Publications by authors named "William K Russell"

78 Publications

Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cell Rep 2021 Apr;35(3):109009

Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.
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http://dx.doi.org/10.1016/j.celrep.2021.109009DOI Listing
April 2021

Poly(ADP-Ribose) polymerase 1 regulates mitochondrial DNA repair in a metabolism-dependent manner.

J Biol Chem 2021 Jan 19:100309. Epub 2021 Jan 19.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA; Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX 77555, USA; Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address:

Mitochondral DNA is located in organelle that house essential metablic reactions and contain high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1 and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD, PARP1 completely inhibits Pol γ, while increasing NAD levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.
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http://dx.doi.org/10.1016/j.jbc.2021.100309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949115PMC
January 2021

Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination.

Mol Cell 2020 10 22;80(2):345-358.e9. Epub 2020 Sep 22.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX 77550, USA. Electronic address:

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.
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http://dx.doi.org/10.1016/j.molcel.2020.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660970PMC
October 2020

A protumorigenic secretory pathway activated by p53 deficiency in lung adenocarcinoma.

J Clin Invest 2021 Jan;131(1)

Department of Thoracic/Head and Neck Medical Oncology, the University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)-myosin IIA-containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a-dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8+ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.
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http://dx.doi.org/10.1172/JCI137186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773359PMC
January 2021

Annexin A2 depletion exacerbates the intracerebral microhemorrhage induced by acute rickettsia and Ebola virus infections.

PLoS Negl Trop Dis 2020 07 20;14(7):e0007960. Epub 2020 Jul 20.

Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections.
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http://dx.doi.org/10.1371/journal.pntd.0007960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392349PMC
July 2020

A cocrystal structure of dengue capsid protein in complex of inhibitor.

Proc Natl Acad Sci U S A 2020 07 15;117(30):17992-18001. Epub 2020 Jul 15.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555;

Dengue virus (DENV) was designated as a top 10 public health threat by the World Health Organization in 2019. No clinically approved anti-DENV drug is currently available. Here we report the high-resolution cocrystal structure (1.5 Å) of the DENV-2 capsid protein in complex with an inhibitor that potently suppresses DENV-2 but not other DENV serotypes. The inhibitor induces a "kissing" interaction between two capsid dimers. The inhibitor-bound capsid tetramers are assembled inside virions, resulting in defective uncoating of nucleocapsid when infecting new cells. Resistant DENV-2 emerges through one mutation that abolishes hydrogen bonds in the capsid structure, leading to a loss of compound binding. Structure-based analysis has defined the amino acids responsible for the inhibitor's inefficacy against other DENV serotypes. The results have uncovered an antiviral mechanism through inhibitor-induced tetramerization of the viral capsid and provided essential structural and functional knowledge for rational design of panserotype DENV capsid inhibitors.
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http://dx.doi.org/10.1073/pnas.2003056117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395448PMC
July 2020

JAK2 regulates Nav1.6 channel function via FGF14 phosphorylation.

Biochim Biophys Acta Mol Cell Res 2020 10 26;1867(10):118786. Epub 2020 Jun 26.

Department of Pharmacology & Toxicology, The University of Texas Medical Branch, Galveston, TX, USA; Center for Addiction Research, The University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

Background: Protein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons.

Methods: Here we present results from an in-cell high-throughput screening (HTS) against the FGF14: Nav1.6 complex using >3000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology.

Results: Compounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14, a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14 mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14.

Conclusions: These studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984254PMC
October 2020

PI4KIIIβ is a therapeutic target in chromosome 1q-amplified lung adenocarcinoma.

Sci Transl Med 2020 01;12(527)

Department of Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.
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http://dx.doi.org/10.1126/scitranslmed.aax3772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702266PMC
January 2020

Peptidoglycan-Associated Cyclic Lipopeptide Disrupts Viral Infectivity.

J Virol 2019 11 29;93(22). Epub 2019 Oct 29.

Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, Texas, USA

Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection. In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.
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http://dx.doi.org/10.1128/JVI.01282-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819921PMC
November 2019

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

Plant J 2018 04 12;94(1):131-145. Epub 2018 Mar 12.

Department of Horticultural Sciences, Texas A&M University, College Station, TX, 77843, USA.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.
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http://dx.doi.org/10.1111/tpj.13847DOI Listing
April 2018

Influence of water and enzyme SpnF on the dynamics and energetics of the ambimodal [6+4]/[4+2] cycloaddition.

