Characterization of pNC1, a small and mobilizable plasmid for use in genetic manipulation of Desulfovibrio africanus

2009 ◽  
Vol 79 (1) ◽  
pp. 23-31 ◽  
Author(s):  
I. Nydia Castañeda-Carrión ◽  
Marvin Whiteley ◽  
Lee R. Krumholz
Keyword(s):  
Genetic Manipulation ◽  
Mobilizable Plasmid

scholarly journals Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Travis J. Wiles ◽  
Elena S. Wall ◽  
Brandon H. Schlomann ◽  
Edouard A. Hay ◽  
Raghuveer Parthasarathy ◽  
...  
Keyword(s):  
Comparative Study ◽  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Functional Characterization ◽  
Symbiotic Bacteria ◽  
Bacterial Isolates ◽  
Major Barrier ◽  
Allelic Exchange ◽  
Genetic Techniques

ABSTRACTCorrelating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis andin vitroassays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCEA great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

The molecular and genetic manipulation of nitrogen fixation

1981 ◽  
Vol 292 (1062) ◽  
pp. 589-599 ◽  
Keyword(s):  
Biological Process ◽  
Genetic Manipulation ◽  
Plant Genome ◽  
Population Pressure ◽  
Major Advance ◽  
Regulated Expression ◽  
Substantial Progress ◽  
World Agriculture ◽  
Physiological Background

The fundamental importance of dinitrogen fixation for world agriculture, in relation to projected energy supplies, population pressure and food requirements over the next decades, obliges scientists to reconsider ways of exploiting this biological process. Genetic manipulation offers several options in principle. Existing symbiotic systems such as the legumes and seemingly inefficient systems such as the grass associations could be improved; new symbioses could be developed by nif gene transfer to rhizosphere commensals or by somatic hybridization of appropriate plants. A major advance would be to render plants independent of microbes by manipulation of expressable nif into the plant genome. This goal is discussed. It requires the complete genetic and physical characterization of nif in particular its regulation, and an understanding of the physiological background within which nif can be expressed, as well as the ability to fuse nif to alien genetic systems. Substantial progress in these directions has been made by using the n if genes of Klebsiella pneumoniae this progress is reviewed. Strategies for the further manipulation of nif towards regulated expression in the plant genome are considered.

scholarly journals Emerging characterization of the role of SIRT3-mediated mitochondrial protein deacetylation in the heart

2011 ◽  
Vol 301 (6) ◽  
pp. H2191-H2197 ◽  
Author(s):  
Michael N. Sack
Keyword(s):  
Posttranslational Modification ◽  
Genetic Manipulation ◽  
Mitochondrial Protein ◽  
Pressure Overload ◽  
Therapeutic Agents ◽  
Redox Stress ◽  
Lysine Residues ◽  
Term Objective

Studies to quantify the protein acetylome show that lysine-residue acetylation rivals phosphorylation in prevalence as a posttranslational modification. Interesting, this posttranslational modification is modified by nutrient flux and by redox stress and targets the vast majority of metabolic pathway proteins in the mitochondria. Furthermore, the mitochondrial deacetylase enzyme SIRT3 appears to be regulated by exercise in skeletal muscle and in response to pressure overload in the heart. The alteration of protein lysine residues by acetylation and the enzymes controlling deacetylation are beginning to be explored as important regulatory events in the control of mitochondrial function and homeostasis. This review focuses on the mitochondrial targets of SIRT3 that are functionally implicated in heart biology and pathology and on the direct cardiac consequences of the genetic manipulation of SIRT3. As therapeutic modulators of other SIRT isoforms have been identified, the longer-term objective of our understanding of this biology would be to identify SIRT3 modulators as putative cardiac therapeutic agents.

scholarly journals The identification and characterization of zebrafish hematopoietic stem cells

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Dongdong Ma ◽  
Jing Zhang ◽  
Hui-feng Lin ◽  
Joseph Italiano ◽  
Robert I. Handin
Keyword(s):  
Danio Rerio ◽  
Genetic Manipulation ◽  
Transplant Recipients ◽  
Adult Zebrafish ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Identification And Characterization

