Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface ...displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass‐sensing RsgI‐type anti‐σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti‐σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn‐Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti‐σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.
Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to ...discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.
(group A
) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections,
uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and ...acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a "cap and release" mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr-Hb complex which function to transiently uncap HID1 from the heme bound to Hb's β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr's NEAT domains. These dynamics may maximize the efficiency of heme scavenging by
, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.
Within these survey images we discovered 65 supernovae (SNe) of all types, out tot ~ 2.5. We classify -24 of these as Type Ia SNe (SNe Ia) based on host galaxy redshifts and SN photometry ...(supplemented by grism spectroscopy of six SNe). Here we present a measurement of the volumetric SN la rate as a function of redshift, reaching for the first time beyond z - 2 and putting new constraints on SN Ia progenitor models. Combining the CANDELS rates with all available SN Ia rate measurements in the literature we find that this prompt SN Ia fraction is f sub(p) = (ProQuest: Formulae and/or non-USASCII text omitted), consistent with a delay time distribution that follows a simple t super(-1) power law for all times t > 40 Myr. In both CANDELS and the sister HST program CLASH (Cluster Lensing And Supernova Survey with Hubble), we find a low rate of SNe Ia at z > 1.
Heme is the most abundant source of iron in the human body and is actively scavenged by bacterial pathogens during infections. Corynebacterium diphtheriae and other species of actinobacteria scavenge ...heme using cell wall associated and secreted proteins that contain Conserved Region (CR) domains. Here we report the development of a fluorescent sensor to measure heme transfer from the C-terminal CR domain within the HtaA protein (CR2) to other hemoproteins within the heme-uptake system. The sensor contains the CR2 domain inserted into the β2 to β3 turn of the Enhanced Green Fluorescent Protein (EGFP). A 2.45 Å crystal structure reveals the basis of heme binding to the CR2 domain via iron-tyrosyl coordination and shares conserved structural features with CR domains present in Corynebacterium glutamicum. The structure and small angle X-ray scattering experiments are consistent with the sensor adopting a V-shaped structure that exhibits only small fluctuations in inter-domain positioning. We demonstrate heme transfer from the sensor to the CR domains located within the HtaA or HtaB proteins in the heme-uptake system as measured by a ∼ 60% increase in sensor fluorescence and native mass spectrometry.
An integral fusion of the C-terminal Conserved Region (CR) domain of the Corynebacterium diphtheriae HtaA protein within Enhanced Green Fluorescent Protein (EGFP) was developed, wherein heme binding to the CR domain quenches fluorescence. This results in a sensor that can measure heme binding and transfer in bacterial receptors. Display omitted
•Development of fluorescent sensor to measure heme transfer from bacterial receptors.•Crystal structure of a Conserved Region domain from Corynebacterium diphtheriae.•Fusion of CR domain within the β2-β3 turn of Enhanced Green Fluorescent Protein (EGFP).•The CR intradomain fusion within EGFP yields a semi-rigid structure.
The Seyfert 1 galaxy Arp 151 was monitored as part of three reverberation mapping campaigns spanning \(2008-2015\). We present modeling of these velocity-resolved reverberation mapping datasets using ...a geometric and dynamical model for the broad line region (BLR). By modeling each of the three datasets independently, we infer the evolution of the BLR structure in Arp 151 over a total of seven years and constrain the systematic uncertainties in non-varying parameters such as the black hole mass. We find that the BLR geometry of a thick disk viewed close to face-on is stable over this time, although the size of the BLR grows by a factor of \(\sim 2\). The dynamics of the BLR are dominated by inflow and the inferred black hole mass is consistent for the three datasets, despite the increase in BLR size. Combining the inference for the three datasets yields a black hole mass and statistical uncertainty of \(\log_{10}(\)M\(_{\rm BH}/\rm{M}_{\odot})=6.82^{+0.09}_{-0.09}\) with a standard deviation in individual measurements of 0.13 dex.
Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), ...exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine-isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from
We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in
uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.
We present an analysis of 507 spectra of 173 stripped-envelope (SE) supernovae (SNe) discovered by the untargeted Palomar Transient Factory (PTF) and intermediate PTF (iPTF) surveys. Our sample ...contains 55 Type IIb SNe (SNe IIb), 45 Type Ib SNe (SNe Ib), 56 Type Ic SNe (SNe Ic), and 17 Type Ib/c SNe (SNe Ib/c). We have compared the SE SN subtypes via measurements of the pseudo-equivalent widths (pEWs) and velocities of the He I λλ5876, 7065 and O I λ7774 absorption lines. Consistent with previous work, we find that SNe Ic show higher pEWs and velocities in O I λ7774 compared to SNe IIb and Ib. The pEWs of the He I λλ5876, 7065 lines are similar in SNe Ib and IIb after maximum light. The He I λλ5876, 7065 velocities at maximum light are higher in SNe Ib compared to SNe IIb. We identify an anticorrelation between the He I λ7065 pEW and O I λ7774 velocity among SNe IIb and Ib. This can be interpreted as a continuum in the amount of He present at the time of explosion. It has been suggested that SNe Ib and Ic have similar amounts of He, and that lower mixing could be responsible for hiding He in SNe Ic. However, our data contradict this mixing hypothesis. The observed difference in the expansion rate of the ejecta around maximum light of SNe Ic (Vm = √2Ek/Mej ≈ 15 000 km s−1Vm=2Ek/Mej≈15 000 km s−1${V_{\rm{m}}} = \sqrt {2{E_{\rm{k}}}/{M_{{\rm{ej}}} \approx 1{\rm{5 \, 000 \, km }} \, {{\rm{s}}^{ - 1}}$) and SNe Ib (Vm ≈ 9000 km s−1) would imply an average He mass difference of ∼1.4 M⊙, if the other explosion parameters are assumed to be unchanged between the SE SN subtypes. We conclude that SNe Ic do not hide He but lose He due to envelope stripping.
Many species of pathogenic gram‐positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are ...assembled by pilus‐specific sortase enzymes that join the pilin components together via lysine‐isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the CdSrtA pilus‐specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)‐T494(SpaA) lysine‐isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N‐terminal domain of SpaA (NSpaA) that is also crosslinked by CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.
Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam ...antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.
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