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Berlin Brandenburg

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  • 1
    Language: English
    In: Journal of bacteriology, March 2013, Vol.195(5), pp.965-76
    Description: Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.
    Keywords: Alcohol Oxidoreductases -- Genetics ; Francisella Tularensis -- Enzymology ; Pantothenic Acid -- Biosynthesis
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 2
    Language: English
    In: Applied and environmental microbiology, October 2012, Vol.78(19), pp.6883-9
    Description: There are a number of genetic tools available for studying Francisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for F. tularensis based on the Tet repressor, TetR. For the inducible system, a tet operator sequence was cloned into a modified F. tularensis groESL promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon, luxCDABE, was cloned under the hybrid Francisella tetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with tetO. Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml(-1). In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of FTRp regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the lux reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring F. tularensis protein function.
    Keywords: Gene Expression Regulation, Bacterial ; Francisella Tularensis -- Genetics ; Genetic Engineering -- Methods ; Transcription Factors -- Genetics
    E-ISSN: 1098-5336
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 3
    Language: English
    In: Journal of Bacteriology, March, 2012, Vol.194(5-6), p.1474(11)
    Description: Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1[beta] (IL-1[beta]) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function. doi: 10.1128/JB.06327-11
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 4
    Language: English
    In: Journal of Bacteriology, 2012, Vol. 194(6), p.1474
    Description: Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1β (IL-1β) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function. ; p. 1474-1484.
    Keywords: Membrane Proteins ; Secretion ; Amino Acid Substitution ; Tularemia ; Crosslinking ; Francisella Tularensis ; Etiological Agents ; Amino Acids ; Interleukin-1beta ; Cytoplasm ; Fluorescent Proteins ; Topology ; Macrophages ; Cell Membranes;
    ISSN: 1098-5530
    ISSN: 10985530
    ISSN: 00219193
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  • 5
    Language: English
    In: Infection and Immunity, 2010, Vol. 78(12), p.5022
    ISSN: 0019-9567
    ISSN: 00199567
    Source: American Society of Microbiology
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  • 6
    Language: English
    In: Infection and immunity, June 2013, Vol.81(6), pp.2028-42
    Description: Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.
    Keywords: Bacterial Vaccines -- Immunology ; Francisella Tularensis -- Genetics ; Tularemia -- Prevention & Control
    E-ISSN: 1098-5522
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 7
    In: The Journal of Bacteriology, 1999, Vol. 181(3), p.941
    Description: One of life's inevitable disappointments—one felt often by scientists and artists, but not only by them—comes from ex- pecting others to share the particularities of one's own sense of awe and wonder. This truth came home to me recently when I picked up Michael Guillen's fine book Five Equations That Changed the World (4) and discovered that my equation—the one that shaped my scientific career—was not considered one of the five. I met this equation in the winter of 1952-1953 when Eman- uel Suter, the bacteriology and immunology instructor in the integrated Medical Sciences program at Harvard Medical School, brought three very young colleagues to help teach the instructional laboratory of this innovative course. In this way we 20 privileged students met Boris Magasanik, Marcus Brooke, and H. Edwin Umbarger and were plunged into bac- terial physiology. They had designed a laboratory experience to introduce us to contemporary issues and cutting-edge techniques in 1950s bacterial physiology. As I remember, one objective was to study diauxic growth by varying the limiting amount of glucose added to a minimal medium containing a secondary carbon source and inoculated with an enteric bacterium. A second objective was to construct the steps in a biosynthetic pathway by examining the abilities of various compounds to satisfy the nutritional needs of auxotrophic mutants. Both experiments required measuring the growth of bacteria, the former as a kinetic process. For me, encountering the bacterial growth curve was a trans- forming experience. As my partner and I took samples of the culture at intervals to measure optical density and plotted the results on semilogarithmic paper, we saw, after the lag period, a straight line developing. . .beautiful in precision and remark- able in speed. As the line extended itself straight-edge true, I imagined what was happening in the flask—living