phylum of Chordata is defined based on the discovery of a coelom-like dorsal notochord in ascidian and amphioxus embryos. Chordata can be classified into three subphylums, Cephalochordata, Urochordata, and Vertebrata, united by the presence of a notochord at some point during development. The origin of the notochord, the signature anatomical structure of chordates, has been under debate since the publication of Alexander Kovalevsky’s work in the mid-19th century that placed ascidians close to the vertebrates on the phylogenetic tree. During the late 20th century, the development of molecular and genetic tools in biology brought about a revival of studies on the evolutionary path of notochord development. Two main hypotheses for the origin of the notochord were proposed, the de novo theory and the axochord theory. The former states that notochord has developed de novo from the mid-dorsal archenteron of a chordate ancestor with simple morphology and no central nervous system nor notochord homolog. The putative notochord along the dorsal side of the animal is proposed to take on the signal functions later from the endoderm and ectoderm. An alternative hypothesis, the axochord theory, proposes that notochord has evolved from the mid-line muscle tissue, the so-called axochord, in annelids. Structural and molecular evidence point to the midline muscle of annelids as a distant homolog of the notochord. This hypothesis thus suggests a notochord-like structure in the urbilaterian ancestor, opposed to the consensus that notochord is a chordate-specific feature. In this review, we introduce the history of the formation of these views and summarize the current understandings of embryonic development, molecular profile, and gene regulatory networks of notochord and notochord-like structures.

Study of the Interaction of Sorption and Catalytic Centers in Carboxypeptidase T by X-ray Analysis

Carboxypeptidase T (CPT; EC 3.4.17.18) from Thermoactinomyces vulgaris is a distant homolog of the highly specific pancreatic carboxypeptidase B; but has a broad substrate specificity; the source of which remains unclear. A previous study of the structural bases of the substrate specificity of CPT using stable sulfamoyl analogs of the transition state of the elimination of leucine; phenylalanine; arginine; and glutamic acid; showed that the binding of the C-terminal residue of the substrate to the primary selectivity pocket of CPT leads to a change in the distance between Zn2+ and the sulfur atom. This value is related to the efficiency of catalysis of the corresponding substrate or the inhibition constant of the corresponding stable analog of the transition state. In this work; we obtained crystallographic and kinetic data of the complex of CPT with N-sulfamoyl-L-valine; confirming the effect of the binding of the ligand’s side group by the primary specificity pocket of CPT on the structure of the catalytic center; which can explain the unusual substrate specificity of CPT.

PGRL2 triggers degradation of PGR5 in the absence of PGRL1

In plants, inactivation of either of the thylakoid proteins PGR5 and PGRL1 impairs cyclic electron flow (CEF) around photosystem I. Because PGR5 is unstable in the absence of the redox-active PGRL1, but not vice versa, PGRL1 is thought to be essential for CEF. However, we show here that inactivation of PGRL2, a distant homolog of PGRL1, relieves the need for PGRL1 itself. Conversely, high levels of PGRL2 destabilize PGR5 even when PGRL1 is present. In the absence of both PGRL1 and PGRL2, PGR5 alters thylakoid electron flow and impairs plant growth. Consequently, PGR5 can operate in CEF on its own, and is the target of the CEF inhibitor antimycin A, but its activity must be modulated by PGRL1. We conclude that PGRL1 channels PGR5 activity, and that PGRL2 triggers the degradation of PGR5 when the latter cannot productively interact with PGRL1.

Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungusPodospora anserinarepresents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents.

Metatranscriptomic Identification of Diverse and Divergent RNA Viruses in Green and Chlorarachniophyte Algae Cultures

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we identified 18 novel RNA viruses in cultured samples from two major groups of microbial algae: the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to the Tombusviridae and Amalgaviridae viral families commonly associated with land plants. This suggests that the evolutionary history of these viruses extends to divergence events between algae and land plants. Seven Ostreobium sp-associated viruses exhibited sequence similarity to the mitoviruses most commonly found in fungi, compatible with horizontal virus transfer between algae and fungi. We also document, for the first time, RNA viruses associated with chlorarachniophytes, including the first negative-sense (bunya-like) RNA virus in microalgae, as well as a distant homolog of the plant virus Virgaviridae, potentially signifying viral inheritance from the secondary chloroplast endosymbiosis that marked the origin of the chlorarachniophytes. More broadly, these data suggest that the scarcity of RNA viruses in algae results from limited investigation rather than their absence.

Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behaviour and distinct patho-phenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and shed new light on key molecular features concerning an important class of pathogenic agents.

A Kayvirus Distant Homolog of Staphylococcal Virulence Determinants and VISA Biomarker Is a Phage Lytic Enzyme

Staphylococcal bacteriophages of the Kayvirus genus are candidates for therapeutic applications. One of their proteins, Tgl, is slightly similar to two staphylococcal virulence factors, secreted autolysins of lytic transglycosylase motifs IsaA and SceD. We show that Tgl is a lytic enzyme secreted by the bacterial transport system and localizes to cell peripheries like IsaA and SceD. It causes lysis of E. coli cells expressing the cloned tgl gene, but could be overproduced when depleted of signal peptide. S. aureus cells producing Tgl lysed in the presence of nisin, which mimics the action of phage holin. In vitro, Tgl protein was able to destroy S. aureus cell walls. The production of Tgl decreased S. aureus tolerance to vancomycin, unlike the production of SceD, which is associated with decreased sensitivity to vancomycin. In the genomes of kayviruses, the tgl gene is located a few genes away from the lysK gene, encoding the major endolysin. While lysK is a late phage gene, tgl can be transcribed by a host RNA polymerase, like phage early genes. Taken together, our data indicate that tgl belongs to the kayvirus lytic module and encodes an additional endolysin that can act in concert with LysK in cell lysis.

A Kayvirus Distant Homolog of Staphylococcal Virulence Determinats and VISA Biomarker is a Phage Lytic Enzyme That Decreases S. aureus Tolerance to Vancomycin

Staphylococcal bacteriophages of Kayvirus genus are candidates for therapeutic applications. One of their proteins, Tgl, is slightly similar to staphylococcal virulence factors, secreted autolysins of lytic transglycosylase motifs, IsaA and SceD. We show that Tgl is also a lytic enzyme secreted by bacterial transport system and localizes to cell peripheries, like IsaA and SceD. It caused lysis of E. coli cells expressing the cloned tgl gene, but could be overproduced when depleted of signal peptide. S. aureus cells producing Tgl lysed in the presence of nisin, which mimics the action of phage holin. In vitro, Tgl protein was able to destruct S. aureus cell walls. The production of Tgl decreased S. aureus tolerance to vancomycin, unlike the production of SceD, which is associated with the decreased sensitivity to vancomycin. In the genomes of kayviruses, the tgl gene is located a few genes away from gene lysK, encoding the major endolysin. While lysK is a late phage gene, tgl can be transcribed by a host RNA polymerase, as are phage early genes. Taken together our data indicate that tgl is a part of kayviruses lytic module and encodes an additional endolysin which can act in concert with LysK in cell lysis.

TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

TheEscherichia coliF element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.