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NaCl-sensitive mutant of Staphylococcus aureus has a Tn917-lacZ insertion in its ars operon

Sarah Scybert, Roger Pechous, Sutthirat Sitthisak, Mathew J. Nadakavukaren, Brian J. Wilkinson, R.K. Jayaswal
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00312-4 171-176 First published online: 1 May 2003


Staphylococcus aureus is a Gram-positive bacterium that is extremely halotolerant. To investigate the molecular mechanisms by which S. aureus can cope with osmotic stress, Tn917-lacZ-induced NaCl-sensitive mutants were isolated. An NaCl-sensitive mutant showed a longer lag period, slower growth rate, and lower final culture turbidity than the parent strain in liquid medium containing 1.5 M NaCl. Electron microscopic observation of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudo-multicellular cells. Addition of exogenous osmoprotectants, such as glycine betaine, choline, l-proline, and proline betaine, did not relieve the NaCl sensitivity of the mutant. The region flanking the transposon insertion site in the NaCl-sensitive S. aureus chromosome was sequenced. The mutated gene was 99% identical to arsR, the arsenic operon regulatory protein present on the pI258 plasmid of S. aureus. The ars operon from pI258 was subcloned into the shuttle vector pLI50 and transferred into the NaCl-sensitive mutant. The ars operon in trans restored NaCl tolerance in the mutant, suggesting that NaCl sensitivity is due to the mutation in arsR.

  • Arsenic operon
  • Osmoregulation
  • NaCl-sensitive mutant
  • Staphylococcus aureus

1 Introduction

An unusual characteristic of Staphylococcus aureus is its ability to grow in the presence of NaCl concentrations as high as 3.5 M [1]. This has practical importance in that S. aureus is the only food-borne bacterial pathogen that can grow at water activity levels below 0.9 (equivalent to 2.6 M NaCl) [2]. Lower water activity in food is an important strategy for the control of microbial growth. Staphylococcal gastroenteritis is a leading cause of food-borne illness [3].

To survive in an environment of high osmotic strength, bacteria respond by increasing the internal pool levels of compatible solutes to maintain water balance within the cell, either by transport or biosynthesis of compatible solutes [46] such as glycine betaine, proline, and proline betaine as compatible solutes [7,8]. The mechanisms involved in high NaCl tolerance in S. aureus have been reviewed by Wilkinson [9]. NaCl provides an osmotic and ionic stress, and the stress of the Na+ ion itself. The cellular level of K+ is high in S. aureus and does not change much upon NaCl stress [8,10,11]. A variety of transport systems that are activated or induced by NaCl are responsible for entry of osmoprotectants into the cell. Besides accumulating compatible solutes to maintain turgor pressure in response to high osmolarity environments, S. aureus also undergoes an extensive program of gene and protein expression in response to NaCl stress [12].

One approach to further understanding NaCl tolerance by S. aureus is to create NaCl-sensitive mutants to identify genes involved in the phenomenon. Vijaranakul et al. [12] created a Tn917-lacZ-induced NaCl-sensitive mutant in S. aureus strain NCTC 8325-4. The NaCl-sensitive phenotype could be rescued by glycine betaine and choline. Subsequent cloning and sequencing of the region surrounding the transposon insertion revealed it was inserted in a branched chain amino acid transporter gene [13].

In the present study, a transposon-induced NaCl-sensitive mutant of S. aureus strain ATCC12600 was characterized. This mutant could not be rescued by osmoprotectants and Tn917-lacZ was inserted into the arsR gene of the ars operon in this strain.

2 Materials and methods

2.1 Growth conditions

S. aureus cells were grown in defined medium (DM) [12] or tryptic soy broth (TSB), and Escherichia coli JM109 cells were grown in Luria–Bertani broth (LB). NaCl-sensitive mutants were derived from S. aureus ATCC12600 containing plasmid pLTV3 [14]. When required, osmoprotectants were added to media at a final concentration of 1 mM. When needed, ampicillin (50 µg ml−1), chloramphenicol (20 µg ml−1), erythromycin (20 µg ml−1), kanamycin (50 µg ml−1), and tetracycline (10 µg ml−1) were added to the growth medium.

