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.

Keywords: Bacillus anthracis.; Burkholderia mallei; Burkholderia pseudomallei; GFP; RFP; Yersinia pestis; fluorescent tagging; select agents.

Figure 1

(A) Plasmid maps of pBKJ258, (B) pBK258-P0253-gfp, and (C) pBK258-P0253-rfp, respectively.

Figure 2

(A) Chromosomally tagged Bacillus anthracis Sterne strains were streaked out on a BHI agar plate. The plate was incubated at 37°C for 24 h and recorded by scanning. The bacteria in each quadrant were Sterne::Pntr-gfp (top left), Sterne::Pntr-rfp (bottom left), Sterne::P0253-gfp (top right), and Sterne::P0253-rfp (bottom right). (B) Fluorescence microscopy of bacteria in each quadrant on the same plate described in (A). Fluorescence images were taken with an exposure time of 1.2 sec for the strains Sterne::Pntr-gfp (top left) and Sterne::Pntr-rfp (bottom left), and with an exposure time of 200 msec for the strains Sterne::P0253-gfp (top right) and Sterne::P0253-rfp (bottom right).

Figure 3

(A) Plasmid maps of pRP1028, (B) pRP1028m, (C) pRP1028-P0253-gfp, and (D) pRP1028m-P0253-rfp, respectively.

Figure 4

Fluorescence micrographs of (A) Bacillus anthracis vegetative cells, (B) B. anthracis spores, and (C) B. anthracis spore expressing GFP within monocyte-derived macrophages.

Figure 5

Plasmid maps of (A) pUC18R6KT-mini-Tn7T-Km, (B) pUC18R6KT-2PcysZK-gfp, and (C) pUC18R6KT-2PcysZK-rfp, respectively.

Figure 6

(A) Escherichia coli DH5α λpir strains harboring reporter plasmid construct pUC18R6KT-PcysZK-gfp, pUC18R6KT-PcysZK-rfp, pUC18R6KT-2PcysZK-gfp, or pUC18R6KT-2PcysZK-rfp were struck on LB-agar plates. (B) Colony fluorescence microscopy of bacteria in each quadrant on the same plate described in (A). (C) Chromosomally integrated Yersina pseudotuberculosis strains were also struck out on L-agar plates and colony micrographs demonstrating GFP and RFP fluorescence are shown in (D).

Figure 7

Fluorescence micrographs of (A) Yersina pseudotuberculosis GFP, (B) Y. pestis CO92 GFP, and (C) Y. pestis CO92 RFP strains with the fusions inserted into the chromosomal Tn7 site.

Figure 8

Plasmids maps of (A) pmini-Tn7-gat, (B) pmini-Tn7-gat-P1-gfp, and (C) pmini-Tn7-gat-P1-rfp, respectively.

Figure 9

(A) Escherichia coli E2072 (pmini-Tn7-gat-P1-gfp) (top of plate) and E. coli E2072(pmini-Tn7-gat-P1-rfp) (bottom of plate) struck out on LB-agar plates. (B) Fluorescence microscopy of the E. coli strains described in (A). (C) Chromosomal glmS1 att-Tn7 site-integrated B. thailandensis Tn7-P1-gfp (top of plate) and B. thailandensis Tn7-P1-rfp (bottom of plate) that struck out on LB-agar plates. (D) Fluorescence microscopy of the B. thailandensis strains described in (C).

Figure 10

Fluorescence microscopy of chromosomal glmS1 att-Tn7 site-integrated Burkholderia GFP and RFP fusion strains. (A) Burkholderia thailandensis GFP, (B) B. thailandensis RFP, (C) B. mallei GFP, (D) B. pseudomallei GFP, and (E) B. mallei expressing GFP within THP-1 macrophages.

Figure 11

Time course of colony forming unit analysis of GFP-tagged B. anthracis (Ba), Y. pestis (Yp), B. mallei (Bm), and B. pseudomallei (Bp) after phagocytosis by THP-1 macrophages (n = 3). The stars (*) above the bars at the 72 h time point indicate a significance level of P < 0.05.

Figure 12

LDH release over a period of 3 days from THP-1 macrophages infected with GFP-tagged B. anthracis, Y. pestis, B. mallei, and B. pseudomallei (infected, dark blue line) versus uninfected (light blue line) (n = 3).