Proc Natl Acad Sci U S A 2018 01 18;115(5):E848-E855. Epub 2018 Jan 18.

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569;

SpnF is the first monofunctional Diels-Alder/[6+4]-ase that catalyzes a reaction leading to both Diels-Alder and [6+4] adducts through a single transition state. The environment-perturbed transition-state sampling method has been developed to calculate free energies, kinetic isotope effects, and quasi-classical reaction trajectories of enzyme-catalyzed reactions and the uncatalyzed reaction in water. Energetics calculated in this way reproduce the experiment and show that the normal Diels-Alder transition state is stabilized by H bonds with water molecules, while the ambimodal transition state is favored in the enzyme SpnF by both intramolecular hydrogen bonding and hydrophobic binding. Molecular dynamics simulations show that trajectories passing through the ambimodal transition state bifurcate to the [6+4] adduct and the Diels-Alder adduct with a ratio of 1:1 in the gas phase, 1:1.6 in water, and 1:11 in the enzyme. This example shows how an enzyme acts on a vibrational time scale to steer post-transition state trajectories toward the Diels-Alder adduct.
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http://dx.doi.org/10.1073/pnas.1719368115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798381PMC
January 2018

Lipofuscin Formation Catalyzed by the Milk Protein β-Lactoglobulin: Lysine Residues in Cycloretinal Synthesis.

Biochemistry 2017 10 10;56(43):5715-5719. Epub 2017 Oct 10.

Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.

Lipofuscins are toxic autofluorescent byproducts of the visual cycle. The accumulation of lipofuscins such as cycloretinal in the retina is thought to play a role in the progression of age-related macular degeneration (AMD). Intriguingly, the milk protein β-lactoglobulin (BLG) can promote the cyclodimerization of all-trans-retinal to cycloretinal both in vitro and in vivo. Here, site-directed mutagenesis of BLG and mass spectrometric analysis with substrate analogues demonstrate that lysine residues play a key role in catalysis. It is also shown that catalytic activity necessitates the presence of a physical binding site and cannot be mediated by a peptide chain. These studies provide insight into the mechanism of the cyclodimerization process and provide a model system for biocatalysis and biosynthesis of cycloretinal in vivo. In the long term, these studies may pave the way for drug development and inhibitor design as an early treatment regimen for AMD.
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http://dx.doi.org/10.1021/acs.biochem.7b00709DOI Listing
October 2017

Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A.

Proc Natl Acad Sci U S A 2017 09 5;114(39):10408-10413. Epub 2017 Sep 5.

Department of Chemistry, University of Texas at Austin, Austin, TX 78712;

The Diels-Alder reaction is one of the most common methods to chemically synthesize a six-membered carbocycle. While it has long been speculated that the cyclohexene moiety found in many secondary metabolites is also introduced via similar chemistry, the enzyme SpnF involved in the biosynthesis of the insecticide spinosyn A in is the first enzyme for which catalysis of an intramolecular [Formula: see text]-cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone [Formula: see text]-cyclases have been reported as potential Diels-Alderases; however, whether their catalytic cycles involve a concerted or stepwise cyclization mechanism has not been addressed experimentally. Here, we report direct experimental interrogation of the reaction coordinate for the [Formula: see text]-carbocyclase SpnF via the measurement of [Formula: see text]-secondary deuterium kinetic isotope effects (KIEs) at all sites of [Formula: see text] rehybridization for both the nonenzymatic and enzyme-catalyzed cyclization of the SpnF substrate. The measured KIEs for the nonenzymatic reaction are consistent with previous computational results implicating an intermediary state between formation of the first and second carbon-carbon bonds. The KIEs measured for the enzymatic reaction suggest a similar mechanism of cyclization within the enzyme active site; however, there is evidence that conformational restriction of the substrate may play a role in catalysis.
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http://dx.doi.org/10.1073/pnas.1710496114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625926PMC
September 2017

The multicomponent antirestriction system of phage P1 is linked to capsid morphogenesis.

Mol Microbiol 2017 Aug 29;105(3):399-412. Epub 2017 May 29.

Center for Phage Technology, Texas A-M University, College Station, TX, 77843, USA.

Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to reconstitute the previously described restriction phenotypes associated with darA and darB. While P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype, implicating several new proteins of previously unknown function in the P1 dar antirestriction system. Interestingly, disruption of ddrB decreased P1's sensitivity to EcoB and EcoK restriction. Proteomic analysis of purified virions suggests that packaging of antirestriction components into P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in addition to DarA and DarB.
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http://dx.doi.org/10.1111/mmi.13705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6011833PMC
August 2017

Grouping of Petroleum Substances as Example UVCBs by Ion Mobility-Mass Spectrometry to Enable Chemical Composition-Based Read-Across.

Environ Sci Technol 2017 Jun 26;51(12):7197-7207. Epub 2017 May 26.

Department of Veterinary Integrative Biosciences, Texas A&M University , College Station, Texas 77843-4461, United States.

Substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs), including many refined petroleum products, present a major challenge in regulatory submissions under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and US High Production Volume regulatory regimes. The inherent complexity of these substances, as well as variability in composition obfuscates detailed chemical characterization of each individual substance and their grouping for human and environmental health evaluation through read-across. In this study, we applied ion mobility mass spectrometry in conjunction with cheminformatics-based data integration and visualization to derive substance-specific signatures based on the distribution and abundance of various heteroatom classes. We used petroleum substances from four petroleum substance manufacturing streams and evaluated their chemical composition similarity based on high-dimensional substance-specific quantitative parameters including m/z distribution, drift time, carbon number range, and associated double bond equivalents and hydrogen-to-carbon ratios. Data integration and visualization revealed group-specific similarities for petroleum substances. Observed differences within a product group were indicative of batch- or manufacturer-dependent variation. We demonstrate how high-resolution analytical chemistry approaches can be used effectively to support categorization of UVCBs based on their heteroatom composition and how such data can be used in regulatory decision-making.
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http://dx.doi.org/10.1021/acs.est.6b06413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627358PMC
June 2017

Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection.

Cell Host Microbe 2017 May;21(5):637-649.e6

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, College Station, Texas 77843, USA; Norman Borlaug Center, Texas A&M University, College Station, Texas 77843, USA; Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA. Electronic address:

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.
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http://dx.doi.org/10.1016/j.chom.2017.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538893PMC
May 2017

CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells.

Protein Expr Purif 2016 12 19;128:101-8. Epub 2016 Aug 19.

Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT.
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http://dx.doi.org/10.1016/j.pep.2016.08.011DOI Listing
December 2016

Polyketide Ring Expansion Mediated by a Thioesterase, Chain Elongation and Cyclization Domain, in Azinomycin Biosynthesis: Characterization of AziB and AziG.

Biochemistry 2016 Feb 19;55(4):704-14. Epub 2016 Jan 19.

Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.

The azinomycins are a family of potent antitumor agents with the ability to form interstrand cross-links with DNA. This study reports on the unusual biosynthetic formation of the 5-methyl naphthoate moiety, which is essential for effective DNA association. While sequence analysis predicts that the polyketide synthase (AziB) catalyzes the formation of this naphthoate, 2-methylbenzoic acid, a truncated single-ring product, is formed instead. We demonstrate that the thioesterase (AziG) acts as a chain elongation and cyclization (CEC) domain and is required for the additional two rounds of chain extension to form the expected product.
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http://dx.doi.org/10.1021/acs.biochem.5b01050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4738070PMC
February 2016

Subunit Interactions within the Carbon-Phosphorus Lyase Complex from Escherichia coli.

Biochemistry 2015 Jun 19;54(21):3400-11. Epub 2015 May 19.

Phosphonates are a large class of organophosphorus compounds with a characteristic carbon-phosphorus bond. The genes responsible for phosphonate utilization in Gram-negative bacteria are arranged in an operon of 14 genes. The carbon-phosphorus lyase complex, encoded by the genes phnGHIJKLM, catalyzes the cleavage of the stable carbon-phosphorus bond of organophosphonates to the corresponding hydrocarbon and inorganic phosphate. Recently, complexes of this enzyme containing five subunits (PhnG-H-I-J-K), four subunits (PhnG-H-I-J), and two subunits (PhnG-I) were purified after expression in Escherichia coli ( Proc. Natl. Acad. Sci., U. S. A. 2011 , 108 , 11393 ). Here we demonstrated using mass spectrometry, ultracentrifugation, and chemical cross-linking experiments that these complexes are formed from a PhnG2I2 core that is further elaborated by the addition of two copies each of PhnH and PhnJ to generate PhnG2H2I2J2. This complex adds an additional subunit of PhnK to form PhnG2H2I2J2K. Chemical cross-linking of the five-component complex demonstrated that PhnJ physically interacts with both PhnG and PhnI. We were unable to demonstrate the interaction of PhnH or PhnK with any other subunits by chemical cross-linking. Hydrogen-deuterium exchange was utilized to probe for alterations in the dynamic properties of individual subunits within the various complexes. Significant regions of PhnG become less accessible to hydrogen/deuterium exchange from solvent within the PhnG2I2 complex compared with PhnG alone. Specific regions of PhnI exhibited significant differences in the H/D exchange rates in PhnG2I2 and PhnG2H2I2J2K.
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http://dx.doi.org/10.1021/acs.biochem.5b00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480679PMC
June 2015

