Skip to main content

Staphylococcus aureus pigmentation is not controlled by Hfq



The golden color of Staphylococcus aureus is due to the synthesis of carotenoid pigments. In Gram-negative bacteria, Hfq is a global posttranscriptional regulator, but its function in S. aureus remains obscure. The absence of Hfq in S. aureus was reported to correlate with production of carotenoid pigment leading to the conclusion that Hfq was a negative regulator of the yellow color. However, we reported the construction of hfq mutants in several S. aureus strains and never noticed any color change; we therefore revisited the question of Hfq implication in S. aureus pigmentation.


The absence or accumulation of Hfq does not affect S. aureus pigmentation.


Staphylococcus aureus is a major pathogen responsible for numerous diseases from minor skin infection to septicemia, affecting humans and other animals. Its name “aureus” comes from the golden color of strains that express carotenoid pigments [1]. These pigments contribute to oxidative stress and neutrophil resistance, and virulence [2]. The carotenoid biosynthetic operon (crtMNOPQ) leading to the synthesis of staphyloxanthin is regulated by σB [3, 4], an alternative σ factor that also controls a large number of general stress genes. σB activity depends on RsbU, its positive regulator [5, 6]. Numerous strains, including the S. aureus model NCTC8325, have rsbU mutations that prevent σB activity and crt operon expression, such that colonies are white. In addition, mutations in 37 genes were shown to result in the loss of a yellow pigmentation [5, 7].

Hfq is an RNA chaperone needed for activity of numerous regulatory RNAs in Gram-negative bacteria [8]. However, its role in Gram-positive bacteria, with the exception of Clostridium difficile [9], remains enigmatic [10]. Hfq functionality from different species is often tested by interspecies complementation tests. However, expression of hfq genes from Gram-positive bacteria S. aureus and Bacillus subtilis in Salmonella could not compensate the absence of endogenous hfq, indicating a functional difference between Gram positive and negative Hfq [11, 12].

We previously compared phenotypes of S. aureus hfq mutants with their isogenic parental strains and observed no detectable difference associated with the absence of Hfq in the tested conditions [13]. However, our results were partly challenged by a publication reporting that carotenoid pigment production was increased in hfq-negative strains [14]. Here we use nine different S. aureus strains to show that Hfq absence or overexpression has no effect on pigment expression.

Main text


Bacterial strains, plasmids and growth conditions

Bacterial strains, plasmids and primers used in this study are listed in Table 1. Allelic replacements of hfq+ by Δhfq::cat were either performed by ϕ11-phage mediated transduction using RN4220 hfq::cat as a donor strain or by homologous recombination using pMADΔhfq::cat [13, 15]. The Δhfq::cat deletion in SAPHB5 was verified by Southern blot and subsequent Δhfq::cat transductants were verified by PCR as described [13].

Table 1 Staphylococcus aureus strains, plasmids and primer used for this study

Engineered plasmids were constructed as described [16]. Conditional hfq expression was obtained by cloning hfq under the xyl/tetO promoter in pRMC2 [17] and pRMC2FLAG (Table 1). pRMC2Hfq allowing hfq conditional expression was obtained as follows: pRMC2 and PCR-amplified hfq (using primers 39/49 on HG003 DNA) were KpnI-EcoRI digested and ligated together. pRMC2FLAG was engineered for conditional expression of 3xFLAG-tagged proteins as followed: pRMC2 and pSUB11 [18] were PCR-amplified using primers 856/918 and 858/919, respectively. The two resulting products, i.e. pRMC2 and 3xflag coding sequence, were assembled using the Gibson method [19]. pRMC2HfqFLAG, allowing conditional expression of Hfq::3xFLAG, was obtained as follows: pRMC2FLAG and hfq HG003 were PCR-amplified using primers 918/865 and 939/940, respectively. The two resulting products were assembled using the Gibson method.

