scholarly journals Development of a High-Efficiency Transformation Method and Implementation of Rational Metabolic Engineering for the Industrial Butanol Hyperproducer Clostridium saccharoperbutylacetonicum Strain N1-4

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Nicolaus A. Herman ◽  
Jeffrey Li ◽  
Ripika Bedi ◽  
Barbara Turchi ◽  
Xiaoji Liu ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Efficiency ◽  
Heterologous Gene Expression ◽  
Transformation Method ◽  
Type I ◽  
Exchange Method ◽  
Content Type ◽  
Solvent Production ◽  
Rational Engineering ◽  
Transformation Methods

ABSTRACT While a majority of academic studies concerning acetone, butanol, and ethanol (ABE) production by Clostridium have focused on Clostridium acetobutylicum, other members of this genus have proven to be effective industrial workhorses despite the inability to perform genetic manipulations on many of these strains. To further improve the industrial performance of these strains in areas such as substrate usage, solvent production, and end product versatility, transformation methods and genetic tools are needed to overcome the genetic intractability displayed by these species. In this study, we present the development of a high-efficiency transformation method for the industrial butanol hyperproducer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT) ATCC 27021. Following initial failures, we found that the key to creating a successful transformation method was the identification of three distinct colony morphologies (types S, R, and I), which displayed significant differences in transformability. Working with the readily transformable type I cells (transformation efficiency, 1.1 × 106 CFU/μg DNA), we performed targeted gene deletions in C. saccharoperbutylacetonicum N1-4 using a homologous recombination-mediated allelic exchange method. Using plasmid-based gene overexpression and targeted knockouts of key genes in the native acetone-butanol-ethanol (ABE) metabolic pathway, we successfully implemented rational metabolic engineering strategies, yielding in the best case an engineered strain (Clostridium saccharoperbutylacetonicum strain N1-4/pWIS13) displaying an 18% increase in butanol titers and 30% increase in total ABE titer (0.35 g ABE/g sucrose) in batch fermentations. Additionally, two engineered strains overexpressing aldehyde/alcohol dehydrogenases (encoded by adh11 and adh5) displayed 8.5- and 11.8-fold increases (respectively) in batch ethanol production. IMPORTANCE This paper presents the first steps toward advanced genetic engineering of the industrial butanol producer Clostridium saccharoperbutylacetonicum strain N1-4 (HMT). In addition to providing an efficient method for introducing foreign DNA into this species, we demonstrate successful rational engineering for increasing solvent production. Examples of future applications of this work include metabolic engineering for improving desirable industrial traits of this species and heterologous gene expression for expanding the end product profile to include high-value fuels and chemicals.

scholarly journals Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Ian R. Monk ◽  
Jai J. Tree ◽  
Benjamin P. Howden ◽  
Timothy P. Stinear ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Plasmid Dna ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Genetic Manipulation ◽  
Bacterial Pathogen ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Adenine Methylation

ABSTRACTStaphylococcus aureusis a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates ofS. aureus. Here, we describe the construction and characterization of the IMXXB series ofEscherichia colistrains that mimic the type I adenine methylation profiles ofS. aureusclonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of majorS. aureuslineages.IMPORTANCEThe genetic manipulation of clinicalS. aureusisolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset ofS. aureusstrains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite ofE. colistrains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer intoS. aureus. The IMXXB series ofE. colistrains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation ofS. aureus.

scholarly journals Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Ian R. Monk ◽  
Ishita M. Shah ◽  
Min Xu ◽  
Man-Wah Tan ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Laboratory Strain ◽  
Small Subset ◽  
Type I ◽  
Major Barrier ◽  
Content Type ◽  
Functional Genomic Analysis

ABSTRACTThe strong restriction barrier present inStaphylococcus aureusandStaphylococcus epidermidishas limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain ofS. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiencyEscherichia colicloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates ofS. aureusandS. epidermidis. We also show that the loss of restriction (both type I and IV) in anS. aureusUSA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCEStaphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding ofStaphylococcus aureusandStaphylococcus epidermidisinfections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

Recent Advances in Heterostructured Photocatalysts for Degradation of Organic Pollutants.

2020 ◽  
Vol 17 ◽  
Author(s):  
Chen-Jing Sun ◽  
Li-Ping Zhao ◽  
Rui Wang
Keyword(s):  
Organic Pollutants ◽  
High Efficiency ◽  
Separation Efficiency ◽  
Organic Pollution ◽  
Utilization Rate ◽  
Type I ◽  
Human Beings ◽  
Global Challenge ◽  
Global Environmental ◽  
High Efficient

