Adaptation to zinc restriction in Streptococcus agalactiae: role of the ribosomal protein and zinc-importers regulated by AdcR

Abstract

Zinc (Zn) is an essential cofactor for numerous bacterial proteins and altering Zn availability is an important component of host innate immunity. During infection, adaptation to both Zn deprivation and excess is critical for pathogenic bacteria development. To understand the adaptive responses to Zn availability of Streptococcus agalactiae, a pathogen causing invasive infections of neonates, global transcriptional profiling was conducted. Results highlight that in response to Zn limitation, genes belonging to the AdcR regulon, the master regulator of Zn homeostasis in streptococci, were overexpressed. Through a combination of in silico analysis and experimental validation, new AdcR-regulated targets were identified. Among them, we identified a duplicated ribosomal protein, RpsNb, and an ABC transporter, and examined the role of these genes in bacterial growth under Zn-restricted conditions. Our results indicated that, during Zn restriction, both the RpsNb protein and a potential secondary Zn transporter are important for S. agalactiae adaptation to Zn deficiency.

Importance: Streptococcus agalactiae is a bacterial human pathobiont causing invasive diseases in neonates. Upon infection, S. agalactiae is presented with Zn limitation and excess but the genetic systems that allow bacterial adaptation to these conditions remain largely undefined. A comprehensive analysis of S. agalactiae global transcriptional response to Zn availability shows that this pathogen manages Zn limitation mainly through upregulation of the AdcR regulon. We demonstrate that several AdcR-regulated genes are important for bacterial growth during Zn deficiency, including human biological fluids. Taken together, these findings reveal new mechanisms of S. agalactiae adaptation under conditions of metal deprivation.

Keywords: Streptococcus agalactiae; duplicated ribosomal proteins; gene regulation; metal ABC-transporter; zinc homeostasis.

Fig 1

Transcriptomic analysis of genes regulated by Zn concentration in S. agalactiae volcano plot of the S. agalactiae A909 strain’s transcriptome at mid-exponential phase comparing bacteria grown in (A) Zn-restricted CDM (0 µM Zn) vs Zn-replete CDM (10 µM ZnSO₄), (B) Zn-restricted CDM (0 µM Zn) vs Zn-excess CDM (300 µM ZnSO₄), and (C) Zn-excess CDM (300 µM Zn) vs Zn-replete CDM (10 µM ZnSO₄). Each dot represents one of the A909 genes with its RNA-seq fold change (FC) and adjusted P-value calculated from three independent replicates. Red dots are genes differentially expressed (|log2 FC| > 1 and < −1; adjusted P-value < 0.05) with selected gene names highlighted. Black dots symbolized non-significant differentially transcribed genes.

Fig 2

AdcR-dependent regulation of selected genes. (A) S. agalactiae A909 (black bars) or its isogenic ΔadcR mutant (white bars) was grown to mid-log phase (OD₆₀₀ = 0.4) in Zn-restricted CDM or inZn-excess CDM. Expression of sak_RS00910, rpsNb, sak_RS08940, adcAII, lmb, sak_RS01090, sak_RS01240, sak_RS07770, adcR, sak_RS05125, sak_RS04000, adhP, sak_RS04420, mvk, and pfkA was assessed either by quantitative real time-PCR (qRT-PCR) or by lacz transcriptional fusions. Gene expression in Zn-excess CDM in the WT and in the ΔadcR mutant strains are presented as a fold change compared to expression of the WT strain grown in Zn-restricted CDM. Red dots delimit significant fold changes (|FC| > 2 and < −0,5). The results are means ± the standard deviations from three independent experiments. (B) AdcR-DNA interactions as assessed by gel mobility shift assay. DNA fragments of similar size were amplified from the promoters of sak_RS01240 (Psak_RS01240), rpsNb (PrpsNb), and the non-specific control promoter of murG (PmurG). Increasing concentrations of AdcR (500 and 1,000 ng) were incubated with the indicated DNA fragments and reaction mixtures were analyzed on a 10% native PAGE.

Fig 3

The three putative AdcR binding sites within the rpsNb promoter are involved in full Zn-dependent promoter repression. Transcriptional lacZ fusions with either the native rpsNb promoter region or the rpsNb promoter region containing point mutations destroying each putative AdcR-box (isolated and combined mutations) were constructed and introduced into the WT strain. The native rpsNb promoter region was also introduced in a ΔadcR mutant strain. The dark red boxes represent native AdcR-boxes, whereas light red represents mutated AdcR-boxes. β-Galactosidase assays were performed as described in Materials and Methods. The activity of the promoters was measured in Zn-excess CDM (300 µM of ZnSO₄) (black bars). The values shown are the means ± standard deviations of three independent assays. The asterisks indicate P values obtained using an unpaired Student t test compared to the promoter activity of the native rpsNb promoter region in the WT strain. *P < 0.05; **P < 0.01; ***P < 0.001. The dark red rectangle indicates native AdcR box and the light red rectangle indicates a mutated AdcR box. The position of the predicted −35 hexamer is indicated by a diamond.