Bacteria were grown in BHI medium (BD Difco, ref: 237500) at 37 °C under vigorous agitation. BHI solid media were obtained by the addition of Bacto Agar 15 g l−1 (BD Difco, ref: 214010). For strains containing pRMC2 and derivatives, chloramphenicol (Sigma-Aldrich, ref: C0378) 5 µg ml−1 was added to media. Expression from pRMC2 and derivatives was achieved by anhydrotetracycline (aTc, Chemodex, ref: CDX-A0197-M500) 250 ng ml−1 addition to growth media.

Protein extraction, Western blotting and staphyloxanthin spectral measurement

Overnight cultures were diluted 1000 times in fresh medium. After 3 h, aTc was added. 10 min and 30 min later, cells were harvested by centrifugation (16,000g for 2 min), resuspended in 400 µl Tris HCl buffer (50 mM, pH 6.8) and lysed using a FastPrep (3 cycles of 45 s at 6.5 m s−1). Cell debris was removed by centrifugation (16,000g for 10 min). Protein concentration was determined by Bradford assays [20]. For each sample, 3 μg of protein extract was separated on a polyacrylamide gel (Blot™ 4–12% Bis–Tris Plus, Invitrogen, ref: NW04122BOX). After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane (iBlot 2 PVDF Mini Stacks, Invitrogen ref: IB24002). For blotting and washing, an iBind™ Flex Western System (ref: SLF2000S) was used according to supplier’s instructions. Membranes were probed with the primary polyclonal ANTI-FLAG antibody produced in rabbit (Sigma, ref: F-7425) at a 1/15,000 dilution. A rabbit secondary antibody conjugated to horseradish peroxidase (Advansta, ref: R-05072-500) was used at a 1/25,000 dilution. Bioluminescent signal was detected with the WesternBright™ ECL-spray (Advansta, ref: K-12040-D50) using a digital camera (ImageQuant™ 350, GE Healthcare).

The S. aureus pigments were extracted as described [21]. In brief, strains were grown in BHI under vigorous agitation for 24 h. Cells were harvested by centrifugation, the pellet was rinsed twice with sterile water and pigments were extracted by methanol. Absorbance between 330 and 550 nm was measured on a microplate reader (CLARIOstar BMG LABTECH).


The absence of Hfq does not alter S. aureus pigmentation

In 2010, Liu et al. reported that “deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments” [14]. Their work was performed using an allele called Δhfq-8325 in which the hfq coding sequence was replaced by a kanamycin cassette. The hfq chromosomal deletion was constructed in strain RN4220 and then transduced into NCTC8325-4, RN6390, COL and ATCC25923 by phage ϕ11. We constructed a similar hfq deletion in RN4220, except that the hfq coding sequence was replaced by a chloramphenicol resistant gene (Δhfq::cat); this allele was transduced into RN6390, COL and Newman by ϕ11-phage mediated transduction [13]. Note that RN4220, RN6390 and COL strains were used for both studies. As we did not notice a change of color when the Δhfq::cat allele was introduced into these strains, this information was not reported [13]. In view of the previous report, we focused this work on the possibility that Hfq could affect S. aureus pigment expression.

NCTC8325 isolated in 1960 from a sepsis patient is the progenitor of numerous strains including NCTC8325-4 (cured of three prophages) which itself gave RN6390 and RN4220 [22]. As these descendants were mutagenized, they carry several mutations that may affect their phenotypes. NCTC8325 has a deletion of 11 bp in rsbU and a point mutation in tcaR. The derivatives HG001 (rsbU restored), HG002 (tcaR restored), HG003 (rsbU and tcaR restored) were constructed to perform physiological studies in a non-mutagenized background [22]. All these NCTC8325 derived strains, except HG001 and HG003 (which have a functional σB factor), give rise to white colonies (Fig. 1). In addition to those reported [13], we constructed Δhfq::cat derivatives in NCTC8325, NCTC8325-4, HG001, HG002 and HG003 (Table 1). In contrast to results reported in Liu et al., deletion of the hfq gene in all tested strain backgrounds had no effect on pigmentation (Fig. 1a). Note that COL, Newman are not NCTC8325 derivatives.

