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  • 1
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 192, No. 6 ( 2010-03-15), p. 1634-1642
    Abstract: The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ . Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
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  • 2
    In: mBio, American Society for Microbiology, Vol. 2, No. 5 ( 2011-11)
    Abstract: Bacteria use RNA to regulate gene expression by a variety of mechanisms. The prgQ and prgX operons of pCF10, a conjugative plasmid of Enterococcus faecalis , have been shown to negatively regulate one another by a variety of mechanisms. One of these mechanisms involves Anti-Q, a small RNA from the prgX operon that prevents gene expression from the prgQ operon. In this work, we find that Qs, an RNA from the prgQ operon, negatively regulates gene expression from the prgX operon. These findings have a number of implications. (i) The Anti-Q and Qs RNAs act by different mechanisms, highlighting the variety of ways in which bacteria can regulate gene expression using RNAs. (ii) Reciprocal regulation between operons mediated by small RNAs has not been previously described, deepening our understanding of how bacteria regulate complex behavior. (iii) Additional roles for Qs have been described, demonstrating the versatility of this RNA.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
    detail.hit.zdb_id: 2557172-2
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  • 3
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 199, No. 12 ( 2017-06-15)
    Abstract: Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis , designated pCIE, utilizing the P Q pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis , par pAD1 of the pAD1 plasmid and par EF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalis . IMPORTANCE E. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the P Q pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2017
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 4
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 63, No. 4 ( 2019-04)
    Abstract: AIDS Clinical Trial Group study A5202 (ClinicalTrials.gov identifier NCT00118898) was a phase 3b, randomized, partially blinded equivalence study of open-label atazanavir/ritonavir or efavirenz, plus either placebo-controlled tenofovir disoproxil fumarate/emtricitabine or abacavir/lamivudine, in treatment-naive adults living with HIV-1, evaluating efficacy, safety, and tolerability. We report an analysis of the contribution of participant characteristics to the disposition of tenofovir plasma concentrations. Tenofovir concentration data from a total of 817 individuals (88% of the total number of eligible patients randomly assigned to receive treatment in the TDF-containing arms of A5202) were available for analysis. Pharmacokinetic analysis was performed using nonlinear mixed-effects modeling. One- and two-compartment models with first-order absorption and first-order elimination were evaluated. An exponential error model was used for examination of interindividual variability (IIV), and a proportional and mixed-error model was assessed for residual variability. The final structural model contained two compartments with first-order absorption and elimination. IIV was estimated for apparent clearance (CL/F) and the first-order absorption rate constant ( k a ), and a proportional residual variability model was selected. The final mean parameter estimates were as follows: k a = 2.87 h −1 , CL/F = 37.2 liters/h, apparent volumes of the central and peripheral compartments = 127 and 646 liters, respectively, and apparent intercompartmental clearance = 107 liters/h. In addition to race/ethnicity, creatinine clearance and assignment to atazanavir/ritonavir or efavirenz were significantly associated with CL/F ( P 〈 0.001). In conclusion, race/ethnicity is associated with tenofovir oral CL in HIV-1 positive, treatment-naive adults. This covariate relationship raises questions about the possibility of differences in efficacy and risk of adverse events in different patient populations and suggests that examining preexposure prophylaxis regimens and tenofovir exposure in different race/ethnicity groups be considered.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
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    SSG: 12
    SSG: 15,3
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  • 5
    In: Journal of Virology, American Society for Microbiology, Vol. 79, No. 13 ( 2005-07), p. 8131-8141
    Abstract: Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8 + lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Δ3 of CD8 + lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8 + lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8 + cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8 + T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01 − animals. Consistent with these findings, the depletion of CD8 + lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01 + than in Mamu-A*01 − animals. The partial control of postchallenge viremia after CD8 + lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2005
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  • 6
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 87, No. 5 ( 2021-02-12)