Abstract HSCs are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. The optical clarity of their embryos and the ease of genetic manipulation make the zebrafish (Danio rerio) an excellent model for studying hematopoiesis. Using flow cytometry, we identified 2 populations of CD41-GFP+ cells (GFPhi and GFPlo) in the whole kidney marrow of Tg(CD41:GFP) zebrafish. Past studies in humans and mice have shown that CD41 is transiently expressed in the earliest hematopoietic progenitors and is then silenced, reappearing in the platelet/thrombocyte lineage. We have transplanted flow-sorted GFPhi and GFPlo cells into irradiated adult zebrafish and assessed long-term hematopoietic engraftment. Transplantation of GFPhi cells did not reconstitute hematopoiesis. In contrast, we observed multilineage hematopoiesis up to 68 weeks after primary and secondary transplantation of GFPlo cells. We detected the CD41-GFP transgene in all major hematopoietic lineages and CD41-GFP+ cells in histologic sections of kidneys from transplant recipients. These studies show that CD41-GFPlo cells fulfill generally accepted criteria for HSCs. The identification of fluorescent zebrafish HSCs, coupled with our ability to transplant them into irradiated adult recipients, provide a valuable new tool to track HSC homing, proliferation, and differentiation into hematopoietic cells.

scholarly journals Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Alvaro Quintanal-Villalonga ◽  
Hirokazu Taniguchi ◽  
Yingqian A. Zhan ◽  
Maysun M. Hasan ◽  
Shweta S. Chavan ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Squamous Cell ◽  
Genetic Manipulation ◽  
Therapeutic Targets ◽  
Xenograft Model ◽  
Driver Mutations ◽  
Preclinical Models ◽  
Patient Derived Xenograft ◽  
Mixed Histology

Abstract Background Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies. Methods We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment. Results Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib. Conclusions Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.

Characterization of the triple-component linoleic acid isomerase inLactobacillus plantarumZS2058 by genetic manipulation

2017 ◽  
Vol 123 (5) ◽  
pp. 1263-1273 ◽  
Author(s):  
B. Yang ◽  
H. Qi ◽  
Z. Gu ◽  
H. Zhang ◽  
W. Chen ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Genetic Manipulation ◽  
Linoleic Acid Isomerase

Genetic manipulation of rumen bacteria: from potential to reality

1996 ◽  
Vol 47 (2) ◽  
pp. 247 ◽  
Author(s):  
K Gregg ◽  
G Allen ◽  
C Beard
Keyword(s):  
Molecular Genetics ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Genetic Material ◽  
Rumen Bacteria ◽  
Butyrivibrio Fibrisolvens ◽  
Prevotella Ruminicola ◽  
The Stability

The development of techniques for manipulating the molecular genetics of bacteria led naturally to suggestions for using this technology to alter rumen function. Despite early difficulties, methods are now available to insert new genetic material into several rumen bacterial species, including Butyrivibrio fibrisolvens, Prevotella ruminicola, and Ruminococcus albus. One strain of B. fibrisolvens has been modified to detoxify a naturally occurring poison that causes major losses of livestock in Australia, Africa, and Central America. The stability of that modified organism has been demonstrated by its recolonization of the rumen and retention of its altered genotype over 5 months in vivo. Many of the persistent doubts about rumen bacterial genetic manipulation and the viability of altered organisms in a competitive environment have been shown to be capable of resolution, and interest in this area of research may be revitalized by these results. Apart from the achievement of specific metabolic improvements, the technology now available will allow extensive characterization of the molecular genetics of rumen bacteria with a precision that was not previously possible.

scholarly journals Temperature sensitive Mutant Proteome Profiling: a novel tool for the characterization of the global impact of missense mutations on the proteome

2019 ◽  
Author(s):  
Sarah A. Peck Justice ◽  
Guihong Qi ◽  
H. R. Sagara Wijeratne ◽  
José F. Victorino ◽  
Ed R. Simpson ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
26S Proteasome ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Sensitive Mutant ◽  
Proteome Profiling ◽  
Missense Mutant ◽  
Biophysical Changes ◽  
Thermal Stability Of

ABSTRACTTemperature sensitive (TS) mutants have been foundational in the characterization of essential genes. However, a high-throughput workflow for characterization of biophysical changes in TS mutants is lacking. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and PPIs. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. TeMPP is a precise approach to measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion.

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