Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300

Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcR_ACME and the arc_ACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcR_ACME protein binds to the promoter region of the arc_ACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

USA300 clone; ACME; transcriptional activator

Authorship

Zayda Lorena Corredor Rozo

Universidad El Bosque, Bacterial Molecular Genetics Laboratory, Bogotá, ColombiaUniversidad El BosqueColombiaBogotá, Colombia, ColombiaUniversidad El Bosque, Bacterial Molecular Genetics Laboratory, Bogotá, Colombia

Ricaurte Alejandro Márquez-Ortiz

Betsy Esperanza Castro

Natasha Vanegas Gómez

University of Technology, Faculty of Science, I3 Institute, Infection, Immunity and Innovation, Sydney, AustraliaUniversity of TechnologyAustraliaSydney, AustraliaUniversity of Technology, Faculty of Science, I3 Institute, Infection, Immunity and Innovation, Sydney, Australia

Javier Escobar-Pérez ⁺+ Corresponding author: escobarjavier@unbosque.edu.co

+ Corresponding author: escobarjavier@unbosque.edu.co

AUTHORS’ CONTRIBUTION

NVG and JEP - Designed the study; ZLCR - conducted the experiments; RAMO - performed the bioinformatics analysis; BEC - contributed to the microbiological experiments; ZLCR, RAMO and JEP - wrote the paper. All the authors analysed data, read and approved the final manuscript.

The authors declare that they have no conflicts of interest.

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Figures

Figures (3)

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Fig. 1
: differential expression of arginine catabolism (arc) operons and transcriptional regulators ArcR. (A) A diagram of arc operons in clone USA300. The inverted arrows indicate the location of primers used for evaluation of the co-transcription from arginine-ornithine antiporter gene (arcD) to carbamate kinase gene (arcC), and arcD to arcR in the arginine catabolism mobile element (arcACME) operon. The fragment indicated by an asterisk (*) is the promoter probe that contains the Crp-binding motif TGTGA-N6-TCACA (dark box). (B) Co-transcription of arcRACME with the other genes of the arcACME operon in strain USA300 under anaerobic conditions, as determined by polymerase chain reaction (PCR) using total cDNA as a template and primers 1 (lane 1) and 2 (lane 2) from panel A. Lane 3 is the same PCR analysis of RNA as a control to rule out DNA remnants. (C) Relative transcription levels of genes arcRACME, arcCACME, arcRcons, and arcCcons genes during anaerobiosis in the stationary phase (20 h; results are expressed as the mean of two different experiments).

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Fig. 2
: expression, purification, and detection of the recombinant argenine transcriptional regulator arginine catabolism mobile element (rArcRACME) protein. (A) The amount of the rArcRACME protein in the insoluble fraction of a total protein extract from Escherichia coli BL21-arcR, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37ºC at different time points. Lane 1, E. coli BL21 (DE3) without recombinant plasmid pET303/CT-His. Lane 2, E. coli BL21 (DE3) with recombinant plasmid pET303/CT-His, not induced. rArcRACME (~27 kDa) was purified from overnight (O.N.) culture of E. coli BL21-arcR by electroelution and visualised by SDS-PAGE (B) and western blotting (C) using an anti-6x-His antibody. Lane C in the western blot is a positive control for the anti-His antibody. NI: not induced.

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Fig. 3
: argenine transcriptional regulator gene (arcR) binding to the regulatory region of arginine catabolism mobile element (arcACME). (A) An electrophoretic mobility shift assay (EMSA) to evaluate mobility of the arcACME promoter probe (61.2 pmol) after interaction with the rArcRACME protein (0.75 μg); the assay included a specific (unbiotinylated probe) and a non-specific competitor (gyrB fragment). (B) UV cross-linking of rArcRACME (0.75 µg) and a USA300 total protein extract (60 μg) with the arcACME promoter region-based biotinylated probe (61.2 pmol). The interaction was visualised by non-denaturing PAGE to assess formation of the protein-probe complex. The non-denaturing PAGE was analysed by western blotting to detect the recombinant protein (with the monoclonal anti-6x-His tag antibody) and by a chemiluminescence assay (with peroxidase streptavidin and luminol) to detect the biotinylated DNA probe. (C) Partial alignment of promoter sequences of the arcACME and arccons operons in clone USA300. Recognition elements are surrounded by black boxes; the transcription start site is indicated as +1.

Fig. 1 : differential expression of arginine catabolism (arc) operons and transcriptional regulators ArcR. (A) A diagram of arc operons in clone USA300. The inverted arrows indicate the location of primers used for evaluation of the co-transcription from arginine-ornithine antiporter gene (arcD) to carbamate kinase gene (arcC), and arcD to arcR in the arginine catabolism mobile element (arcACME) operon. The fragment indicated by an asterisk () is the promoter probe that contains the Crp-binding motif TGTGA-N6-TCACA (dark box). (B) Co-transcription of arcRACME* with the other genes of the arcACME operon in strain USA300 under anaerobic conditions, as determined by polymerase chain reaction (PCR) using total cDNA as a template and primers 1 (lane 1) and 2 (lane 2) from panel A. Lane 3 is the same PCR analysis of RNA as a control to rule out DNA remnants. (C) Relative transcription levels of genes arcRACME, arcCACME, arcRcons, and arcCcons genes during anaerobiosis in the stationary phase (20 h; results are expressed as the mean of two different experiments).

Fig. 2 : expression, purification, and detection of the recombinant argenine transcriptional regulator arginine catabolism mobile element (rArcRACME) protein. (A) The amount of the rArcRACME protein in the insoluble fraction of a total protein extract from Escherichia coli BL21-arcR, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37ºC at different time points. Lane 1, E. coli BL21 (DE3) without recombinant plasmid pET303/CT-His. Lane 2, E. coli BL21 (DE3) with recombinant plasmid pET303/CT-His, not induced. rArcRACME (~27 kDa) was purified from overnight (O.N.) culture of E. coli BL21-arcR by electroelution and visualised by SDS-PAGE (B) and western blotting (C) using an anti-6x-His antibody. Lane C in the western blot is a positive control for the anti-His antibody. NI: not induced.

Fig. 3 : argenine transcriptional regulator gene (arcR) binding to the regulatory region of arginine catabolism mobile element (arcACME). (A) An electrophoretic mobility shift assay (EMSA) to evaluate mobility of the arcACME promoter probe (61.2 pmol) after interaction with the rArcRACME protein (0.75 μg); the assay included a specific (unbiotinylated probe) and a non-specific competitor (gyrB fragment). (B) UV cross-linking of rArcRACME (0.75 µg) and a USA300 total protein extract (60 μg) with the arcACME promoter region-based biotinylated probe (61.2 pmol). The interaction was visualised by non-denaturing PAGE to assess formation of the protein-probe complex. The non-denaturing PAGE was analysed by western blotting to detect the recombinant protein (with the monoclonal anti-6x-His tag antibody) and by a chemiluminescence assay (with peroxidase streptavidin and luminol) to detect the biotinylated DNA probe. (C) Partial alignment of promoter sequences of the arcACME and arccons operons in clone USA300. Recognition elements are surrounded by black boxes; the transcription start site is indicated as +1.

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Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300

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[1] + Corresponding author: escobarjavier@unbosque.edu.co