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Journal of Bacteriology logoLink to Journal of Bacteriology
. 2004 Dec;186(24):8407–8423. doi: 10.1128/JB.186.24.8407-8423.2004

Effect of Mild Acid on Gene Expression in Staphylococcus aureus

Brian Weinrick 1, Paul M Dunman 2,, Fionnuala McAleese 2, Ellen Murphy 2, Steven J Projan 2, Yuan Fang 1, Richard P Novick 1,*
PMCID: PMC532443  PMID: 15576791

Abstract

During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, ∼5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.


Facultative pathogens such as staphylococci produce a wide variety of accessory proteins. Many of these are involved in, or required for, infectivity and are collectively referred to as the virulon. It is widely believed that the genes encoding these proteins are regulated according to the exigencies of the local sites in which the organism is able to establish an infection. The evidence in support of this very logical view is, at best, rather sketchy, largely for two reasons. Firstly, it has, until very recently, been unapproachably difficult to determine the putative locale-specific gene expression patterns. Secondly, it has been no easy matter to identify the specific environmental factors that would determine these patterns. As a preamble to the ultimate objective of defining in vivo environmental factors that influence the expression of accessory genes involved in local pathogenesis, we have begun to reexamine the effect of certain nutrients and other chemicals, including glucose, acidic pH, salt, salicylic acid, and subinhibitory concentrations of antibiotics, on the expression of virulence genes during growth in vitro. These substances have widespread effects on the transcription of virulence genes and other accessory genes, which are presumably initiated through signaling elements and mediated through the complex regulatory network that governs the expression of all accessory genes (34). Our entrée into the transduction of environmental signals was provided by the observation that all of these substances affected transcription of a key signaling locus, sae, which is involved in the regulation of the expression of many exoproteins (36). In that study, which was initiated by a determination of the effect of glucose, it was shown that sae has a complex transcriptional pattern that undergoes a very striking switch during in vitro growth at neutral pH, in which a 2.1-kb transcript, present initially, is replaced by three other, of 3.2, 2.6, and 0.5 kb (36). When the Staphylococcus aureus strain tested is grown in the presence of glucose, the switch occurs but the new transcripts are quickly turned off. At this point the glucose is exhausted and the pH has dropped from 7.5 to 5.5, suggesting that the drop in pH rather than the presence of glucose is responsible for the turnoff (36). Here, we confirm that pH affects the expression of sae and show that a modest reduction in pH, at which the organisms survive and grow, probably without activating the full-scale acid stress response, affects the expression of many sae-regulated genes, as well as many genes that do not belong to the sae regulon. To define this newly identified mild acid stimulon (MAS), we have used transcriptome profiling by microarray. The MAS does not represent an acid-shock response; the cultures used were pregrown in pH-adjusted media such that at the first time point the cultures were in steady-state exponential growth. As described here, the MAS consists of all S. aureus genes whose transcript level differs by at least twofold during growth at pH 5.5 versus that at pH 7.5.

MATERIALS AND METHODS

Bacterial strains and plasmids.

Bacterial strains and plasmids used in this study are listed in Table 1. The standard strain used in most of the work is RN6734, a φ13 lysogen of RN6390B. These two agr group I strains are in the NCTC8325 lineage and therefore have an rsbU defect, leading to a partial σB-negative phenotype (23).

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid Genotype or description Reference or source
Strains
    DH5α F φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK mK+) Promega
    RN4220 Restriction-deficient mutant of strain 8325-4 Kreiswirth et al. (27a)
    RN6734 phi13 lysogen of RN6390B Vojtov et al. (52)
    PC1839 8325-4 sarA::Km Chan & Foster (8)
    RN9388 RN6734 sarA::Km; transductant of PC1839 Tegmark et al. (48a)
    KT201 8325-4 sarH1::pKT200 Em (aka sarS) This work
    RN9897 RN6734 sarS::Em; transductant of KT201 This work
    ALC1905 RN6390 sarT::Em Schmidt et al. (45a)
    RN9899 RN6734 sarT::Em; transductant of ALC1905 This work
    RN9375 8325-4 sigB::Tc Nicholas et al. (33a)
    RN9898 RN6734 sigB::Tc; transductant of RN9375 This work
    CYL807 COL arlR::Tn551; gift of C. Lee (Kansas City, Kans.) This work
    RN9896 RN6734 arlR::Tn551; transductant of CYL807 This work
    PM614 PM466 rot::Tn917 McNamara et al. (32)
    RN9886 RN6734 rot::Tn917; transductant of PM614 This work
    RN9360 Replacement of rsbU deletion in 8325-4, adjacent Tc; gift of I. Kullik Novick and Jiang (36)
    RN10029 RN6734 rsbU+ Tc; transductant of RN9360 This work
    RN7206 phi13 lysogen of RN6911 (Δagr::tetM) Novick et al. (36a)
Plasmids
    pRN7044 Ptst::blaZ transcriptional fusion Vojtov et al. (52)
    pRN7166 pRN7044 with Emr replaced by Cmr This work
    pRN6827 pSA3800 Phla::blaZ Vandenesch and Novick (unpub.)
    pRN7041 PsspA::blaZ transcriptional fusion Vojtov et al.
    pCN50 (Source of Cm cassette, 1-kb ApaI/XhoI fragment) Charpentier et al. (submitted)

Media and growth conditions.

S. aureus was cultured in Casamino Acids-yeast extract-glycerophosphate (CYGP) broth (35), without glucose, and which was adjusted to pH 5.5 or 7.5. Broth was inoculated to an initial density of 5 × 107 CFU ml−1 with S. aureus from GL agar plates with appropriate antibiotic and grown overnight at 37°C. Cultures were grown in Erlenmeyer flasks at a 1/10 volume and incubated with shaking at ∼220 rpm at 37°C. Cultures were monitored turbidometrically with a Klett-Summerson (New York, N.Y) colorimeter at 540 nm. After reaching ∼3 times the initial inoculum (growth from ∼15 to ∼50 Klett units), cultures were diluted twofold and grown again to 50 Klett units. Cultures were again diluted twofold. The time zero (T0) time point for all time course experiments was defined when the density reached 50 Klett units for the third time (∼1.5 × 108 CFU ml−1). For cultures grown without glucose, the pH at the end of 6 h was always within 0.2 units of the starting pH.

RNA purification.

Cells were harvested by centrifugation at specified time points and treated with RNA Protect reagent from QIAGEN (Valencia, Calif.). Cells were mechanically disrupted by agitation with glass beads using a Fast-Prep apparatus (Q-Biogene, Carlsbad, Calif.), and RNA was purified using the RNeasy kit from QIAGEN. RNA integrity was checked by agarose gel electrophoresis in Tris-acetate buffer, and RNA was quantitated by determination of absorbance at 260 nm.

Northern blot hybridization.

RNA from equal numbers of cells was separated by denaturing gel electrophoresis (morpholinepropanesulfonic acid-formaldehyde) on 1% agarose gels. RNA was transferred to Hybond N+ membranes from Amersham by vacuum and UV cross-linked. [α-32P]dATP-labeled (Amersham) probes were generated by PCR and hybridized to the blots overnight. