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Search Results: 1 - 10 of 1906 matches for " Milton Saier "
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Biophysical Studies of the Membrane-Embedded and Cytoplasmic Forms of the Glucose-Specific Enzyme II of the E. coli Phosphotransferase System (PTS)
Mohammad Aboulwafa, Milton H. Saier
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024088
Abstract: The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-IIGlc-YFP and MBP-IIGlc-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET-) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET+). The monomeric species could form a heterodimeric species (FRET+) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-IIGlc activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-IIGlc retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-IIGlc indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-IIGlc.
Functional Promiscuity of Homologues of the Bacterial ArsA ATPases
Rostislav Castillo,Milton H. Saier Jr.
International Journal of Microbiology , 2010, DOI: 10.1155/2010/187373
Abstract: The ArsA ATPase of E. coli plays an essential role in arsenic detoxification. Published evidence implicates ArsA in the energization of As(III) efflux via the formation of an oxyanion-translocating complex with ArsB. In addition, eukaryotic ArsA homologues have several recognized functions unrelated to arsenic resistance. By aligning ArsA homologues, constructing phylogenetic trees, examining ArsA encoding operons, and estimating the probable coevolution of these homologues with putative transporters and auxiliary proteins unrelated to ArsB, we provide evidence for new functions for ArsA homologues. They may play roles in carbon starvation, gas vesicle biogenesis, and arsenic resistance. The results lead to the proposal that ArsA homologues energize four distinct and nonhomologous transporters, ArsB, ArsP, CstA, and Acr3.
A Novel Mechanism of Transposon-Mediated Gene Activation
Zhongge Zhang,Milton H. Saier Jr
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000689
Abstract: Transposable Insertion Sequences (IS elements) have been shown to provide various benefits to their hosts via gene activation or inactivation under stress conditions by appropriately inserting into specific chromosomal sites. Activation is usually due to derepression or introduction of a complete or partial promoter located within the element. Here we define a novel mechanism of gene activation by the transposon IS5 in Escherichia coli. The glycerol utilization operon, glpFK, that is silent in the absence of the cAMP-Crp complex, is activated by IS5 when inserted upstream of its promoter. High-level expression is nearly constitutive, only mildly dependent on glycerol, glucose, GlpR, and Crp, and allows growth at a rate similar to or more rapid than that of wild-type cells. Expression is from the glpFK promoter and dependent on (1) the DNA phase, (2) integration host factor (IHF), and (3) a short region at the 3′ end of IS5 harboring a permanent bend and an IHF binding site. The lacZYA operon is also subject to such activation in the absence of Crp. Thus, we have defined a novel mechanism of gene activation involving transposon insertion that may be generally applicable to many organisms.
Human survival: evolution, religion and the irrational
Milton H. Saier, jr,Jack T. Trevors
Dialogues in Philosophy, Mental and Neuro Sciences , 2010,
Abstract: Is there a possible biological explanation for religion? That is, is there a genetic basis for believing in mystical, supernatural beings when there is no scientifi c evidence for their existence? Can we explain why some people prefer to accept myth over science? Why do so many people still accept creation and refuse to embrace evolution? Is there an evolutionary basis for religious beliefs? It is certainly true that religions have been part of human civilization throughout most of its recent history, at least for the last 5,000 years, and probably for much longer. Even great nonmystical philosophers such as Confucius, Buddha, and Lao Tzu have had their teachings evolve into mystical religions with spiritual ancestors, gods and reincarnation. On the other hand, religion is largely absent in modern Chinese culture, and of diminishing importance in Japanese and European cultures. In all cultures, the degrees of education gained by individuals correlate inversely with attachments to mystical deities. Atheists abound although they may be reluctant to come out of the closet and affi rm their rational convictions. In this article, we seek explanations for human irrationality.
Science, religious tolerance and freedom of expression
Jack T. Trevors,Milton H. Saier, jr.
Dialogues in Philosophy, Mental and Neuro Sciences , 2010,
Abstract: In this article we offer a perspective on the immense number of problems and challenges confronting humanity in our common biosphere. As our human population grows and urbanization increases globally, billions of humans with diverse beliefs and opinions are living in large urban areas without the basic needs of life. The way forward in our biosphere is not violence and disrespect. It is working to maintain and improve our common biosphere and solve our common global problems. Religion and religious believers will need science, so humans can survive and sustain our biosphere.
Functional Promiscuity of Homologues of the Bacterial ArsA ATPases
Rostislav Castillo,Milton H. Saier Jr.