    Abstract: This study describes three closely related proteins cloned from Brevibacillus laterosporus strains that are lethal upon feeding to Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR). Mpp75Aa1, Mpp75Aa2, and Mpp75Aa3 were toxic to WCR larvae when the larvae were fed purified protein. Transgenic plants expressing each mMpp75Aa protein were protected from feeding damage and showed a significant reduction in adult emergence from infested plants by both susceptible Cry3Bb1 and Cry34Ab1/Cry35Ab1-resistant WCR. These results demonstrate that proteins from B. laterosporus are as efficacious as the well-known Bacillus thuringiensis insecticidal proteins in controlling major insect pests such as WCR. The deployment of transgenic maize expressing mMpp75Aa, along with other active molecules lacking cross-resistance, has the potential to be a useful tool for control of WCR populations resistant to current B. thuringiensis traits. IMPORTANCE Insects feeding on roots of crops can damage the plant roots, resulting in yield loss due to poor water and nutrient uptake and plant lodging. In maize, the western corn rootworm (WCR) can cause severe damage to the roots, resulting in significant economic loss for farmers. Genetically modified (GM) plants expressing Bacillus thuringiensis insect control proteins have provided a solution for control of these pests. In recent years, populations of WCR resistant to the B. thuringiensis proteins in commercial GM maize have emerged. There is a need to develop new insecticidal traits for the control of WCR populations resistant to current commercial traits. New proteins with commercial-level efficacy on WCR from sources other than B. thuringiensis are becoming more critical. The Mpp75Aa proteins from B. laterosporus , when expressed in maize, are efficacious against the resistant populations of WCR and have the potential to provide solutions for control of resistant WCR.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
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    SSG: 12
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  • 7
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 52, No. 5 ( 2014-05), p. 1617-1621
    Abstract: Tigecycline is one of the few remaining therapeutic options for extensively drug-resistant (XDR) Gram-negative bacilli (GNB). MICs of tigecycline to Acinetobacter baumannii have been reported to be elevated when determined by the Etest compared to determinations by the broth microdilution (BMD) method. The study aim was to compare the susceptibility of GNB to tigecycline by four different testing methods. GNB were collected from six health care systems (25 hospitals) in southeast Michigan from January 2010 to September 2011. Tigecycline MICs among A. baumannii , carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae , and susceptible Enterobacteriaceae isolates were determined by Etest, BMD, Vitek-2, and MicroScan. Nonsusceptibility was categorized as a tigecycline MIC of ≥4 μg/ml for both A. baumannii and Enterobacteriaceae . The study included 4,427 isolates: 2,065 ESBL-producing Enterobacteriaceae , 1,105 A. baumannii , 888 susceptible Enterobacteriaceae , and 369 CRE isolates. Tigecycline nonsusceptibility among A. baumannii isolates was significantly more common as determined by Etest compared to that determined by BMD (odds ratio [OR], 10.3; P 〈 0.001), MicroScan (OR, 12.4; P 〈 0.001), or Vitek-2 (OR, 9.4; P 〈 0.001). These differences were not evident with the other pathogens. Tigecycline MICs varied greatly according to the in vitro testing methods among A. baumannii isolates. Etest should probably not be used by laboratories for tigecycline MIC testing of A. baumannii isolates, since MICs are significantly elevated with Etest compared to those determined by the three other methods.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
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    SSG: 12
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  • 8
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 188, No. 6 ( 2006-03-15), p. 2115-2125
    Abstract: Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these α-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-α-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of α-glucan substrates by P. furiosus .
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 9
    Online Resource
    Online Resource
    American Society for Microbiology ; 2004
    In:  Applied and Environmental Microbiology Vol. 70, No. 10 ( 2004-10), p. 6098-6112
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 70, No. 10 ( 2004-10), p. 6098-6112
    Abstract: Thermotoga maritima , a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli , and Staphylococcus epidermidis ). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several β-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased β-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 10
    In: Journal of Virology, American Society for Microbiology, Vol. 77, No. 4 ( 2003-02-15), p. 2400-2409
    Abstract: Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested ΔphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01 + macaques were inoculated with three oral doses of recombinant Salmonella , followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag 181-189 epitope were detected following each dose of Salmonella . After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag 181-189 -specific CD8 + T-cell responses in peripheral blood. A significant percentage of the Gag 181-189 -specific T-cell population in each animal also expressed the intestinal homing receptor α4β7. Additionally, Gag 181-189 -specific CD8 + T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella -primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 1495529-5
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