title: Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.
abstract: Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267.
authors.forename: Shengchang
authors.initials: S
authors.lastname: Su
authors.forename: Hansraj
authors.initials: H
authors.lastname: Bangar
authors.forename: Roland
authors.initials: R
authors.lastname: Saldanha
authors.forename: Adin
authors.initials: A
authors.lastname: Pemberton
authors.forename: Bruce
authors.initials: B
authors.lastname: Aronow
authors.forename: Gary E
authors.initials: GE
authors.lastname: Dean
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lamkin
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Hassett
mesh_terms: D001408:Bacillus anthracis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D001424:Bacterial Infections / Q000382:microbiology; D042726:Burkholderia mallei / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D016957:Burkholderia pseudomallei / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D002460:Cell Line; D002876:Chromosomes, Bacterial / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000382:microbiology; D014774:Virulence; D015010:Yersinia pestis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Bacillus anthracis.; Burkholderia mallei; Burkholderia pseudomallei; GFP; RFP; Yersinia pestis; fluorescent tagging; select agents