Fig. 1

Absence of Hfq does not affect S. aureus pigmentation. The indicated strains were grown overnight in BHI and then a streaked on BHI agar or b assayed for spectral profiles as described [21]

Spectral profiles highlighting S. aureus carotenoid production were determined as described [21] for three strains and their hfq derivatives after growth for 24 h in BHI. HG003 and HG003 Δhfq::cat gave equivalent profiles with three pics characteristic of carotenoid production. In contrast, NCTC8325-4 and RN1 had spectra characteristic of no or very little carotenoid production. As expected from our visual observation (Fig. 1a), the spectra of Δhfq derivatives did not differ from those of their respective parental strains (Fig. 1b).

Hfq overexpression does not alter S. aureus pigmentation

In the above-described strains, hfq is possibly poorly expressed, in which case hfq deletions would not lead to detectable phenotypes. We therefore tested the effects of an inducible Hfq expression system on pigment production. If the absence of Hfq leads to yellow colonies as proposed [14], the presence of Hfq could lower pigment production and lead to white colonies. To address this point, hfq was cloned under the control of the Pxyl/tetO promoter in multi-copy plasmid pRMC2 [17] leading to pRMC2Hfq. hfq expression in strains harboring pRMC2Hfq was induced upon aTc addition to media. To confirm that Pxyl/tetO was effectively driving hfq expression, a pRMC2Hfq derivative was engineered harboring a 3xflag sequence inserted in frame at the end of the hfq open reading frame. The resulting plasmid, pRMC2HfqFLAG is a proxy for expression from pRMC2Hfq. HG003 was transformed with pRMC2, pRMC2Hfq and pRMC2HfqFLAG. The protein Hfq::3xFLAG was detected upon aTc induction by western blotting using FLAG antibodies (Fig. 2a). We inferred from this result that addition of aTc to strains harboring pRMC2Hfq lead to Hfq synthesis. The RN4220 white and HG003 yellow colors were not affected by the presence of either pRMC2, pRMC2Hfq or pRMC2HfqFLAG and remained identical upon aTc addition to growth medium (Fig. 2b).

Fig. 2

Accumulation of Hfq does not affect S. aureus pigmentation. The indicated strains were grown in BHI supplemented with chloramphenicol. They were then a assayed by Western blotting for hfq::3xflag expression (see “Methods”) and b streaked on BHI agar supplemented with aTc when indicated


Our results show that neither the absence, nor the accumulation of Hfq affects pigmentation of S. aureus: Hfq does not appear to regulate staphyloxanthin synthesis. Our conclusions are supported by Tarrant PhD dissertation showing an NCTC8325 hfq mutant that remained unpigmented [23]. Of note, Pseudomonas aeruginosa reportedly induces pigment production of a non-pigmented phenotypic variant of S. aureus, however, this effect was independent of hfq transcription [24]. In addition, color variation in USA300 strain was screened in a genome-wide transposon mutant library, and the hfq inactivation was not reported to affect S. aureus pigmentation [7].

While the hfq gene is absent in some Firmicutes (e.g. Lactobacillales), it is conserved in all S. aureus, suggesting that it plays a crucial function, however not related to pigment expression. The quest to find the Staphylococcal Hfq function remains open.


Our conclusion is in contradiction with Liu et al. results concerning the effect of Hfq on S. aureus pigmentation [14]. We cannot rule out that our observation is limited to specific S. aureus strains. However, we used an NCTC8325-4 hfq derivative similar the one used in the previous study. Furthermore, the present results are strengthened by the construction of hfq mutants in numerous S. aureus backgrounds. The discrepancy between our and Liu et al. 2010 [14] results, is a possible inadvertent selection of mutants with altered color patterns (as shown in [23]) in the former study.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. Strains and plasmids are available from the corresponding author on reasonable request.