Fig 4

Expression of the rpsNb and sak_RS01240 genes is gradually repressed by Zn. The rpsNb and sak_RS01240 promoter activity was measured in Zn-restricted CDM supplemented with various amounts of added metals (0 to 300 µM). Cells containing the P_rpsNb-lacZ transcriptional fusions were grown until the mid-exponential phase of growth (OD₆₀₀ = 0.4), and β-galactosidase assays were performed as described in Materials and Methods. The reference value (100%) corresponds to the rpsNb or sak_RS01240 promoter activity in Zn-restricted CDM (0 µM Zn). The values shown are mean results ± standard deviations. The asterisks indicate P values obtained using an unpaired Student t test, comparing the promoter activity of cells grown in Zn-restricted CDM and cells grown in CDM with the various added Zn concentrations. **P < 0.01; ***P < 0.001.

Fig 5

Effect on zinc repression of rpsNb of different mismatches in the AdcR box. Transcriptional lacZ fusions with either the rpsNb promoter region containing only the native AdcR-Box1 or the rpsNb promoter region with substitution of selected nucleotide were constructed and introduced into the WT strain. The dark red boxes represent functional AdcR-boxes, whereas the light red box represents inactivated AdcR-box. Bacteria were grown either in Zn-restricted CDM or in CDM supplemented with 300 µM Zn until the mid-exponential phase of growth (OD₆₀₀ = 0.4). β-Galactosidase assays were performed as described in Materials and Methods. Results are expressed as a relative promoter activity with the reference value (100%) corresponding to the promoter activity in Zn-restricted CDM. The values shown are the means ± standard deviations of three independent assays. The asterisks indicate P values obtained using an unpaired Student t test between the promoter activity of the native AdcR-Box1 compared to AdcR boxes carrying mismatches. *P < 0.05; **P < 0.01.

Fig 6

Comparative expression of the RpsNa and RpsNb proteins. Cells containing the lacZ transcriptional fusions or proteins fused to the 3×Flag were grown until the mid-exponential phase of growth (OD₆₀₀ = 0.4) in Zn-restricted CDM or in CDM supplemented with 100 µM of Zn. (A) The rpsNa and rpsNb promoter activity was measured by β-galactosidase assays. The values shown are mean results ± standard deviations. The asterisks indicate P values obtained using unpaired Student’s t test, comparing promoter activity of cells grown in Zn-restricted CDM and cells grown in CDM with 100 µM added Zn concentrations. ***P < 0.001. (B) Expression of RpsNa-3×FLAG and RpsNb-3×FLAG of an expected size of 9.77 and 13.2 kDa, respectively. Protein extract from S. agalactiae with empty plasmid (left lane) was used as a specificity control. Immunoblot analysis was performed with anti-Flag antibody. Brilliant Blue staining was done as a protein loading control.

Fig 7

RpsNb is important for bacteria growth in Zn-restricted CDM. The S. agalactiae WT, ΔrpsNb strains and the two complemented strains ΔrpsNb + P(rpsNb) (plasmidic pTcvPtet complementation) and ΔrpsNb^c (chromosomic complementation) were grown in Zn-restricted CDM (0 µM ZnSO₄). The empty vector pTcvPtet (ᴓ) was inserted in the WT, ΔrpsNb, and ΔrpsNb^c strains as a control. Growth was monitored by OD₆₀₀ measurements every 30 min for 15 h. The data are presented as mean OD₆₀₀ measurements ± the standard deviations from three independent experiments.

Fig 8

The RpsNb ribosomal protein participates in S. agalactiae survival in human biological fluids. A909 WT and ΔrpsNb mutant strains were inoculated in equivalent numbers into human serum, cerebrospinal fluid (CSF), or amniotic fluid. Cocultures were incubated at 37°C for 24 h without agitation. The proportion of each strain was monitored by plating diluted cultures on TH agar containing erythromycin (10 µg mL⁻¹) and X-Gal (60 µg mL⁻¹) (see Materials and Methods). Results are presented as the means ± standard deviations for three independent cocultures. The asterisks indicate P values obtained using an unpaired Student t test to compare the proportion of the strain at T0 and its proportion at the indicated times. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 9

SAK_RS01240-RS01260 is a potential secondary Zn-transporter in S. agalactiae. The S. agalactiae WT, ∆sak_RS01240-RS01260, ∆adc/lmb single mutant, and ∆adc/lmb ∆sak_RS01240-RS01260 double mutant strains were grown overnight in CDM with 25 µM ZnSO₄ and inoculated at an OD₆₀₀ of 0.005 either in Zn-restricted (0 µM ZnSO₄) (A), Zn-replete CDM (10 µM ZnSO₄) (B), or Zn-excess (300 µM ZnSO₄) (C) conditions. Growth was monitored by OD₆₀₀ measurements every hour for 12 h. The data are presented as mean OD₆₀₀ measurements ± the standard deviations from three independent experiments.