Cells were grown with shaking (200 rpm) at 37°C in 300 ml nephelometer flasks containing 50 ml of medium. A 2% (v/v) inoculum from an overnight culture in TSB or DM was the standard inoculum. Growth was measured turbidimetrically by optical density measurements at 600 nm with a Spectronic-20 spectrophotometer (Bausch and Lomb, Rochester, NY, USA).

2.2 Transposon mutagenesis and screening for NaCl-sensitive mutants

Transposon Tn917-lacZ is a 5.4-kb transposable element that is present in plasmid pLTV3. This plasmid carries tetracycline resistance and a temperature-sensitive replicon outside of the transposon, so that transposition can be forced to occur at 43°C [14]. To induce random transposition, the S. aureus strain harboring plasmid pLTV3 was grown in TSB containing erythromycin and tetracycline at 43°C for 16–18 h in a shaking incubator. Erythromycin-resistant colonies were screened for the NaCl-sensitive phenotype by replica patching on tryptic soy agar containing erythromycin (20 µg ml−1), with or without 1.5 M NaCl. Colonies that failed to grow or showed poor growth on the NaCl-containing agar were designated as NaCl-sensitive mutants. One of the NaCl-sensitive mutants was designated as MB900.

2.3 Transmission electron microscopy

NaCl-sensitive mutant MB900 and the parent strain were examined by transmission electron microscopy (TEM) as described by Vijaranakul et al. [15].

2.4 Cloning and nucleotide sequencing of the flanking region of the mutated gene

Cloning of the Tn917-lacZ (left junction) flanking region was accomplished essentially as described by Camilli et al. [14]. The chromosomal DNA from the NaCl-sensitive mutant was isolated and digested with XbaI, self-ligated, and transformed into E. coli JM109. The transformants were selected on LB agar containing kanamycin. The plasmid was isolated from an overnight culture of transformant using the Qiagen plasmid mini-prep kit. The flanking sequences of the mutant were determined using a specific primer, 5′-GTTAAATGTACAAAATAACAGCGA-3′, which was derived from the sequence of Tn917-LTV3, on the proximal side of lacZ and about 70 bp from its end. Nucleotide sequencing was performed by dye terminator cycle sequencing protocol with AmpliTaq DNA polymerase and an automated sequencer, ABI Prism 310 genetic analyzer (Perkin Elmer, Foster City, CA, USA). The nucleotide sequence was used to search S. aureus databases (http://www.ncbi.nlm.nih.gov/; http://www.tigr.org/tdb/mdb/mdbcomplete.html; http://www.genome.ou.edu/staph.html; (http://www.sanger.ac.uk/Projects/S_aureus/) using the BLAST program.

2.5 Complementation of the mutant phenotype

To complement the mutation of the NaCl-sensitive mutant, a 2.7-kb KpnI–XbaI fragment containing arsRBC was cloned into the shuttle vector pLI50 [16] as shown in Fig. 1. First a tetracycline gene was subcloned into the HindIII site of pLI50 from pT181 plasmid [17]. Two primers (forward primer, 5′-ATGGTACCAATGCTAAAGACCCAC-3′, reverse primer, 5′CGTCTAGAGGAAAAAAAGAAAGCGTTT-3′) were designed to amplify the S. aureus wild-type copy of the arsRBC operon in a polymerase chain reaction using pGJ103 [18] as a template. The amplified fragment was cloned into a pGEM-T vector (Promega, Madison, WI, USA). The ars operon was subsequently subcloned into pLI50-tet plasmid within KpnI–XbaI sites. The resulting recombinant plasmid, pLI50-tet-ars, was transformed by electroporation [19] into S. aureus RN4220. The plasmid was re-isolated from S. aureus RN4220 and transformed into the NaCl-sensitive mutant. The transformants were tested for their ability to grow in TSB containing 1.5 M NaCl and TSB containing 5 mM potassium arsenate.

