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Photoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B 12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide visualizations of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter −35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B 12 and provide fundamental insight into a new mode of light-dependent gene regulation. Crystal structures are presented of Thermus thermophilus CarH, a photoreceptor that uses a vitamin B 12 derivative, in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. New insights into light-dependent gene regulation CarH is a photoreceptor that mediates light-dependent gene regulation in Myxococcus xanthus and Thermus thermophilus , using the vitamin B 12 derivative, adenosylcobalamin, as the light-sensing chromophore. Catherine Drennan and colleagues have solved X-ray crystal structures of CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. The structures reveal how exposure to light triggers large conformational changes that lead to the disassociation of CarH from DNA and relief of CarH-mediated transcriptional repression of carotenoid biosynthetic genes.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern 1 . For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings 2 . Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the m 2 6 A 2058 nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance. Structure-guided design and component-based synthesis are used to produce iboxamycin, a novel ribosome-binding antibiotic with potent activity against Gram-positive and Gram-negative bacteria.

Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus (‘wide’ and ‘narrow’), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility. Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. Here, Neuhaus et al. show that the bacterium Thermus thermophilus produces two forms of type IV pilus, differing in structure, protein composition, and function.

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-Å resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the β ′-subunit ‘lid’ loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination. RNA polymerase up close Two complementary papers this week focus on the structure and function of bacterial RNA polymerase. In the first, the enzyme is bound to the DNA template and RNA product, to give a close-up of the transcription elongation complex. The structure reveals details of the DNA-to-RNA transcription process, vital to all living cells. In the second paper, the RNA polymerase elongation complex is pictured bound to various substrate analogues and to an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition. Comparisons between the structures of human and bacteria RNA polymerase should aid in drug design: RNA polymerase is a target of antibiotics, including rifamycin and its derivatives. Crystal structure of bacterial RNA polymerase bound to the DNA template and RNA product, revealing a detailed view of the transcription elongation complex.

We report on crystal structures of ternary Thermus thermophilus Argonaute (Tt Ago) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg ²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for Tt Ago-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.

Bacteria experience a continual array of environmental stresses, necessitating adaptive mechanisms crucial for their survival. Thermophilic bacteria, such as Thermus thermophilus , face constant environmental challenges, particularly high temperatures, which requires robust adaptive mechanisms for survival. Studying these extremophiles provides valuable insights into the intricate molecular and physiological processes used by extremophiles to adapt and survive in harsh environments. Through meta-analysis of microarray data, we revealed the key genes in T. thermophilus HB8 that respond to various environmental stresses. The analysis revealed 20 differentially expressed genes (DEGs), including 13 upregulated and seven downregulated genes, with a threshold of|log fold change| > 1 and an adjusted p-value < 0.05. Several genes identified as up-regulated in our analysis belonged to the CRISPR-associated protein (Cas) family. To validate these findings, we further evaluated the relative expression levels of TTHB188 ( cas1/casA ), TTHB189 ( cas2/casB ), TTHB190 ( cas7 / casC ), TTHB191 ( cas5/casD ), TTHB192 ( cas6 / casE ), and TTHB193 ( cas1e ) using RT-qPCR under H 2 O 2 and salt stress conditions. The RT-qPCR analysis revealed significant up-regulation of transcripts, casA , casB , casC , casD , c asE , and cas1e under salt stress. However, under H 2 O 2 stress, only, casA , casB , and c asC exhibited substantial increases in expression. Our findings may indicate that the CRISPR-associated proteins significantly impact the adaptive response of T. thermophilus HB8 to various environmental stresses, particularly salt stress, highlighting its significance in extremophile survival and adaptation. This research offers an important understanding of the complex strategies used by extremophiles to survive in challenging conditions.

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli . The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus . Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σ A , and a promoter DNA fragment corresponding to the transcription bubble and downstream double-stranded DNA of the RNAP-promoter open complex. The structures show that σ recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases and that RNAP recognizes the -4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand single-stranded DNA (ssDNA) preorganize template-strand ssDNA to engage the RNAP active center.

Structural and biochemical studies reveal how the antimicrobial peptide Onc112 binds to bacterial ribosomes and show that Onc112 blocks and destabilizes the translation-initiation complex. The increasing prevalence of multidrug-resistant pathogenic bacteria is making current antibiotics obsolete. Proline-rich antimicrobial peptides (PrAMPs) display potent activity against Gram-negative bacteria and thus represent an avenue for antibiotic development. PrAMPs from the oncocin family interact with the ribosome to inhibit translation, but their mode of action has remained unclear. Here we have determined a structure of the Onc112 peptide in complex with the Thermus thermophilus 70S ribosome at a resolution of 3.1 Å by X-ray crystallography. The Onc112 peptide binds within the ribosomal exit tunnel and extends toward the peptidyl transferase center, where it overlaps with the binding site for an aminoacyl-tRNA. We show biochemically that the binding of Onc112 blocks and destabilizes the initiation complex, thus preventing entry into the elongation phase. Our findings provide a basis for the future development of this class of potent antimicrobial agents.