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. 2017 Mar 7;7:43889.
doi: 10.1038/srep43889.

Fluorescent Reporters for Markerless Genomic Integration in Staphylococcus Aureus

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Free PMC article

Fluorescent Reporters for Markerless Genomic Integration in Staphylococcus Aureus

Nienke W M de Jong et al. Sci Rep. .
Free PMC article

Abstract

We present integration vectors for Staphylococcus aureus encoding the fluorescent reporters mAmetrine, CFP, sGFP, YFP, mCherry and mKate. The expression is driven either from the sarA-P1 promoter or from any other promoter of choice. The reporter can be inserted markerless in the chromosome of a wide range of S. aureus strains. The integration site chosen does not disrupt any open reading frame, provides good expression, and has no detectable effect on the strains physiology. As an intermediate construct, we present a set of replicating plasmids containing the same fluorescent reporters. Also in these reporter plasmids the sarA-P1 promoter can be replaced by any other promoter of interest for expression studies. Cassettes from the replication plasmids can be readily swapped with the integration vector. With these constructs it becomes possible to monitor reporters of separate fluorescent wavelengths simultaneously.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Genomic region of S. aureus strain Newman where the fluorescent contrusts were integrated.
(A) Schematic overview of the insertion site for the fluorescent protein expression construct in the chromosome of S. aureus Newman (NC_009641.1). The construct is introduced by a double cross over event between the genes NWMN0029 and NWMN0030. (B) Plasmid pRN1112 used for integration by double cross over of PsarA_P1-mAmetrineA construct in the chromosome of S. aureus strains. CmR = chloramphenicol resistance gene; TetR: Tetracycline resistance gene; AmpR = Ampicillin resistance gene.
Figure 2
Figure 2. Spectral separation and intensity of all fluorescent constructs obtained by integration in the chromosome of S. aureus strains USA300.
Data points show mean +/− SD is shown of 4 technical replicates.
Figure 3
Figure 3. Simultaneous imaging of a mix of 4 separate S. aureus strains expressing fluorescent proteins from replicative plasmids using confocal microscopy.
Acquisition settings: (A) CFP: excitation laser 458 nm, detection 465–502 nm; (B) GFP: excitation laser 476 nm, detection 500–510 nm; (C) YFP: excitation laser 514 nm, detection 525–600 nm; (D) DsRed: excitation laser 543 nm, detection 598–709 nm. (E) Composite image of (A–D).
Figure 4
Figure 4. Excitation and emission spectra for mAmetrine when expressed in the cytosol of S. aureus USA300.
Figure 5
Figure 5. Growth of S. aureus strains USA300 and MW2 with chromosomal integration of different fluorescent markers.
No effect on growth dynamics of the genomic integration of the various fluorescent constructs is visible.

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