Characterization of an algal phosphomannose isomerase gene and its application as a selectable marker for genetic manipulation of tomato

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.

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The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Metabolites ◽

10.3390/metabo9030049 ◽

2019 ◽

Vol 9 (3) ◽

pp. 49 ◽

Cited By ~ 1

Author(s):

Ana Molina-Márquez ◽

Marta Vila ◽

Javier Vigara ◽

Ana Borrero ◽

Rosa León

Keyword(s):

Genetic Transformation ◽

Selectable Marker ◽

Genetic Manipulation ◽

Plant Cells ◽

Phytoene Desaturase ◽

Carotenoid Content ◽

Great Promise ◽

Encoding Gene ◽

Average Transformation ◽

Selection Of

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

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The molecular and genetic manipulation of nitrogen fixation

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1981.0053 ◽

1981 ◽

Vol 292 (1062) ◽

pp. 589-599 ◽

Cited By ~ 15

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.

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Isolation and characterization of human hepatoma cells with targeted insertions of agpt selectable marker in the α1—Antitrypsin locus

Somatic Cell and Molecular Genetics ◽

10.1007/bf02369569 ◽

1996 ◽

Vol 22 (4) ◽

pp. 311-327 ◽

Cited By ~ 2

Author(s):

Mary Beth Porter ◽

R. E. K. Fournier

Keyword(s):

Selectable Marker ◽

Hepatoma Cells ◽

Human Hepatoma ◽

Human Hepatoma Cells ◽

Isolation And Characterization

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Phosphomannose-isomerase (pmi) gene as a selectable marker for rice transformation via Agrobacterium

Plant Science ◽

10.1016/s0168-9452(03)00338-8 ◽

2004 ◽

Vol 166 (1) ◽

pp. 17-22 ◽

Cited By ~ 41

Author(s):

Zhengquan He ◽

Yaping Fu ◽

Huamin Si ◽

Guocheng Hu ◽

Shihong Zhang ◽

...

Keyword(s):

Selectable Marker ◽

Phosphomannose Isomerase ◽

Rice Transformation

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Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

Applied and Environmental Microbiology ◽

10.1128/aem.00820-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6062-6075 ◽

Cited By ~ 32

Author(s):

Michael H. Norris ◽

Yun Kang ◽

Diana Lu ◽

Bruce A. Wilcox ◽

Tung T. Hoang

Keyword(s):

Burkholderia Pseudomallei ◽

Selectable Marker ◽

Genetic Manipulation ◽

Chromosome 1 ◽

Chromosome 2 ◽

Gene Encoding ◽

Selectable Markers ◽

Genetic Manipulations ◽

Semialdehyde Dehydrogenase ◽

Allelic Replacement

ABSTRACT Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

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Retraction : The Use of Phosphomannose-isomerase as a Selectable Marker to Recover Transgenic Cauliflower

Korean Journal of Horticultural Science and Technology ◽

10.7235/hort.2014.11042 ◽

2014 ◽

Vol 32 (2) ◽

pp. 277-277

Author(s):

Byung Whan Min ◽

Suk Weon Kim

Keyword(s):

Selectable Marker ◽

Phosphomannose Isomerase

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TRANSFORMATION OF APPLE (MALUS DOMESTICA BORKH.) USING THE PHOSPHOMANNOSE ISOMERASE GENE AS A SELECTABLE MARKER

Acta Horticulturae ◽

10.17660/actahortic.2006.725.113 ◽

2006 ◽

pp. 811-816 ◽

Cited By ~ 4

Author(s):

J. Degenhardt ◽

I. Szankowski

Keyword(s):

Malus Domestica ◽

Selectable Marker ◽

Phosphomannose Isomerase ◽

Malus Domestica Borkh

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Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

Applied and Environmental Microbiology ◽

10.1128/aem.06095-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 568-574 ◽

Cited By ~ 33

Author(s):

Tao Zheng ◽

Qihong Huang ◽

Changyi Zhang ◽

Jinfeng Ni ◽

Qunxin She ◽

...

Keyword(s):

Selectable Marker ◽

Gene Knockout ◽

Plasmid Vector ◽

Selection Marker ◽

Content Type ◽

Sulfolobus Islandicus ◽

Gene Coding ◽

High Level ◽

Coa Reductase

ABSTRACTWe report here a novel selectable marker for the hyperthermophilic crenarchaeonSulfolobus islandicus. The marker cassette is composed of thesac7dpromoter and thehmggene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Psac7d-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting thepyrEFgene of the expression vector pSeSD for Psac7d-hmgwith which theSulfolobusexpression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization ofSulfolobustransformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His6-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEFandhmg) was subsequently exploited for genetic analysis. AherAmerodiploid strain ofS. islandicuswas constructed usingpyrEFmarker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from theherAmerodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability ofS. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

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