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

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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Genetic Tools for Allelic Replacement in Burkholderia Species

Applied and Environmental Microbiology ◽

10.1128/aem.00531-08 ◽

2008 ◽

Vol 74 (14) ◽

pp. 4498-4508 ◽

Cited By ~ 70

Author(s):

Ashley R. Barrett ◽

Yun Kang ◽

Ken S. Inamasu ◽

Mike S. Son ◽

Joseph M. Vukovich ◽

...

Keyword(s):

Selectable Marker ◽

Clinical Importance ◽

The United States ◽

Gene Replacement ◽

Betaine Aldehyde Dehydrogenase ◽

Selectable Markers ◽

Α Subunit ◽

Genetic Traits ◽

Allelic Replacement ◽

Resistance Markers

ABSTRACT Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the α-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Telr) and trimethoprim (Tpr) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Telr-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tpr), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Telr) and pFLP-AB5 (Tpr). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the ΔbetBA::Telr-FRT and ΔbetBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.

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Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

10.1101/606590 ◽

2019 ◽

Author(s):

Erin A. Mack ◽

Yu-Ping Xiao ◽

David R. Allred

Keyword(s):

Antigenic Variation ◽

Genetic Manipulation ◽

Artificial Chromosome ◽

Babesia Bovis ◽

Chromosome 1 ◽

Gene Encoding ◽

Persistent Infections ◽

Essentially Normal ◽

Regulated Gene Expression

AbstractBabesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

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Reclamation of Ampicillin Sensitivity for the Genetic Manipulation of Legionella pneumophila

Applied and Environmental Microbiology ◽

10.1128/aem.00669-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5457-5459 ◽

Cited By ~ 1

Author(s):

Molly C. Sutherland ◽

Joseph P. Vogel

Keyword(s):

Mutant Strain ◽

Causative Agent ◽

Legionella Pneumophila ◽

Selectable Marker ◽

Genetic Manipulation ◽

Selectable Markers ◽

Content Type ◽

Enhanced Sensitivity ◽

Legionnaires Disease

ABSTRACTResearch onLegionella pneumophila, the causative agent of Legionnaires' disease, has been hampered due to the lack of selectable markers for genetic manipulation. We report the construction of a mutant strain ofL. pneumophilalackingloxA, a chromosomally encoded β-lactamase, that has enhanced sensitivity to ampicillin. Also described are a method for convertingLegionellastrains to ampicillin sensitivity and conditions for utilizingblaas a selectable marker.

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Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

Journal of Fungi ◽

10.3390/jof7070506 ◽

2021 ◽

Vol 7 (7) ◽

pp. 506

Author(s):

Clara Baldin ◽

Alexander Kühbacher ◽

Petra Merschak ◽

Luis Enrique Sastré-Velásquez ◽

Beate Abt ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Selectable Marker ◽

Genetic Manipulation ◽

Inducible Promoter ◽

Fungal Species ◽

Marker Genes ◽

Selectable Markers ◽

Simple Modification ◽

Selectable Marker Genes

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

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EVOLUTION AND VARIATION OF RENIN GENES IN MICE

Genetics ◽

10.1093/genetics/108.3.651 ◽

1984 ◽

Vol 108 (3) ◽

pp. 651-667

Author(s):

Douglas P Dickinson ◽

Kenneth W Gross ◽

Nina Piccini ◽

Carol M Wilson

Keyword(s):

Gene Expression ◽

Submandibular Gland ◽

Regulation Of Gene Expression ◽

Inbred Strains ◽

Duplication Event ◽

Comparative Sequence Analysis ◽

Chromosome 1 ◽

Gene Encoding ◽

Prior Estimate ◽

Low Levels

ABSTRACT Inbred strains of mice carry Ren-1, a gene encoding the thermostable Renin-1 isozyme. Ren-1 is expressed at relatively low levels in mouse submandibular gland and kidney. Some strains also carry Ren-2, a gene encoding the thermolabile Renin-2 isozyme. Ren-2 is expressed at high levels in the mouse submandibular gland and at very low levels, if at all, in the kidney. Ren-1 and Ren-2 are closely linked on mouse chromosome 1, show extensive homology in coding and noncoding regions and provide a model for studying the regulation of gene expression. An investigation of renin genes and enzymatic activity in wild-derived mice identified several restriction site polymorphisms as well as putative variants in renin gene expression and protein structure. The number of renin genes carried by different subpopulations of wild-derived mice is consistent with the occurrence of a gene duplication event prior to the divergence of M. spretus (2.75-5.5 million yr ago). This conclusion is in agreement with a prior estimate based upon comparative sequence analysis of Ren-1 and Ren-2 from inbred laboratory mice.

