Fig 1.
Tn-Seq identifies genes required or detrimental for the growth of S. aureus under low pH.
(A) Schematic representation of the Tn-Seq experimental protocol. The transposon library was grown in TSB at pH 7.3, pH 5.5, or pH 4.5 for approximately 10 generations, bacteria harvested and transposon insertion sites identified by sequencing. (B) Bacterial growth curves. The growth of two transposon mutant pools (library A and library B) in TSB pH 7.3, pH 5.5, or pH 4.5 medium was monitored by taking OD600 readings at defined time points. The dotted line represents the 10-generation cut off point at which the bacteria were harvested. The culture of Library A grown at pH 4.5 did not reach the desired OD600 value due to clumping and was instead harvested after 11 h of growth. (C and D) Volcano plots. As a visual representation of the number of essential and detrimental genes obtained following growth of S. aureus in (C) TSB pH 4.5 or (D) TSB pH 5.5 compared to pH 7.3 volcano plots were generated. On the x-axis the fold change in the number of transposon insertions per gene was plotted on a log2 scale with negative values indicating essential genes and positive values indicating detrimental genes. Vertical green, orange or red lines indicate 2-, 5- or 10-fold changes, respectively. The y-axes are the q-values (Benjamini–Hochberg corrected p-values) and the black horizontal line indicates a q-value of 0.1. Each dot represents one gene and colouring follows the fold-change scheme whenever the q-value threshold was met. Very small q-values were truncated to fit onto the graph. (E and F) Venn diagrams. The list of genes with a 2-fold decrease (essential genes) or a 2-fold increase (detrimental genes) in the number of transposon insertions and a Benjamini–Hochberg value of ≤ 0.1 were compared between libraries A and B and displayed in Venn diagrams for (E) pH 4.5 and (F) pH 5.5 stress conditions. The overlapping 31 essential and 10 detrimental genes for the pH 4.5 growth condition are listed in Table 1. The image in Fig 1A was modified from Figure 1(a) published in Schuster et al. [25] under a CC BY 4.0 deed license (https://creativecommons.org/licenses/by/4.0/).
Table 1.
Genes identified by Tn-Seq as essential or detrimental for growth of S. aureus in TSB pH 4.5 medium.
Fig 2.
A number of factors involved in the cell wall remodelling were identified as essential in the TnSeq experiment.
Schematic representation of the S. aureus cell wall with proteins highlighted in this study as essential for growth in TSB pH 4.5 and involved in cell wall synthesis or remodelling depicted. If the proteins are part of complexes, the proteins identified in the Tn-seq experiment are shown in colour while the others are shown in grey.
Fig 3.
Growth plate and survival analysis of S. aureus mutant strains with transposon insertions in genes identified as essential or detrimental for growth at pH 4.5.
(A-G) Bacterial growth on TSA pH 4.5 plates. Overnight cultures of the indicated WT and mutant strains were serially diluted and aliquots spotted onto TSA pH 4.5 plates. Images were taken following 24 h incubation at 37°C. Each image is a representative of three experiments. (H-J) Acid stress survival curves. Overnight cultures of WT JE2 as well as (H) JE2 lepA::Tn, (I) JE2 noc::Tn or (J) JE2 lytH::Tn were washed and diluted into TSB pH 2.5 medium. Immediately afterwards (T = 0 h) and at 2 h intervals up to 8 h aliquots were removed and CFUs determined by plating dilutions onto TSA plates. The CFU count at T = 0 h was set to 100% for each strain and % survival at the subsequent time points calculated. The average and standard deviations of the % survival from three (H and J) or four (I) experiments were plotted.
Fig 4.
Genetic complementation restores the acid growth ability of the 0846::Tn mutant strain.