International Journal of Microbiology , 2010, DOI: 10.1155/2010/187373
Abstract: The ArsA ATPase of E. coli plays an essential role in arsenic detoxification. Published evidence implicates ArsA in the energization of As(III) efflux via the formation of an oxyanion-translocating complex with ArsB. In addition, eukaryotic ArsA homologues have several recognized functions unrelated to arsenic resistance. By aligning ArsA homologues, constructing phylogenetic trees, examining ArsA encoding operons, and estimating the probable coevolution of these homologues with putative transporters and auxiliary proteins unrelated to ArsB, we provide evidence for new functions for ArsA homologues. They may play roles in carbon starvation, gas vesicle biogenesis, and arsenic resistance. The results lead to the proposal that ArsA homologues energize four distinct and nonhomologous transporters, ArsB, ArsP, CstA, and Acr3. 1. Introduction Arsenical species present threats to all organisms. The two predominant states of inorganic arsenic are arsenate [As(V)] and arsenite [As(III)]. As(V) disrupts the cellular energy machinery as a phosphate analog, uncoupling energy production. The even more toxic As(III) binds the sulfhydryl groups of cysteine residues and inactivates proteins [1]. A variety of defenses have evolved against arsenic toxicity. Plasmid- and chromosome-borne ars operons encode genes including arsA, -B, -C, -D, -H, -I, -P, and -R. arsC encodes an arsenate reductase that transfers electrons to As(V), reducing it to As(III), the efflux pump substrate [2]. ArsD is a metallochaperone which sequesters arsenite and antimonite and transfers them to the ArsA ATPase increasing the apparent affinity of ArsA for its substrates and lowering the concentration of free As(III) and Sb(III) in the cytosol [3]. ArsH, which is related to NADPH-dependent FMN reductases, has been implicated in arsenic resistance, but its precise biochemical function is not yet known [4]. arsP encodes a putative transmembrane permease which may play an ill-defined role in arsenic resistance in some organisms [5]. ArsB is a 12 -helix transmembrane spanning (TMS) pump extruding As(III) and Sb(III) [6]. Transport via ArsB can be energized by the pmf or by forming an oxyanion-translocating complex with the catalytic ArsA subunit, coupling ATP hydrolysis to efflux [7]. The characterized E. coli ArsA, a 583-amino acid (aa) ATPase, is composed of two homologous domains, A1 and A2, joined by a flexible linker. A1 and A2 are undoubtedly the products of a tandem intragenic duplication event. As(III) and Sb(III) form covalent bonds at the metal binding sites in A1 and A2, bringing the two
Genomic Analyses of Transport Proteins in Ralstonia metallidurans
Torsten von Rozycki,Dietrich H. Nies,Milton H. Saier Jr.
Comparative and Functional Genomics , 2005, DOI: 10.1002/cfg.454
Abstract: Ralstonia (Wautersia, Cupriavidus) metallidurans (Rme) is better able to withstand high concentrations of heavy metals than any other well-studied organism. This fact renders it a potential agent of bioremediation as well as an ideal model organism for understanding metal resistance phenotypes. We have analysed the genome of Rme for genes encoding homologues of established and putative transport proteins; 13% of all genes in Rme encode such homologues. Nearly one-third of the transporters identified (32%) appear to function in inorganic ion transport with three-quarters of these acting on cations. Transporters specific for amino acids outnumber sugar transporters nearly 3 : 1, and this fact plus the large number of uptake systems for organic acids indicates the heterotrophic preferences of these bacteria. Putative drug efflux pumps comprise 10% of the encoded transporters, but numerous efflux pumps for heavy metals, metabolites and macromolecules were also identified. The results presented should facilitate genetic manipulation and mechanistic studies of transport in this remarkable bacterium.
Bacterial Adaptor Membrane Fusion Proteins and the Structurally Dissimilar Outer Membrane Auxiliary Proteins Have Exchanged Central Domains in -Proteobacteria
Anthony Y. Xiao,Jing Wang,Milton H. Saier Jr.
International Journal of Microbiology , 2010, DOI: 10.1155/2010/589391
Abstract: Transport systems frequently include auxiliary proteins that perform subfunctions within the transporter protein complex. Two such proteins found in Gram-negative bacteria are the Membrane Fusion Proteins (MFPs) and the Outer Membrane Auxiliary (OMA) proteins. We here demonstrate that OMAs present in -proteobacteria (but not in other bacterial types) contain a long -helical region that is homologous to corresponding regions in the MFPs. The results suggest that during their evolution, OMAs, specifically from -proteobacteria, exchanged their own -helical domain for one derived from an MFP. The structural and functional implications of these findings are discussed.
Transcriptional Regulation of the rsbV Promoter Controlling Stress Responses to Ethanol, Carbon Limitation, and Phosphorous Limitation in Bacillus subtilis
Soo-Keun Choi,Milton H. Saier Jr.
International Journal of Microbiology , 2010, DOI: 10.1155/2010/263410
Abstract: The B-dependent promoter in front of the rsbV gene of Bacillus subtilis is induced ~5-fold in response to (1) the addition of 4% ethanol, (2) carbon starvation, and (3) phosphorous starvation. Binding sites for the global carbon and nitrogen regulators, CcpA and TnrA, were mutated, and the consequences of their loss and that of CcpA or TnrA were studied using rsbV-lacZ fusions. These responses proved to be dependent on CcpA, TnrA, and their putative binding sites upstream of the promoter. Induction in response to glucose limitation was largely abolished by loss of CcpA or the upstream region, while induction in response to phosphorous limitation was largely abolished only by the upstream mutations. The results suggest that CcpA directly influences the carbon starvation response and that both proteins exert indirect effects on all three stress responses. The integrity of the DNA sequence is important for all three responses.
A new direction for directed mutation?
Zhongge Zhang,Ming Ren Yen,Milton H. Saier Jr.
Trends in Evolutionary Biology , 2011, DOI: 10.4081/eb.2011.e3
Abstract: Directed mutation is a controversial process that allows mutations to occur at higher frequencies when they are beneficial. Here we review evidence for transposon-mediated directed mutation. crp deletion mutants (Glp–) of Escherichia coli (E. coli) mutate specifically to glycerol utilization (Glp+) at rates that are enhanced by glycerol or the loss of the glycerol repressor (GlpR), and depressed by glucose or glpR overexpression. Of the four tandem GlpRbinding sites (O1-O4), O4 specifically controls glpFK expression while O1 controls mutation rate. Mutation is due to insertion of the IS5 transposon into a specific site upstream of the glpFK promoter. Mutational control by GlpR is independent of the selection and assay procedures, and IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish an example of transposon-mediated directed mutation, identify the protein responsible for its regulation, and define essential features of the mechanism involved. We discuss this phenomenon from an evolutionary standpoint and provide examples of analogous switch mechanisms that may or may not be directed.
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