Figure 1

Cloning of ars operon into pLI50 shuttle vector.

2.6 Northern blot analysis

Total RNA from the NaCl-sensitive mutant and parent strains was isolated as described by Vijaranakul et al. [12]. 10 µg of total RNA was electrophoresed on formaldehyde agarose gels (1.0%) and transferred to nitrocellulose membranes. The blot was probed with radiolabeled ars genes.

2.7 Molecular genetic procedures

Plasmid and chromosomal DNA isolation, DNA manipulations, digestion of DNA with restriction enzymes, DNA ligation, oligolabeling, polymerase chain reactions, and Northern blotting were performed as described by Novick [20] and Sambrook et al. [21]. All enzymes were used as directed by the manufacturer.

3 Results and discussion

3.1 Isolation and characterization of an NaCl-sensitive mutant

Among 6300 erythromycin-resistant colonies patched on tryptic soy agar (TSA) plates containing erythromycin (20 µg ml−1) with and without 1.5 M NaCl, only one colony showed a clear inhibition of growth in the presence of NaCl. This mutant was designated as MB900 and used for further studies.

3.2 Growth characteristics of the NaCl-sensitive mutant

The growth of the NaCl-sensitive mutant in TSB containing erythromycin was similar to the parent strain (Fig. 2). Addition of 1.5 M NaCl to the medium severely inhibited the growth of the mutant compared to the parent strain (Fig. 2). Clumping also occurred, which increased later in the growth cycle. The mutant was unable to grow in 2.0 M NaCl.

Figure 2

Growth of S. aureus strains ATCC12600 (■), MB900 (▲) in TSB and ATCC12600 (□), MB900 (▵) in TSB containing 1.5 M NaCl. Overnight cultures were diluted 100-fold in 25 ml medium and at various intervals the optical density at 600 nm was measured with a spectrophotometer.

To determine if the sensitivity was limited to NaCl, TSB containing KCl or LiCl was tested. The mutant strain showed a slightly decreased growth rate in the presence of 1.5 M KCl and 1.5 M LiCl, but very minimal cellular clumping (data not shown). The mutant strain also showed sensitivity to 1.5 M NaNO3 and formed clumps. Addition of non-ionic osmotic agents such as 1.0 M sucrose had no effect on the growth of the mutant. These data suggested that the mutant phenotype was specifically sensitive to Na+ rather than to other ions or uncharged osmolytes.

The growth characteristics of the mutant were also determined in chemically defined media. As expected, the growth of the mutant in DM was similar to the parent strain in the absence of NaCl. However, growth of the mutant in DM containing 1.0 M NaCl was significantly reduced and severe clumping was obvious during mid-exponential phase. The mutant was unable to reach an optical density above 0.9 even after 60 h.

Several different osmoprotectants have been shown to stimulate growth of S. aureus in high osmotic-strength media. Glycine betaine, choline, and l-proline are known to alleviate the osmotic stress of S. aureus and encourage growth [8]. Addition of glycine betaine (1 mM) decreased the lag phase and increased the growth rate of the parent strain, but had little impact on the lag period or growth rate of the mutant strain in either complex or DM medium.

3.3 TEM of the NaCl-sensitive mutant under NaCl stress conditions

As mentioned above, the mutant grown in the presence of 1.5 M NaCl aggregated to form clumps. As shown in Fig. 3, there were clear differences between the parent and mutant strains when grown in TSB containing 1.5 M NaCl. The parent strain shows normal cell division with typical septal formation and separation of daughter cells (Fig. 3A). The cell walls of the parent strain also appear normal. However, many cells of the mutant strain appear to remain unseparated after cell division, with abnormal septa and cell walls thicker than those of the parental strain (Fig. 3B). These cells appear similar to the NaCl-sensitive mutant of S. aureus reported earlier [12,15]. Even some of the mutant cells that appeared to divide normally remained trapped within the thick cell wall.

