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.

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

scholarly journals EXPRESSION ANALYSIS OF THE COFFE (Coffea arabica L.) FRIGIDA4-like GENE (CaFRL4)

2018 ◽  
Vol 5 (Especial) ◽  
pp. 204-213
Author(s):  
Matheus Martins Daude ◽  
André Almeida Lima ◽  
Antonio Chalfun Junior ◽  
Horllys Gomes Barreto
Keyword(s):  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Foreign Market ◽  
Expression Levels ◽  
Future Studies ◽  
Coffea Arabica L ◽  
Production Losses ◽  
Flowering Process

ABSTRACT Coffee is one of the most economically important commodities. In Brazil, this crop is responsible for generating more than eight million jobs. In the foreign market, Brazil is the largest producer and exporter of coffee. Due to its economic importance, several studies aiming the improvement of coffee are conducted, but there are still problems related to its productivity and quality of the beverage, such as sequential flowering, which causes production losses and a low quality drink. Thus, understanding the molecular mechanisms involved in the flowering process is essential to elucidate how flowering occurs in the coffee crop. The FRI gene is one of the main genes involved in flowering, as it positively regulates the FLC gene at expression levels that inhibit flowering. Thus, the objective of this work was to identify and analyze the expression of the FRIGIDA4-like gene (FRL4) through Bioinformatics and real-time PCR (RT-qPCR). The CaFRL4 gene was identified and showed high expression levels in leaf during flowering, which corroborates with the literature. The results obtained provide the basis for future studies involving genetic transformation in model plants and coffee, permitting the functional characterization of this gene.

scholarly journals Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7

2019 ◽  
Vol 151 (4) ◽  
pp. 505-517 ◽  
Author(s):  
Paola Bartoccioni ◽  
Joana Fort ◽  
Antonio Zorzano ◽  
Ekaitz Errasti-Murugarren ◽  
Manuel Palacín
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Functional Characterization ◽  
Physiological Role ◽  
Cell Uptake ◽  
Amino Acid Transporters ◽  
Inhibitor Selectivity ◽  
Substrate Binding Sites

Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

Genome sequencing and functional characterization of Xanthomonas cucurbitae, the causal agent of bacterial spot disease of cucurbits

2021 ◽  
Author(s):  
Rikky Rai ◽  
Julius Pasion ◽  
Tanvi Majumdar ◽  
Cory E Green ◽  
Sarah Refi Hind
Keyword(s):  
Molecular Mechanisms ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Whole Genome Sequence ◽  
Leaf Spot Disease ◽  
Transcriptional Responses ◽  
Spot Disease ◽  
Xanthomonas Species ◽  
Reference Quality

Bacterial leaf spot disease caused by X. cucurbitae has severely affected the pumpkin industries in the Midwestern region of United States, with the bacteria mainly infecting pumpkin leaves and fruits, and leading to significant yield losses. In this study, we utilized genomics and genetics approaches to elucidate Xanthomonas cucurbitae molecular mechanisms of pathogenesis during interaction with its host. We generated the first reference-quality whole-genome sequence of the X. cucurbitae type isolate and compared to other Xanthomonas species, X. cucurbitae has a smaller genome size with fewer virulence-related genes. RNA-seq analysis of X. cucurbitae under plant-mimicking media conditions showed altered transcriptional responses, with upregulation of virulence genes and downregulation of cellular homeostasis genes. Additionally, characterization of key virulence genes using gene deletion methods revealed that both type II enzymes and type III effectors are necessary for X. cucurbitae to cause infection in the pumpkin host.

scholarly journals Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

2008 ◽  
Vol 105 (46) ◽  
pp. 17730-17735 ◽  
Author(s):  
Boguslaw Nocek ◽  
Samvel Kochinyan ◽  
Michael Proudfoot ◽  
Greg Brown ◽  
Elena Evdokimova ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Molecular Mechanisms ◽  
Catalytic Mechanism ◽  
Functional Characterization ◽  
High Energy ◽  
Inorganic Polyphosphate ◽  
The Family ◽  
Adp Crystal ◽  
Intracellular Energy

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer α/β/α sandwich fold with an α-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

scholarly journals Functional characterization of α1 -adrenoceptor subtypes in vascular tissues using different experimental approaches:a comparative study

2003 ◽  
Vol 138 (2) ◽  
pp. 359-368 ◽  
Author(s):  
Regina Gisbert ◽  
Yolanda Madrero ◽  
Valentina Sabino ◽  
M Antonia Noguera ◽  
M Dolores Ivorra ◽  
...  
Keyword(s):  
Comparative Study ◽  
Functional Characterization ◽  
Vascular Tissues

scholarly journals Comparative study of T84 and T84SF human colon carcinoma cells: in vitro and in vivo ultrastructural and functional characterization of cell culture and metastasis

2006 ◽  
Vol 449 (1) ◽  
pp. 48-61
Author(s):  
Riccardo Alessandro ◽  
Maria Antonietta Di Bella ◽  
Anna Maria Flugy ◽  
Simona Fontana ◽  
Francesca Damiani ◽  
...  
Keyword(s):  
Cell Culture ◽  
Comparative Study ◽  
Functional Characterization ◽  
Human Colon ◽  
Carcinoma Cells ◽  
Colon Carcinoma Cells ◽  
Human Colon Carcinoma

scholarly journals Network Analysis of Plasmidomes: The Azospirillum brasilense Sp245 Case

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Valerio Orlandini ◽  
Giovanni Emiliani ◽  
Marco Fondi ◽  
Isabel Maida ◽  
Elena Perrin ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Molecular Mechanisms ◽  
Gene Loss ◽  
Functional Characterization ◽  
Evolutionary Relationships ◽  
Computational Pipeline ◽  
Wheat Roots ◽  
Source Organism ◽  
Evolutionary Pathways

Azospirillum brasilense is a nitrogen-fixing bacterium living in association with plant roots. The genome of the strain Sp245, isolated in Brazil from wheat roots, consists of one chromosome and six plasmids. In this work, the A. brasilense Sp245 plasmids were analyzed in order to shed some light on the evolutionary pathways they followed over time. To this purpose, a similarity network approach was applied in order to identify the evolutionary relationships among all the A. brasilense plasmids encoded proteins; in this context a computational pipeline specifically devoted to the analysis and the visualization of the network-like evolutionary relationships among different plasmids molecules was developed. This information was supplemented with a detailed (in silico) functional characterization of both the connected (i.e., sharing homology with other sequences in the dataset) and the unconnected (i.e., not sharing homology) components of the network. Furthermore, the most likely source organism for each of the genes encoded by A. brasilense plasmids was checked, allowing the identification of possible trends of gene loss/gain in this microorganism. Data obtained provided a detailed description of the evolutionary landscape of the plasmids of A. brasilense Sp245, suggesting some of the molecular mechanisms responsible for the present-day structure of these molecules.

Sign in / Sign up

Export Citation Format

Share Document