Fluorescent probes for tracking the transfer of iron-sulfur cluster and other metal cofactors in biosynthetic reaction pathways.

J Am Chem Soc 2015 Jan 24;137(1):390-8. Epub 2014 Dec 24.

Department of Biochemistry and Biophysics and ‡Department of Chemistry, Texas A&M University , College Station, Texas 77842-3012, United States.

Iron-sulfur (Fe-S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe-S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe-S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe-2S] and [4Fe-4S] clusters), ligand environments ([2Fe-2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe-S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe-S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe-2S]-DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe-S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe-S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. We anticipate that this cluster detection methodology will transform the study of Fe-S cluster pathways and potentially other metal cofactor biosynthetic pathways.
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http://dx.doi.org/10.1021/ja510998sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675328PMC
January 2015

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Plant J 2014 Oct 5;80(1):27-39. Epub 2014 Aug 5.

Molecular and Environmental Plant Sciences, Department of Horticultural Sciences, Vegetable and Fruit Development Center, Texas A&M University, College Station, TX, 77843, USA.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.
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http://dx.doi.org/10.1111/tpj.12612DOI Listing
October 2014

Mechanistic consequences of chiral radical clock probes: analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates.

J Am Chem Soc 2014 Feb 13;136(8):2944-7. Epub 2014 Feb 13.

Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, University of Texas at Austin , Austin, Texas 78712, United States.

(S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.
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http://dx.doi.org/10.1021/ja4100035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004275PMC
February 2014

Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

PLoS One 2013 26;8(11):e80509. Epub 2013 Nov 26.

Department of Horticultural Sciences, Texas A&M University, College Station, Texas, United States of America ; Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Korea.

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080509PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841200PMC
October 2014

Crystal structure of Mycobacterium tuberculosis polyketide synthase 11 (PKS11) reveals intermediates in the synthesis of methyl-branched alkylpyrones.

J Biol Chem 2013 Jun 24;288(23):16484-16494. Epub 2013 Apr 24.

Departments of Biochemistry and Biophysics, College Station, Texas 77843. Electronic address:

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.
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http://dx.doi.org/10.1074/jbc.M113.468892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675584PMC
June 2013

Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein.

Anal Chem 2013 Mar 6;85(6):3229-37. Epub 2013 Mar 6.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Metalation and demetalation of human metallothionein-2A (MT) with Cd(2+) is investigated by using chemical labeling and "bottom-up" and "top-down" proteomics approaches. Both metalation and demetalation of MT-2A by Cd(2+) are shown to be domain specific and occur as two distinct processes. Metalation involves sequential addition of Cd(2+) to the α-domain resulting in formation of an intermediate, Cd4MT. Chemical labeling with N-ethylmaleimide (NEM) and tandem mass spectrometry experiments clearly show that the four metal ions are located in the α-domain. In the presence of excess Cd(2+), the Cd4MT intermediate reacts to add Cd(2+) to the β-domain to yield the fully metalated Cd7MT. Demetalation occurs in the reverse order, i.e., Cd(2+) is removed (by EDTA) first from the β-domain followed by Cd(2+) removal from the α-domain. Metalation of human MT-2A is shown to be metal ion specific by comparing relative metal ion binding constants for Cd(2+) and Zn(2+).
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http://dx.doi.org/10.1021/ac303522hDOI Listing
March 2013

CAPA-gene products in the haematophagous sandfly Phlebotomus papatasi (Scopoli)--vector for leishmaniasis disease.

Peptides 2013 Mar 21;41:2-7. Epub 2012 Dec 21.

Zoological Institute, Biocenter University of Cologne, Cologne, Germany.

