PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for
            <i>Streptococcus pneumoniae</i>

Belanger, Aimee E.; Lai, Angel; Brackman, Marcia A.; LeBlanc, Donald J.

doi:10.1128/aac.46.8.2507-2512.2002

Cited by 18 publications

(14 citation statements)

References 25 publications

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“…The morphology change was reversible because transformation of AB7 with genomic DNA from R6 resulted in the isolation of R colonies at approximately the same genetic frequency. The frequencies at which R to S and S to R morphology changes took place were consistent with the allelic exchange of a single marker (2). The unknown gene associated with the new S2 phenotype was not linked to the cps2 gene cluster because DNA derived from the AB28 strain still mediated the morphology change.…”

Section: Type 2 Capsule Alone Does Not Determine S2 Morphologysupporting

confidence: 56%

“…S. pneumoniae genomic DNA was isolated according to previously published methods (2). The PCR-based ordered genomic library was constructed as described previously (2) except that the DNA was amplified under high-fidelity conditions with Platinum Taq High Fidelity polymerase (Invitrogen, Carlsbad, Calif.). Routine transformations were performed as described (2) except that the concentration of DNA in the transformation mix was 1 g/ml.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR-based ordered genomic library was constructed as described previously (2) except that the DNA was amplified under high-fidelity conditions with Platinum Taq High Fidelity polymerase (Invitrogen, Carlsbad, Calif.). Routine transformations were performed as described (2) except that the concentration of DNA in the transformation mix was 1 g/ml. Gene insertions, deletions, and substitutions were accomplished by transforming S. pneumoniae with DNA amplicons generated with Platinum Taq High Fidelity polymerase under the PCR conditions suggested by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

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Pyruvate Oxidase Is a Determinant of Avery's Rough Morphology

et al. 2004

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. They demonstrated, by means of variation in colony morphology, that this substance could transform their rough type 2 Streptococcus pneumoniae strain R36A into a smooth type 3 strain. It has become accepted as fact, from modern textbook accounts of these experiments, that smooth pneumococci make capsule, while rough strains do not. We found that rough-to-smooth morphology conversion did not occur in rough strains R36A and R6 when the ability to synthesize native type 2 capsule was restored. The continued rough morphology of these encapsulated strains was attributed to a second, since-forgotten, morphology-affecting mutation that was sustained by R36A during strain development. We used a new genome-PCR-based approach to identify spxB, the gene encoding pyruvate oxidase, as the mutated locus in R36A and R6 that, with unencapsulation, gives rise to rough colony morphology, as we know it. The variant spxB allele of R36A and R6 is associated with increased cellular pyruvate oxidase activity relative to the ancestral strain D39. Increased pyruvate oxidase activity alters colony shape by mediating cell death. R36A requires a wild-type spxB allele for the expression of smooth type 2 morphology but not for the expression of smooth type 3 morphology, the phenotype monitored by Avery et al. Thus, the mutated spxB allele did not impact their use of smooth morphology to identify the transforming principle.The bacterium Streptococcus pneumoniae owns the important distinction of having introduced the world to the concept that DNA is the hereditary material. In their landmark studies, Avery, MacLeod, and McCarty identified DNA as the substance that could convert or transform one type of S. pneumoniae into another (1). The indicator of the transformation event was the ability of an unencapsulated strain of S. pneumoniae with rough colony morphology (R) to acquire a new encapsulated smooth (S) appearance (1).The R and S nomenclature has long been used to describe the colony morphology of S. pneumoniae: R colonies appear rough and irregular on solid media, while S colonies appear smooth and shiny. In the celebrated work of Griffith, these terms were originally used to correlate colony morphology with virulence properties (4). Later, R and S came to describe the encapsulation state of the bacterium because it was found that, in contrast to S colonies, R colonies do not produce typespecific polysaccharide capsule. The R designation is actually an umbrella term that has been used to describe many morphologically distinct unencapsulated S. pneumoniae mutants. Although all strains producing R colonies have lost their ability to make capsule, not all R colonies look the same, because the strains in question may have accumulated additional mutations that further alter colony morphology.One R variant that has sustained multiple colony morphology-altering mutations is S. pneumoniae R36A, the strain used by Avery et al. to demonstrate transformation (1). The history of R36A indicates that the R phenotype is the result of at least two inde...

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Section: Type 2 Capsule Alone Does Not Determine S2 Morphologysupporting

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pyruvate Oxidase Is a Determinant of Avery's Rough Morphology

et al. 2004

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“…In several organisms transformation frequencies have been found to be proportional to the length of DNA fragments carrying a mutation (7,(25)(26)(27)(28)57). We therefore hypothesized that the physical location of restriction enzyme sites relative to the location of a mutation could be predicted from differences in transformation frequencies observed with restriction enzyme digests of genomic DNA.…”

Section: Resultsmentioning

confidence: 99%

Novel Approach to Mapping of Resistance Mutations in Whole Genomes by Using Restriction Enzyme Modulation of Transformation Efficiency

Lerner

Kakavas

Wagner

et al. 2005

Antimicrob Agents Chemother

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Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using Haemophilus influenzae strains with mutations in gyrA, gyrB, and rpsE that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds: acrB, which encodes a subunit of the AcrAB-TolC efflux pump; and fadL, which encodes a long-chain fatty acid transporter.

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“…Generally, these can be classified into three categories: genetic, proteomic, and chemical. Genetic approaches include comparing transcriptomes of treated and untreated samples using microarray analysis (83,84) or transcriptome sequencing (RNA-seq) (85), sequencing a resistant mutant (86)(87)(88), and error-prone PCR-based target identification (89). Proteomic methods include comparing the proteomes of treated and untreated samples using affinity pulldown analysis (90), stable isotope labeling by amino acids in cell culture (SILAC) (91,92), or isobaric tags for relative and absolute quantification (iTRAQ) (93,94).…”

Section: Advances In Target Identificationmentioning

confidence: 99%

Chemical Biology Applied to the Study of Bacterial Pathogens

Anthouard

DiRita

2015

Infect Immun

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In recent years, chemical biology and chemical genomics have been increasingly applied to the field of microbiology to uncover new potential therapeutics as well as to probe virulence mechanisms in pathogens. The approach offers some clear advantages, as identified compounds (i) can serve as a proof of principle for the applicability of drugs to specific targets; (ii) can serve as conditional effectors to explore the function of their targets in vitro and in vivo; (iii) can be used to modulate gene expression in otherwise genetically intractable organisms; and (iv) can be tailored to a narrow or broad range of bacteria. This review highlights recent examples from the literature to illustrate how the use of small molecules has advanced discovery of novel potential treatments and has been applied to explore biological mechanisms underlying pathogenicity. We also use these examples to discuss practical considerations that are key to establishing a screening or discovery program. Finally, we discuss the advantages and challenges of different approaches and the methods that are emerging to address these challenges.

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PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for Streptococcus pneumoniae

Cited by 18 publications

References 25 publications

Pyruvate Oxidase Is a Determinant of Avery's Rough Morphology

Pyruvate Oxidase Is a Determinant of Avery's Rough Morphology

Novel Approach to Mapping of Resistance Mutations in Whole Genomes by Using Restriction Enzyme Modulation of Transformation Efficiency

Chemical Biology Applied to the Study of Bacterial Pathogens

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