Characterization of the Bifunctional Enzyme BioDA Involved in Biotin Synthesis and Pathogenicity in Aspergillus flavus.

Biotin is an important enzyme cofactor that plays a key role in all three domains. The classical bifunctional enzyme BioDA in eukaryotes (such as Aspergillus flavus and Arabidopsis thaliana) is involved in the antepenultimate and penultimate steps of biotin biosynthesis. In this study, we identified a A. flavus bifunctional gene bioDA which could complement both Escherichia coli ΔEcbioD and ΔEcbioA mutants. Interestingly, the separated domain of AfBioD and AfBioA could, respectively, fuse with EcBioA and EcBioD well and work together. What is more, we found that BioDA was almost localized to the mitochondria in A. flavus, as shown by N-terminal red fluorescent protein tag fusion. Noteworthy, the subcellular localization of AfBioDA is never affected by common environmental stresses (such as hyperosmotic stress or oxidative stress). The knockout strategy demonstrated that the deletion of AfbioDA gene from the chromosome impaired the biotin de novo synthesis pathway in A. flavus. Importantly, this A. flavus mutant blocked biotin production and decreased its pathogenicity to infect peanuts. Based on the structural comparison, we found that two inhibitors (amiclenomycin and gemcitabine) could be candidates for antifungal drugs. Taken together, our findings identified the bifunctional AfbioDA gene and shed light on biotin biosynthesis in A. flavus.

Biochemical and structural characterization of the BioZ enzyme engaged in bacterial biotin synthesis pathway.

Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes "BioC-BioH" pathway in Escherichia coli. Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales, protein phylogeny infers that BioZ is domesticated to gain an atypical role of ß-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5'-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.

Carriage of Distinct mcr-1-Harboring Plasmids by Unusual Serotypes of Salmonella.

Colistin acts as a last-resort antibiotic against lethal infections by carbapenem-resistant Enterobacterial pathogens. Enterobacteriaceae carrying mobile colistin resistance (MCR) genes are rapidly emerging and threatening human health and food safety. Despite mcr-1 being prevalent in Escherichia coli, its dissemination in Salmonella is not well characterized. Herein, two unusual serotypes of colistin-resistant Salmonella isolates are reported first, namely serotype Ngor (ST5399) and Goldcoast (ST2529). Using whole genome sequencing, it is shown that mcr-1 is located on the IncHI2-like plasmid pTB501 (188,527 bp) of strain SSDFZ54 and the IncX4-type plasmid pTB602 (33,303 bp) in strain SSDFZ69, respectively. Furthermore, the backbone, tra- and antimicrobial resistance genes relative variable regions in the mcr-1-bearing IncHI2 plasmids are systematically characterized. Phylogenetic analysis shows that all IncHI2-type plasmids from non-Chinese regions are clustered together, suggesting a possible Chinese origin. Taken together, these findings extend the understanding of Salmonella as a vehicle of mcr-1 carriage and distribution.

Yeast Sphingolipid Phospholipase Gene ISC1 Regulates the Spindle Checkpoint by a CDC55-Dependent Mechanism.

Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, and GIM4) as synthetically lethal with ISC1 The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, and KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1 Reciprocally, ISC1 deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions.

A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation.

Nicotinamide adenine dinucleotide (NAD+) is an indispensable cofactor in all domains of life, and its homeostasis must be regulated tightly. Here we report that a Nudix-related transcriptional factor, designated MsNrtR (MSMEG_3198), controls the de novo pathway of NAD+biosynthesis in M. smegmatis, a non-tuberculosis Mycobacterium. The integrated evidence in vitro and in vivo confirms that MsNrtR is an auto-repressor, which negatively controls the de novo NAD+biosynthetic pathway. Binding of MsNrtR cognate DNA is finely mapped, and can be disrupted by an ADP-ribose intermediate. Unexpectedly, we discover that the acetylation of MsNrtR at Lysine 134 participates in the homeostasis of intra-cellular NAD+ level in M. smegmatis. Furthermore, we demonstrate that NrtR acetylation proceeds via the non-enzymatic acetyl-phosphate (AcP) route rather than by the enzymatic Pat/CobB pathway. In addition, the acetylation also occurs on the paralogs of NrtR in the Gram-positive bacterium Streptococcus and the Gram-negative bacterium Vibrio, suggesting that these proteins have a common mechanism of post-translational modification in the context of NAD+ homeostasis. Together, these findings provide a first paradigm for the recruitment of acetylated NrtR to regulate bacterial central NAD+ metabolism.