Polymerase chain reaction


Brain heart infusion




  1. 1.

    Marshall JH, Wilmoth GJ. Pigments of Staphylococcus aureus, a series of triterpenoid carotenoids. J Bacteriol. 1981;147(3):900–13.

    CAS  Article  Google Scholar 

  2. 2.

    Liu GY, Essex A, Buchanan JT, Datta V, Hoffman HM, Bastian JF, Fierer J, Nizet V. Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J Exp Med. 2005;202(2):209–15.

    CAS  Article  Google Scholar 

  3. 3.

    Bischoff M, Dunman P, Kormanec J, Macapagal D, Murphy E, Mounts W, Berger-Bachi B, Projan S. Microarray-based analysis of the Staphylococcus aureus sigmaB regulon. J Bacteriol. 2004;186(13):4085–99.

    CAS  Article  Google Scholar 

  4. 4.

    Pelz A, Wieland KP, Putzbach K, Hentschel P, Albert K, Gotz F. Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus. J Biol Chem. 2005;280(37):32493–8.

    CAS  Article  Google Scholar 

  5. 5.

    Palma M, Cheung AL. sigma(Beta) activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth. Infect Immun. 2001;69(12):7858–65.

    CAS  Article  Google Scholar 

  6. 6.

    Giachino P, Engelmann S, Bischoff M. Sigma(B) activity depends on RsbU in Staphylococcus aureus. J Bacteriol. 2001;183(6):1843–52.

    CAS  Article  Google Scholar 

  7. 7.

    Fey PD, Endres JL, Yajjala VK, Widhelm TJ, Boissy RJ, Bose JL, Bayles KW. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio. 2013;4(1):e00537-12.

    Article  Google Scholar 

  8. 8.

    Vogel J, Luisi BF. Hfq and its constellation of RNA. Nat Rev Microbiol. 2011;9(8):578–89.

    CAS  Article  Google Scholar 

  9. 9.

    Boudry P, Gracia C, Monot M, Caillet J, Saujet L, Hajnsdorf E, Dupuy B, Martin-Verstraete I, Soutourina O. Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile. J Bacteriol. 2014;196(18):3234–48.

    CAS  Article  Google Scholar 

  10. 10.

    Bouloc P, Repoila F. Fresh layers of RNA-mediated regulation in Gram-positive bacteria. Curr Opin Microbiol. 2016;30:30–5.

    CAS  Article  Google Scholar 

  11. 11.

    Rochat T, Bouloc P, Yang Q, Bossi L, Figueroa-Bossi N. Lack of interchangeability of Hfq-like proteins. Biochimie. 2012;94(7):1554–9.

    CAS  Article  Google Scholar 

  12. 12.

    Rochat T, Delumeau O, Figueroa-Bossi N, Noirot P, Bossi L, Dervyn E, Bouloc P. Tracking the elusive function of Bacillus subtilis Hfq. PLoS ONE. 2015;10(4):e0124977.

    Article  Google Scholar 

  13. 13.

    Bohn C, Rigoulay C, Bouloc P. No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus. BMC Microbiol. 2007;7:10.

    Article  Google Scholar 

  14. 14.

    Liu Y, Wu N, Dong J, Gao Y, Zhang X, Mu C, Shao N, Yang G. Hfq is a global regulator that controls the pathogenicity of Staphylococcus aureus. PLoS ONE. 2010;5(9):e13069.

    Article  Google Scholar 

  15. 15.

    Le Lam TN, Morvan C, Liu W, Bohn C, Jaszczyszyn Y, Bouloc P. Finding sRNA-associated phenotypes by competition assays: an example with Staphylococcus aureus. Methods. 2017;117:21–7.

    Article  Google Scholar 

  16. 16.