: With the development of industrialization, the global environmental pollution and energy crisis are becoming increasingly serious. Organic pollutants pose a serious health threat to human beings and other organisms. The removal of organic pollutants in environment has become a global challenge. The photocatalytic technology has been widely used in the degradation of organic pollutants with its characteristics of simple process, high efficiency, thorough degradation and no secondary pollution. However, the single photocatalyst represented by TiO2 has disadvantages of low light utilization rate and high recombination rate of photocarriers. Building heterojunction is considered one of the most effective methods to enhance the photocatalytic performance of single photocatalyst, which can improve the separation efficiency of photocarriers and utilization of visible light. The classical heterojunction can be divided into four different cases: type I, typeⅡ, p–n heterojunctions and Z-scheme junction. In this paper, the recent progress in the treatment of organic pollution by heterostructure photocatalysts is summarized and the mechanism of heterostructure photocatalysts for the treatment of organic pollutants is reviewed. It is expected that this paper can deepen the understanding of heterostructure photocatalysts and provide guidance for high efficient photocatalytic degradation of organic pollutants in the future.

scholarly journals Modulation of Chicken Intestinal Immune Gene Expression by Small Cationic Peptides as Feed Additives during the First Week Posthatch

2013 ◽  
Vol 20 (9) ◽  
pp. 1440-1448 ◽  
Author(s):  
Michael H. Kogut ◽  
Kenneth J. Genovese ◽  
Haiqi He ◽  
Christina L. Swaggerty ◽  
Yiwei Jiang
Keyword(s):  
Gene Expression ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Feed Additives ◽  
Feed Additive ◽  
Cationic Peptides ◽  
Type I ◽  
Immune Gene ◽  
Content Type ◽  
Functional Efficiency

ABSTRACTWe have been investigating modulation strategies tailored around the selective stimulation of the host's immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soil bacterium,Brevibacillus texasporus. These peptides have immune modulatory properties that enhance both leukocyte functional efficiency and leukocyte proinflammatory cytokine and chemokine mRNA transcription activitiesin vitro. In addition, when provided as a feed additive for just 4 days posthatch, BT peptides significantly induce a concentration-dependent protection against cecal and extraintestinal colonization bySalmonella entericaserovar Enteritidis. In the present studies, we assessed the effects of feeding BT peptides on transcriptional changes on proinflammatory cytokines, inflammatory chemokines, and Toll-like receptors (TLR) in the ceca of broiler chickens with and withoutS. Enteritidis infection. After feeding a BT peptide-supplemented diet for the first 4 days posthatch, chickens were then challenged withS. Enteritidis, and intestinal gene expression was measured at 1 or 7 days postinfection (p.i.) (5 or 11 days of age). Intestinal expression of innate immune mRNA transcripts was analyzed by quantitative real-time PCR (qRT-PCR). Analysis of relative mRNA expression showed that a BT peptide-supplemented diet did not directly induce the transcription of proinflammatory cytokine, inflammatory chemokine, type I/II interferon (IFN), or TLR mRNA in chicken cecum. However, feeding the BT peptide-supplemented diet primed cecal tissue for increased (P≤ 0.05) transcription of TLR4, TLR15, and TLR21 upon infection withS. Enteritidis on days 1 and 7 p.i. Likewise, feeding the BT peptides primed the cecal tissue for increased transcription of proinflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-18, type I and II IFNs) and inflammatory chemokine (CxCLi2) in response toS. Enteritidis infection 1 and 7 days p.i. compared to the chickens fed the basal diet. These small cationic peptides may prove useful as alternatives to antibiotics as local immune modulators in neonatal poultry by providing prophylactic protection againstSalmonellainfections.

scholarly journals A new method for identifying the role of marital preferences at shaping marriage patterns

2021 ◽  
pp. 1-27
Author(s):  
Anna Naszodi ◽  
Francisco Mendonca
Keyword(s):  
Contingency Table ◽  
Transformation Method ◽  
New Method ◽  
Marriage Patterns ◽  
The Us ◽  
Analytical Properties ◽  
The Matrix ◽  
Transformation Methods

Abstract We develop a method which assumes that marital preferences are characterized either by the scalar-valued measure proposed by Liu and Lu, or by the matrix-valued generalized Liu–Lu measure. The new method transforms an observed contingency table into a counterfactual table while preserving its (generalized) Liu–Lu value. After exploring some analytical properties of the new method, we illustrate its application by decomposing changes in the prevalence of homogamy in the US between 1980 and 2010. We perform this decomposition with two alternative transformation methods as well where both methods capture preferences differently from Liu and Lu. Finally, we use survey evidence to support our claim that out of the three considered methods, the new transformation method is the most suitable for identifying the role of marital preferences at shaping marriage patterns. These data are also in favor of measuring assortativity in preferences à la Liu and Lu.

scholarly journals Random numbers from the tails of probability distributions using the transformation method

2013 ◽  
Vol 16 (2) ◽  
Author(s):  
Daniel Fulger ◽  
Enrico Scalas ◽  
Guido Germano
Keyword(s):  
Probability Distributions ◽  
Transformation Method ◽  
Fractional Diffusion ◽  
Random Numbers ◽  
Stochastic Solution ◽  
Probability Densities ◽  
Speed Up ◽  
Time Fractional Diffusion Equation ◽  
Transformation Methods ◽  
Very High