(A) Bacterial growth on TSA pH 4.5 plates. Overnight cultures of LAC* pCL55 (WT) LAC* 0846::Tn pCL55 (0846::Tn), and the complementation strain LAC* 0846::Tn pCL55-0846 (0846::Tn comp.) were serially diluted and aliquots spotted on TSA pH 4.5. Images were taken following 24 h incubation at 37°C. Each image is representative of three experiments. (B-C) Bacterial growth curves. The same strains as in (A) were grown in (B) TSB pH 7.3 or (C) TSB pH 4.5 medium in 96-well plates and OD600 measurements taken at the indicated time points. The average readings and standard deviations of three independent repeats were plotted.
Fig 5.
0846 is a main histidine transporter in S. aureus.
(A) Bacterial growth on TSA pH 4.5 plates. Overnight cultures of JE2 (WT), 0846::Tn, cpa1-1::Tn, cpa1-2::Tn, cpa2::Tn, nhaC1::Tn, nhaC2::Tn strains were serially diluted and spotted on TSA pH 4.5 plates. Images were taken following 24 h incubation at 37°C. Each image is representative of three biological experiments. (B) Schematic representation of the S. aureus genes and operons suggested to be regulated by the proposed histidine transcription factor HisR. (C). Histidine uptake assay. 3H radiolabelled L-histidine was added to washed mid-log phase cultures of LAC* pCL55 (WT), LAC* 0846::Tn pCL55 (0846::Tn), and the complementation strain LAC* 0846::Tn pCL55-0846 (0846::Tn comp.). At the indicated time points, culture aliquots were removed, filtered, washed and the accumulated radioactivity in each sample measured as counts per minute (CPM) using a scintillation counter. The CPM values were normalised to OD600 values, and the average CPM / OD600 and standard deviations of three independent experiments were plotted.
Fig 6.
Histidine and its uptake are important for the growth of S. aureus under acid stress conditions.
(A-C). Bacterial growth curves in CDM pH 7.2. S. aureus strains (A) LAC* pCL55 (WT), (B) LAC* 0846::Tn pCL55 (0846::Tn), and (C) LAC* 0846::Tn pCL55-0846 (0846::Tn comp.) were grown in CDM pH 7.2 with or without 130 μM histidine and OD600 readings taken at timed intervals. The average readings and standard deviation of three independent repeats were plotted. (D-F) Bacterial growth curves in CDM pH 4.3. The growth of (D) LAC* pCL55 (WT), (E) LAC* 0846::Tn pCL55 (0846::Tn), and (F) LAC* 0846::Tn pCL55-0846 (0846::Tn comp.) was monitored and plotted as described for panels A-C, but using CDM pH 4.3 with or without 130 μM histidine.
Fig 7.
The acid-sensitive phenotype of the 0846::Tn mutant strain can be bypassed by activating the stringent response.
(A) Bacterial growth on TSA pH 4.5 plates. Overnight cultures of LAC* (WT), the 0846::Tn mutant or the suppressor strains 0846::Tn-S1, 0846::Tn-S2, 0846::Tn-S3, 0846::Tn-S4, 0846::Tn-S5, 0846::Tn-S6, 0846::Tn-S7, 0846::Tn-S8 were serially diluted and aliquots spotted on TSA pH 4.5 plates. Images were taken following 24 h incubation at 37°C. This experiment was only performed once, but the presence of genomic mutations was confirmed by whole genome sequencing (B). Schematic representation of the stringent response. Under nutrient and amino acid surplus, CodY is in the GTP-bound form, interacts with DNA and prevents gene expression. Under conditions of nutrient or amino acid limitation, Rel produces (p)ppGpp from GTP, decreasing intracellular concentrations of GTP. At low cellular GTP levels, CodY will be in a GTP-unbound state and will no longer bind to DNA, thus allowing expression of the stringent response genes. (C) Bacterial growth curves. LAC* (WT), LAC* codY::Tn, LAC* 0846::Tn mutant and the indicated LAC* 0846::Tn suppressor strains were grown in (C) TSB pH 4.5 medium and OD600 readings taken at timed intervals. The average readings and standard deviations of three independent repeats were plotted.