    Rochat T, Bohn C, Morvan C, Le Lam TN, Razvi F, Pain A, Toffano-Nioche C, Ponien P, Jacq A, Jacquet E, et al. The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus. Nucleic Acids Res. 2018;46(17):8803–16.

    CAS  Article  Google Scholar 

  17. 17.

    Corrigan RM, Foster TJ. An improved tetracycline-inducible expression vector for Staphylococcus aureus. Plasmid. 2009;61(2):126–9.

    CAS  Article  Google Scholar 

  18. 18.

    Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L. Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci USA. 2001;98(26):15264–9.

    CAS  Article  Google Scholar 

  19. 19.

    Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6(5):343–5.

    CAS  Article  Google Scholar 

  20. 20.

    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    CAS  Article  Google Scholar 

  21. 21.

    Morikawa K, Maruyama A, Inose Y, Higashide M, Hayashi H, Ohta T. Overexpression of sigma factor, sigma(B), urges Staphylococcus aureus to thicken the cell wall and to resist beta-lactams. Biochem Biophys Res Commun. 2001;288(2):385–9.

    CAS  Article  Google Scholar 

  22. 22.

    Herbert S, Ziebandt AK, Ohlsen K, Schafer T, Hecker M, Albrecht D, Novick R, Gotz F. Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates. Infect Immun. 2010;78(6):2877–89.

    CAS  Article  Google Scholar 

  23. 23.

    Tarrant EJ. The role of Hfq in S. aureus gene regulation. PhD. University of Leicester; 2013.

  24. 24.

    Antonic V, Stojadinovic A, Zhang B, Izadjoo MJ, Alavi M. Pseudomonas aeruginosa induces pigment production and enhances virulence in a white phenotypic variant of Staphylococcus aureus. Infect Drug Resist. 2013;6:175–86.

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Nair D, Memmi G, Hernandez D, Bard J, Beaume M, Gill S, Francois P, Cheung AL. Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain. J Bacteriol. 2011;193(9):2332–5.

    CAS  Article  Google Scholar 

  26. 26.

    Novick RP, Richmond MH. Nature and interactions of the genetic elements governing penicillinase synthesis in Staphylococcus aureus. J Bacteriol. 1965;90:467–80.

    CAS  Article  Google Scholar 

  27. 27.

    Novick R. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology. 1967;33(1):155–66.

    CAS  Article  Google Scholar 

  28. 28.

    Novick RP. Molecular biology of the staphylococci. New York: VCH Publishers; 1990.

    Google Scholar 

  29. 29.

    Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, Ravel J, Paulsen IT, Kolonay JF, Brinkac L, Beanan M, et al. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol. 2005;187(7):2426–38.

    CAS  Article  Google Scholar 

  30. 30.

    Baba T, Bae T, Schneewind O, Takeuchi F, Hiramatsu K. Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands. J Bacteriol. 2008;190(1):300–10.

    CAS  Article  Google Scholar 

Download references


We thank Sandy Gruss, Annick Jacq and Yan Jing for critical reading of the manuscript, helpful discussions and warm support.


This work was funded by the Agence Nationale pour la Recherche (ANR) (Grant # ANR-15-CE12-0003-01 “sRNA-Fit”) and by the Fondation pour la Recherche Médicale (FRM) (Grant # DBF20160635724 “Bactéries et champignons face aux antibiotiques et antifongiques”). WL was the recipient of fellowships from the Chinese scholarship council. PiB was the recipient of fellowships from the FRM.

Author information




PhB designed the experiments and wrote the manuscript. WL, PiB and CB performed the experiments, analyzed data and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Philippe Bouloc.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, W., Boudry, P., Bohn, C. et al. Staphylococcus aureus pigmentation is not controlled by Hfq. BMC Res Notes 13, 63 (2020).

Download citation


  • Staphylococcus aureus
  • Hfq
  • Pigmentation
  • Staphyloxanthin
  • Regulation