Sandflies (Phlebotominae, Nematocera, Diptera) are responsible for transmission of leishmaniasis and other protozoan-borne diseases in humans, and these insects depend on the regulation of water balance to cope with the sudden and enormous intake of blood over a very short time period. The sandfly inventory of neuropeptides, including those that regulate diuretic processes, is completely unknown. Direct MALDI-TOF/TOF mass spectrometric analysis of dissected ganglia of Phlebotomus papatasi, combined with a data-mining of sandfly genome 'contigs', was used to identify native CAPA-peptides, a peptide class associated with the regulation of diuresis in other hematophagous insects. The CAPA-peptides identified in this study include two CAPA-PVKs, differentially processed CAPA-PK, and an additional CAPA precursor peptide. The mass spectrometric analysis of different parts of the neuroendocrine system of the sandfly indicate that it represents the first insect which accumulates CAPA-PVKs exclusively in hormone release sites of abdominal ganglia and CAPA-PK (nearly) exclusively in the corpora cardiaca. Additionally, sandflies feature the smallest abdominal ganglia (~35 μm) where CAPA-peptides could be detected so far. The small size of the abdominal ganglia does not appear to affect the development of the median neurosecretory system as it obviously does in another comparably small insect species, Nasonia vitripennis, in which no capa-gene expression was found. Rather, immunocytochemical analyses confirm that the general architecture in sandflies appears identical to that of much larger mosquitoes.
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http://dx.doi.org/10.1016/j.peptides.2012.12.009DOI Listing
March 2013

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage.

BMC Genomics 2012 Oct 10;13:542. Epub 2012 Oct 10.

Center for Phage Technology, 2128 TAMU, Texas A&M University, College Station, Texas, TX 77843, USA.

Background: The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives.

Results: Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell's replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level.

Conclusions: Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus.
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http://dx.doi.org/10.1186/1471-2164-13-542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556154PMC
October 2012

Proteomic methods for biomarker discovery in a rat model of alcohol steatosis.

Methods Mol Biol 2012 ;909:259-77

Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX, USA.

Understanding changes in the expression of specific proteins and/or alterations in their posttranslational modifications is crucial to elucidating the molecular mechanisms underlying disease states such as alcoholic liver disease. Protein separation and analysis techniques such as two-dimensional electrophoresis and mass spectrometry can be used for identifying biomarker proteins that are altered during progression of alcoholic liver disease. In this chapter, we outline methods for resolving liver tissue proteins from a rodent model of alcoholic liver disease using two-dimensional electrophoresis and identifying differentially expressed proteins using mass spectrometry. In addition, since oxidative stress strongly correlates with alcoholic liver disease, we also describe methods for identifying oxidatively modified proteins from liver tissue. We specifically focus on identifying proteins that are carbonylated as protein carbonylation is a permanent modification and considered deleterious to cells. The combination of two-dimensional electrophoresis for protein resolution, mass spectrometry for protein identification, and affinity-based methods for enriching and identifying carbonylated proteins is a powerful methodology for protein biomarker identification.
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http://dx.doi.org/10.1007/978-1-61779-959-4_17DOI Listing
December 2012

Imaging secondary metabolism of Streptomyces sp. Mg1 during cellular lysis and colony degradation of competing Bacillus subtilis.

Antonie Van Leeuwenhoek 2012 Oct 10;102(3):435-45. Epub 2012 Jul 10.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, USA.

Soil streptomycetes are saprotrophic bacteria that secrete numerous secondary metabolites and enzymes for extracellular functions. Many streptomycetes produce antibiotics thought to protect vegetative mycelia from competing organisms. Here we report that an organism isolated from soil, Streptomyces sp. Mg1, actively degrades colonies and causes cellular lysis of Bacillus subtilis when the organisms are cultured together. We predicted that the inhibition and degradation of B. subtilis colonies in this competition depends upon a combination of secreted factors, including small molecule metabolites and enzymes. To begin to unravel this complex competitive phenomenon, we use a MALDI imaging mass spectrometry strategy to map the positions of metabolites secreted by both organisms. In this report, we show that Streptomyces sp. Mg1 produces the macrolide antibiotic chalcomycin A, which contributes to inhibition of B. subtilis growth in combination with other, as yet unidentified factors. We suggest that efforts to understand competitive and cooperative interactions between bacterial species benefit from assays that pair living organisms and probe the complexity of metabolic exchanges between them.
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http://dx.doi.org/10.1007/s10482-012-9769-0DOI Listing
October 2012