Figure 3

Transmission electron micrographs of S. aureus ATCC12600 (A), MB900 (B) strains grown in TSB containing 1.5 M NaCl to mid-exponential phase. Bar represents 1 µm.

3.4 Cloning and identification of the mutated gene

Nucleotide sequencing and analysis of the flanking region of the Tn917-lacZ insertion site of the mutated gene (submitted to GenBank under Accession no. AY237169) showed 99% sequence homology to arsR, a repressor gene of the arsenic operon, present on the staphylococcal plasmids pI258 (GenBank Accession no. M86824.1), EDINA (GenBank Accession no. AP003089.1), and N315 (GenBank Accession no. AP003139.1). The transposon Tn17-lacZ was localized 109 nucleotides downstream from the start codon of the arsR gene in MB900. Also, sequence analysis of the chromosomal region of methicillin-resistant S. aureus strain EMRSA-16 showed the presence of two genes, arsB and arsC, following arsR. In bacteria the ars operon endows the cell with resistance to arsenate, arsenite, and antimonite [2226]. The arsR, arsB, and arsC genes encode an inducer-dependent repressor of transcription, an inner membrane arsenite extrusion pump, and a reductase enzyme that converts arsenate to arsenite, respectively. In the presence of arsenic, the ars operon is induced. ArsC converts arsenate into arsenite, which is effluxed from the cells by ArsB [25]. The chromosomal ars operon and that on the pI258 plasmid [18] are almost identical. The mutated arsR gene also showed about 75% sequence identity with another region of the MRSA chromosome. In the database these open reading frames are designated as homologs of arsR. Further analysis of the arsR homolog region showed the presence of the arsB homolog but not arsC.

To test whether the ars operon is functional in S. aureus ATCC12600, we determined its susceptibility to arsenate. As shown in Fig. 4A, S. aureus ATCC12600 can grow in TSB containing 5 mM arsenate. However, the NaCl-sensitive mutant MB900 was unable to grow in TSB containing even 1 mM arsenate. Inactivation of the arsR gene has been shown to result in constitutive expression of the ars operon [27,28]. For this reason, full resistance to arsenate was expected in the NaCl-sensitive mutant. However, the arsR mutant showed sensitivity to arsenate, indicating a probable polar effect of transposon insertion on the entire ars operon. RNA isolated from the mutant and the parent strains grown in TSB with and without 1 mM arsenate was probed with ars genes in Northern blot analysis (Fig. 5). An ars transcript was detected in the parent cells grown in the presence of arsenate (Fig. 5, lane 2); however, no transcripts of any size were detected in the mutant cells grown in the presence of arsenate (Fig. 5, lane 4). These results confirm our assumption that the transposon insertion in the arsR caused a polar effect on the expression of the ars operon, abolishing its expression.

Figure 4

Growth of S. aureus ATCC12600 (■), MB900 (▲), and MB900 complemented strain (◇) in TSB containing 5 mM potassium arsenate (A), TSB containing 1.5 M NaCl (B). In each case the overnight grown cultures were diluted 100-fold in 25 ml and at various intervals the optical density at 600 nm was measured with a spectrophotometer.

Figure 5

Northern blotting analysis of the ars transcript. Total RNA (5 µg) isolated from samples was separated electrophoretically and transferred by blotting onto a membrane. The blot was probed with the 32P-labeled ars genes. Lanes: 1, RNA from the parent grown in TSB; 2, parent grown in TSB + 1 mM potassium arsenate; 3, MB900 grown in TSB; 4, MB900 grown in TSB + 1 mM potassium arsenate; 5, complemented strain grown in TSB; 6, complemented strain grown in TSB + 1 mM potassium arsenate.