Table 2.
Genomic alterations identified in the LAC*0846::Tn suppressor strains.
Fig 8.
The histidine transporter gene 0846 is induced in WT while the histidine biosynthesis genes are induced in the 0846 mutant strain under acid stress.
The expression of the histidine biosynthesis genes hisD and hisG and the histidine transporter gene 0846 were determined by qPCR in different S. aureus strains following growth in TSB pH 7.3 or TSB 4.5 medium. (A-D) To determine if histidine biosynthesis or transport genes are activated during acid stress, hisD, hisG and where applicable 0846 expression levels in TSB pH7.3 versus TSB 4.5 medium were determined for (A) LAC* (WT), (B) LAC* codY::Tn, (C) LAC* 0846::Tn and (D) the suppressor strain LAC* 0846::Tn / codYE163*. (E-F) To determine differences in histidine biosynthesis or transport gene transcription levels between WT and mutant strains, the hisD, hisG and 0846 levels were compared between WT and the indicated mutant strains following growth in (E) TSB pH7.3 medium and (F) TSB pH 4.5 medium. The relative expression of hisD, hisG and SAUSA300_0846 were calculated using the ΔΔCt method and using gyrB as assay reference gene. The averages values of the fold change in expression level (2-ΔΔCt) and standard deviations from three replicates were plotted. For statistical analysis student’s t-tests were performed to compare in panels A-D the transcript levels of the indicated gene at pH 7.3 versus pH 4.5. An asterisk (*) indicates a p≤ 0.05 and ns = not significant. For statistical analysis in panels E and F, student’s t-tests were performed for the comparison of the 0846 transcript levels between WT and the codY::Tn. An asterisk (*) indicates a p≤0.05 and ns = not significant. Student’s t-tests with Bonferroni multiple comparison correction were performed to compare the transcript level between WT and all three mutant strains. In this case, the asterisk (*) indicates a p≤ 0.0167.
Fig 9.
Assessing the importance of the histidine utilization pathway, cellular histidine levels and cytosolic pH for the survival of S. aureus under acid stress.
(A) Bacterial growth on TSA pH 7.3 and 4.5 plates. Overnight cultures of JE2 (WT), the JE2 0846::Tn mutant and two histidine utilization pathway mutants JE2 hutH::Tn and JE2 hutU::Tn were serially diluted and aliquots spotted on TSA pH 7.3 (left panel) or TSA pH 4.5 plates (right panel). Images were taken following 24 h incubation at 37°C. The experiment was repeated three times, and a representative image is shown. (B) Cellular histidine levels. LAC* (WT), LAC* 0846::Tn and the complementation strain LAC* 0846::Tn compl. were grown to mid-log phase in TSB pH 4.5 medium, extracts prepared as described in the method section and cellular histidine levels determined by LC-MS. The experiment was performed with 5 biological replicates and the average values and standard deviation of the histidine specific signal normalized for the sample protein content was plotted. For statistical analysis a one-way ANOVA test was performed followed by Turkey’s multiple comparison test. An asterisk (*) indicates p≤0.05 and ns = not significant. (C-D) Intracellular pH assay. S. aureus strains (C) LAC* (WT) and LAC* 0846::Tn and (D) LAC* 0846::Tn and the suppressor strain LAC* 0846::Tn / codYE163* were grown in TSB pH 7.3 or TSB pH 4.5 to mid-log phase and the intracellular pH determined using the pHrodo Red dye as described in the materials and method section. The average values and standard deviations of three independent experiments were plotted. (E) The change in cytosolic pH (ΔpH) for LAC* (WT), LAC* 0846::Tn, and the suppressor strain LAC* 0846::Tn / codYE163* following growth in TSB pH 4.5 medium versus pH 7.3 medium derived from the data shown in panels C and D was plotted.