Functional definition of NrtR, a remnant regulator of NAD+ homeostasis in the zoonotic pathogen Streptococcus suis.

NAD+ is an enzyme cofactor required for the 3 domains of life. However, little is known about the NAD+ biosynthesis and salvage pathways in the opportunistic pathogen Streptococcus suis. A genome-wide search allows us to identify the NAD+ salvage pathway encoded by an operon of nadR-pnuC-nrtR (from SSU05_1973 to SSU05_1971 on the reverse strand) in the S. suis 05ZYH33 that causes streptococcal toxin shock-like syndrome. The regulator of this pathway is Nudix-related transcriptional regulator (NrtR), a transcription regulator of the Nudix family comprising an N-terminal Nudix-like effector domain, and a C-terminal DNA-binding winged helix-turn-helix-like domain. Intriguingly, the S. suis NrtR naturally contains a single amino acid substitution (K92E) in the catalytic site of its Nudix domain that renders it catalytically inactive but does not influence its ability to bind DNA. Despite its lack of enzymatic activity, DNA-binding activity of NrtR is antagonized by the effector ADP-ribose. Furthermore, nrtR knockout in S. suis serotype 2 reduces its capacity to form biofilms and attenuates its virulence in a mouse infection model. Genome mining indicates that nrtR appears in a strain-specific manner whose occupancy is correlated to bacterial infectivity. Unlike the paradigmatic member of NrtR family having 2 unrelated functions (Nudix hydrolase and DNA binding), S. suis 2 retains a single regulatory role in the modulation of NAD+ salvage. This control of NAD+ homeostasis contributes to S. suis virulence.-Wang, Q., Hassan, B. H., Lou, N., Merritt, J., Feng, Y. Functional definition of NrtR, a remnant regulator of NAD+ homeostasis in the zoonotic pathogen Streptococcus suis.

Formation of Arabidopsis Cryptochrome 2 photobodies in mammalian nuclei: application as an optogenetic DNA damage checkpoint switch.

Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA damage checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA damage signaling pathway in the absence of DNA damage.

Direct role for the replication protein treslin (Ticrr) in the ATR kinase-mediated checkpoint response.

TopBP1 (topoisomerase IIß-binding protein 1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA replication has been partially elucidated; however, its role in the checkpoint response remained elusive. In this study, we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin at Ser-1000 is important for its checkpoint activity. Overall, our results indicate that, like TopBP1, Treslin is a dual replication/checkpoint protein that directly participates in ATR-mediated checkpoint signaling.

Protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism.

Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification, the dehydrogenases are inactive, and aerobic metabolism is blocked. In Escherichia coli, two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, an S-adenosyl-L-methionine radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic, and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast, LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid-requiring enzyme of Escherichia coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains but that the enzymes that catalyze the assembly are also present "on site."

Engineering cooperative tecto-RNA complexes having programmable stoichiometries.

High affinity and specificity RNA-RNA binding interfaces can be constructed by combining pairs of GNRA loop/loop-receptor interaction motifs. These interactions can be fused using flexible four-way junction motifs to create divalent, self-assembling scaffolding units ('tecto-RNA') that have favorable properties for nanomedicine and other applications. We describe the design and directed assembly of tecto-RNA units ranging from closed, cooperatively assembling ring-shaped complexes of programmable stoichiometries (dimers, trimers and tetramers) to open multimeric structures. The novelty of this work is that tuning of the stoichiometries of self-assembled complexes is achieved by precise positioning of the interaction motifs in the monomer units rather than changing their binding specificities. Structure-probing and transmission electron microscopy studies as well as thermodynamic analysis support formation of closed cooperative complexes that are highly resistant to nuclease digestion. The present designs provide two helical arms per RNA monomer for further functionalization aims.