Resistance to arsenate in the parent S. aureus ATCC12600 strain is due to the presence of an intact arsenic operon present on the chromosome which, as mentioned above, showed 99% sequence identity to the arsenic operon found on the S. aureus pI258 plasmid and in various MRSA strains. Interestingly, the ars operon is not present in several S. aureus strains, including S. aureus NCTC-8325. Instead, two genes showing about 75% sequence identity to arsR and arsB are found in the chromosome of these strains. It seems these genes do not contribute to arsenite resistance, as S. aureus NCTC-8325 shows sensitivity to arsenite at a concentration of 0.1 mM. The presence of ion transport systems both on plasmids and the bacterial chromosome and their mode of regulation have been reviewed [29]. The molecular evolution of an arsenate detoxification pathway has been proposed [30]. However, the origin of these arsR and arsB homologs in methicillin-susceptible strains and their relationship to the resistance ars operon found in the chromosome of MRSA remain unclear.

3.5 Is the arsR mutation responsible for the NaCl-sensitive phenotype?

As previously mentioned, in addition to reducing arsenic tolerance, the arsR mutation significantly decreased the ability of the mutant MB900 to grow in the presence of NaCl. Efforts to mobilize the arsR mutation from MB900 to other S. aureus strains by bacteriophage Ø11 and 80α were unsuccessful due to the phage-resistant phenotype of S. aureus ATCC12600. Therefore, an alternative complementation method was used to provide genetic evidence that the mutation in the arsR is the cause of the NaCl-sensitive phenotype of the mutant. A 2.7-kb fragment containing the ars operon was cloned into a shuttle vector pLI50 and transferred into the mutant MB900 (Fig. 1). Transformants were selected on TSA containing tetracycline and erythromycin and tested for the ability to grow in TSB containing 1.5 M NaCl or 5 mM arsenate. All of the transformants were able to grow in medium containing 1.5 M NaCl (Fig. 4B) or arsenate (Fig. 4A) similar to the parent strain. The results also indicated that the NaCl stress was indeed alleviated and no clumping was observed during growth.

The levels of arsenic and antimony in NaCl solutions were determined by inductively coupled plasma atomic emission spectroscopy in order to see whether the NaCl sensitivity might have a trivial explanation such as contamination of NaCl with these ions. Analysis showed that the concentrations of arsenic and antimony in 1.5 M NaCl were 1.5 and 4 µM, respectively. These concentrations are about 1000-fold less than is required for growth inhibition of the mutant MB900. However, these micromolar concentrations of arsenate and antimony are enough to induce the ars expression as reported earlier [18,31].

The finding of a transposon insertion in the arsR gene of the NaCl-sensitive mutant raises the question of how abolishing the activity of the ars operon leads to NaCl sensitivity. Broer et al. [32] studied the ars operon of plasmid pI258. ArsB is a membrane efflux protein that pumps out arsenic and arsenite by an energy-dependent process [18], possibly energized by a chemiosmotic mechanism. Bacterial sodium stress has been reviewed by Padan and Krulwich [33]. The Bacillus subtilis Tet (L) protein and the closely related S. aureus plasmid-encoded Tet (K) protein are antiporters that can efflux a tetracycline-divalent complex, Na+ and K+. Perhaps the S. aureus ars operon, which results in the efflux of arsenic and arsenite also, has Na+ efflux activities, and low expression of the ars operon impedes the ability of S. aureus to rid itself of cytoplasmic Na+ in NaCl-stressed cells. In fact, a similar observation has been reported earlier involving Bacillus firmus CadC, which is an ArsR homolog. CadC was proposed to bind intracellular Na+ and deliver it to the residual nhaB-encoded Na+/H+ antiporter when cadC was overexpressed in an E. coli mutant lacking the nha-encoded Na+/H+ antiporter [34].


We thank Simon Silver for providing the pGJ103 plasmid containing the ars operon, and John Baur for determining the concentrations of arsenic and antimony. We thank Mindy Brown, Sangeeth Krishnachattier and Shalaka Kotkar for technical assistance during the initial phase of this study. This work was supported by grants from the National Institutes of Health and the American Heart Association — Midwest Affiliate.


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