protoplasm being made from glucose and salts as the initial cells (Klebsiella aerogenes, they were called then) grew and divided. The liquid in the flask progressed from having a barely discernible haze to a milky whiteness thick with the stuff of life, all within a very brief Boston winter afternoon. Mutably specific autocatalysis, the physicist Erwin Schrodinger had declared a few years ear- lier (28), was the defining characteristic of living systems, and I had just witnessed the working out of the mathematical state- ment of that property, dN/dt 5 kN (where N is the number of cells or any extensive property thereof, t is time, and k is the first-order rate constant (in reciprocal time units)). I had never before seen such a clear display of autocatalysis. Its mathematical elegance and simplicity—but more impor- tantly, its invitation to explore—affected me profoundly. The first-order rate constant k in the growth equation seemed to me the ideal tool by which to assess the state of a culture of cells, i.e., the rate at which they were performing life, as it were. I elected to pursue my Ph.D. studies with Boris Ma- gasanik, studying the molecular basis of diauxic growth. Over the ensuing half-century, close analysis of growth curves was to be a central feature of my work, as I followed my intense curiosity (read obsession) about the processes that form living matter from salts and sugar. Catabolite repression, the growth rate-related regulation of stable RNA synthesis, the isolation and use of temperature-sensitive mutants in essential functions (particularly aminoacyl-tRNA synthetases), and the molecular responses of bacteria to heat and other stresses—all these studies depended on inferences and deductions from the growth behavior of bacterial cultures. For anyone interested in the synthesis of protoplasm, bac- teria are the system to study (reviewed in reference 17). With four billion years of practice they have perfected the art of growing in many environments, and they outclass all other known forms of life in their rate of metabolism geared for autocatalysis. The first-order rate constant k is most conve-
    Keywords: Biology;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 8
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(12), p.e115225
    Description: The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an "obesogenic" bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Journal of Biological Chemistry, 09/11/1998, Vol.273(37), pp.24030-24036
    Description: H-NS is an Escherichia coli nucleoid protein known only to function as a modulator of gene expression. In this study, we found that specific single amino acid substitutions in H-NS caused an approximately 50% increase in flagellum rotational speed. In fluorescence anisotropy and chemical cross-linking assays, H-NS interacted with the flagellar torque-generating rotor protein FliG to form a complex with a Kd of 2.15 microM. Furthermore, one of the altered H-NS proteins that exhibited high speed flagellum rotation bound FliG 50% tighter than wild-type H-NS. These results demonstrate the first non-regulatory role for H-NS and provide a direct correlation between H-NS-FliG binding affinities, flagellar rotation, and motor torque generation.
    Keywords: Chemistry ; Anatomy & Physiology;
    ISSN: 0021-9258
    E-ISSN: 1083-351X
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  • 10
    Language: English
    In: eLife, 23 January 2016, Vol.5
    Description: Macrophages are myeloid-derived phagocytic cells and one of the first immune cell types to respond to microbial infections. However, a number of bacterial pathogens are resistant to the antimicrobial activities of macrophages and can grow within these cells. Macrophages have other immune surveillance roles including the acquisition of cytosolic components from multiple types of cells. We hypothesized that intracellular pathogens that can replicate within macrophages could also exploit cytosolic transfer to facilitate bacterial spread. We found that viable Francisella tularensis, as well as Salmonella enterica bacteria transferred from infected cells to uninfected macrophages along with other cytosolic material through a transient, contact dependent mechanism. Bacterial transfer occurred when the host cells exchanged plasma membrane proteins and cytosol via a trogocytosis related process leaving both donor and recipient cells intact and viable. Trogocytosis was strongly associated with infection in mice, suggesting that direct bacterial transfer occurs by this process in vivo.
    Keywords: Cell to Cell Spread ; Francisella Tularensis ; Immunology ; Infectious Disease ; Microbiology ; Mouse ; Salmonella Typhimurium ; Trogocytosis ; Cell Communication ; Cytoplasm -- Microbiology ; Francisella Tularensis -- Isolation & Purification ; Immunological Synapses -- Microbiology ; Macrophages -- Immunology ; Salmonella Enterica -- Isolation & Purification
    E-ISSN: 2050-084X
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