AbstractThe speed of many one-line transformation methods for the production of, for example, Lévy alpha-stable random numbers, which generalize Gaussian ones, and Mittag-Leffler random numbers, which generalize exponential ones, is very high and satisfactory for most purposes. However, fast rejection techniques like the ziggurat by Marsaglia and Tsang promise a significant speed-up for the class of decreasing probability densities, if it is possible to complement them with a method that samples the tails of the infinite support. This requires the fast generation of random numbers greater or smaller than a certain value. We present a method to achieve this, and also to generate random numbers within any arbitrary interval. We demonstrate the method showing the properties of the transformation maps of the above mentioned distributions as examples of stable and geometric stable random numbers used for the stochastic solution of the space-time fractional diffusion equation.

scholarly journals A Putative Cro-Like Repressor Contributes to Arylomycin Resistance in Staphylococcus aureus

2015 ◽  
Vol 59 (6) ◽  
pp. 3066-3074 ◽  
Author(s):  
Arryn Craney ◽  
Floyd E. Romesberg
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Transcriptional Regulator ◽  
Signal Peptidase ◽  
Health Concern ◽  
Resistant Bacteria ◽  
Type I ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Wide Range

ABSTRACTAntibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogenStaphylococcus aureus. To understand the origins of the limited activity againstS. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand howS. aureusresponds to the arylomycins, we profiled the transcriptional response ofS. aureusNCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistanceex vivodemonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate howS. aureuscopes with secretion stress and how it evolves resistance to the inhibition of SPase.

Split spinal cord malformations in children

1998 ◽  
Vol 88 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Yusuf Ersşahin ◽  
Saffet Mutluer ◽  
Sevgül Kocaman ◽  
Eren Demirtasş
Keyword(s):  
Spinal Cord ◽  
Neurological Deficits ◽  
Single Type ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Spinal Lesion ◽  
The Mean ◽  
Fine Print

Object. The authors reviewed and analyzed information on 74 patients with split spinal cord malformations (SSCMs) treated between January 1, 1980 and December 31, 1996 at their institution with the aim of defining and classifying the malformations according to the method of Pang, et al. Methods. Computerized tomography myelography was superior to other radiological tools in defining the type of SSCM. There were 46 girls (62%) and 28 boys (38%) ranging in age from less than 1 day to 12 years (mean 33.08 months). The mean age (43.2 months) of the patients who exhibited neurological deficits and orthopedic deformities was significantly older than those (8.2 months) without deficits (p = 0.003). Fifty-two patients had a single Type I and 18 patients a single Type II SSCM; four patients had composite SSCMs. Sixty-two patients had at least one associated spinal lesion that could lead to spinal cord tethering. After surgery, the majority of the patients remained stable and clinical improvement was observed in 18 patients. Conclusions. The classification of SSCMs proposed by Pang, et al., will eliminate the current chaos in terminology. In all SSCMs, either a rigid or a fibrous septum was found to transfix the spinal cord. There was at least one unrelated lesion that caused tethering of the spinal cord in 85% of the patients. The risk of neurological deficits resulting from SSCMs increases with the age of the patient; therefore, all patients should be surgically treated when diagnosed, especially before the development of orthopedic and neurological manifestations.

scholarly journals A Novel Line Immunoassay Based on Recombinant Virulence Factors Enables Highly Specific and Sensitive Serologic Diagnosis of Helicobacter pylori Infection

2013 ◽  
Vol 20 (11) ◽  
pp. 1703-1710 ◽  
Author(s):  
Luca Formichella ◽  
Laura Romberg ◽  
Christian Bolz ◽  
Michael Vieth ◽  
Michael Geppert ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Immune Responses ◽  
Virulence Factors ◽  
Gastrointestinal Disease ◽  
Individual Risk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Serologic Test ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloricolonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome.H. pylorivirulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to importantH. pylorivirulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed inEscherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) wereH. pylorinegative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), therecomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, therecomLine assay provides a valuable tool for the diagnosis ofH. pyloriinfection.

scholarly journals Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

2015 ◽  
Vol 83 (10) ◽  
pp. 3890-3901 ◽  
Author(s):  
Prasun Moitra ◽  
Hong Zheng ◽  
Vivek Anantharaman ◽  
Rajdeep Banerjee ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Apical Membrane ◽  
The United States ◽  
Host Cells ◽  
Health Concern ◽  
Babesia Microti ◽  
Type I ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Extracellular Region

The intraerythrocytic apicomplexanBabesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-lengthB. microtiAMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including otherBabesiaandTheileriaspecies, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed inEscherichia colireacted with a mouse anti-BmAMA1 antibody using Western blotting.In vitrobinding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation ofB. microtiparasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

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