Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry - PubMed (original) (raw)

Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry

L H Hansen et al. Appl Environ Microbiol. 2001 Jan.

Abstract

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated P(tet) promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

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Figures

FIG. 1

Composition of pTGFP2. A928-bp _Eco_RI-Sph_I fragment encompassing tetR, P_tet, and the E. coli atpE translation initiation region (in black), all obtained by PCR, was inserted into pJBA27. The bla gene encodes β-lactamase conferring resistance to ampicillin. ori colE1, origin of replication originating from pUC18-Not_I; lacZ′, partially deleted lacZ gene; gfp_mut3, FACS-optimized gfp gene encoding GFP. The two T's are transcriptional terminator sequences from pJBA27. P_tet is the tet promoter and tetR encodes the tet repressor protein, both originating from Tn_10.

FIG. 2

GFP in cultures of MC4100 harboring pTGFP1 or pTGFP2. Strains were grown overnight in LB4 containing increasing concentrations of oxytetracycline. Diamonds represent MC4100/pTGFP1, and squares represent MC4100/pTGFP2. Vertical bars show the standard deviations (n = 3).

FIG. 3

Flow cytometric analysis of E. coli MC4100/pTGFP2 cells (induced with 50 ng of oxytetracycline per ml) within soil. Region 1 defines where bacteria lie according to size (left). The second dot plot (right) is gated on bacteria (region 1 [R1]). Region 2 (R2) defines the population of GFP-expressing bacteria.

FIG. 4

Enumeration of induced biosensor bacteria (E. coli MC4100/pTGFP2). Soil microcosms were extracted with 10 ml of PBS, sedimented for 1 h, and filtered through a 38-μm-pore-Size mesh. Soil extracts were then analyzed on a FACScalibur flow cytometer, and only bacteria lying in the R2 region (shown and defined in Fig. 3) are counted as positive. Values and standard deviations in the negative area are not shown. Series A, B, C, and D were initially inoculated with 8.6 × 104, 8.6 × 102, 8.6, and 0 spores per g of wet soil, respectively.

FIG. 5

Flow cytometric analysis at day 0 (left) and day 2 (right) of soil extract from microcosms inoculated with a high density of S. rimosus spores (series A).

FIG. 6

Enumeration of biosensor bacteria (E. coli MC4100/ pTGFP2) on LB4 containing ampicillin. Open diamonds, microcosms containing undiluted S. rimosus spore inoculum (A series); open squares, 100×-diluted spore inoculum (B series); open triangles, 10,000×-diluted spore inoculum (C series); multiplication signs, no spores added (D series).

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Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry - PubMed (original) (raw)