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Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

Applied and Environmental Microbiology ◽

10.1128/aem.00759-14 ◽

2014 ◽

Vol 80 (13) ◽

pp. 3868-3878 ◽

Cited By ~ 11

Author(s):

Ana Yepes ◽

Gudrun Koch ◽

Andrea Waldvogel ◽

Juan-Carlos Garcia-Betancur ◽

Daniel Lopez

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Biology ◽

Genetic Manipulation ◽

Protein Localization ◽

Antibiotic Resistance Genes ◽

Wall Material ◽

Content Type ◽

Genetic Manipulations ◽

Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

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The Optimal Design on Connection Order of Network in Hydraulic Manifold Block

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.201-203.2190 ◽

2011 ◽

Vol 201-203 ◽

pp. 2190-2194

Author(s):

Jun Jun Zhang ◽

Ji Sheng Wang ◽

Jiang Yong Wang ◽

Gang Liu ◽

Jie Wang

Keyword(s):

Genetic Algorithm ◽

Optimal Design ◽

Genetic Manipulation ◽

Optimal Algorithm ◽

Single Point ◽

Adaptive Mutation ◽

Gene Encoding ◽

Mutation Strategy ◽

The Common

As one of the important questions in the design of hydraulic manifold block — connection order of network, give a solution based on genetic algorithm. Genetic algorithm is the common effective intelligent optimal algorithm and suitable for solving a large combinatorial optimal problems. Gene encoding of ordinal representation, single-point crossover strategy and adaptive mutation strategy are used in the design of genetic manipulation.

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Methylome-wide analysis reveals epigenetic marks associated with resistance to tuberculosis in HIV-infected individuals from East Africa

10.1101/2020.07.14.20153395 ◽

2020 ◽

Author(s):

Catherine Stein ◽

Penelope Bencheck ◽

Jacquelaine Bartlett ◽

Robert P Igo ◽

Rafal S Sobota ◽

...

Keyword(s):

Genome Wide Association Study ◽

Sub Saharan Africa ◽

Chromosome 1 ◽

Chromosome 2 ◽

Chromosome 5 ◽

Epigenetic Marks ◽

Genome Wide ◽

A Genome ◽

Immunodeficiency Virus ◽

Sub Saharan

Background: Tuberculosis (TB) is the most deadly infectious disease globally and highly prevalent in the developing world, especially sub-Saharan Africa. Even though a third of humans are exposed to Myocbacterium tuberculosis (Mtb), most infected immunocompetent individuals do not develop active TB. In contrast, for individuals infected with both TB and the human immunodeficiency virus (HIV), the risk of active disease is 10% or more per year. Previously, we identified in a genome-wide association study a region on chromosome 5 that was associated with resistance to TB. This region included epigenetic marks that could influence gene regulation so we hypothesized that HIV-infected individuals exposed to Mtb, who remain disease free, carry epigenetic changes that strongly protect them from active TB. To test this hypothesis, we conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania. Results: In 221 HIV-infected adults from Uganda and Tanzania, we identified 3 regions of interest that included markers that were differentially methylated between TB cases and LTBI controls, that also included methylation QTLs and associated SNPs: chromosome 1 (RNF220, p=4x10-5), chromosome 2 (between COPS8 and COL6A3 genes, p=2.7x10-5), and chromosome 5 (CEP72, p=1.3x10-5). These methylation results colocalized with associated SNPs, methylation QTLs, and methylation x SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung. Conclusion: Epigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

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Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

Genetics ◽

10.1093/genetics/136.1.107 ◽

1994 ◽

Vol 136 (1) ◽

pp. 107-118 ◽

Cited By ~ 5

Author(s):

T A Harkness ◽

R L Metzenberg ◽

H Schneider ◽

R Lill ◽

W Neupert ◽

...

Keyword(s):

Neurospora Crassa ◽

Target Gene ◽

Protein Import ◽

Selectable Marker ◽

Mitochondrial Protein ◽

Mutant Gene ◽

Mitochondrial Protein Import ◽

Gene Encoding ◽

Precursor Proteins ◽

Import Receptor

Abstract We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

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The Essential Genome of Burkholderia cenocepacia H111

Journal of Bacteriology ◽

10.1128/jb.00260-17 ◽

2017 ◽

Vol 199 (22) ◽

Cited By ~ 7

Author(s):

Steven Higgins ◽

Maria Sanchez-Contreras ◽

Stefano Gualdi ◽

Marta Pinto-Carbó ◽

Aurélien Carlier ◽

...

Keyword(s):

Minimal Medium ◽

Essential Genes ◽

Burkholderia Cenocepacia ◽

Chromosome 1 ◽

Biological Research ◽

Chromosome 2 ◽

Powerful Method ◽

Rich Medium ◽

Content Type ◽

Autonomous Growth

ABSTRACT The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera. IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of “core essential genes” that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

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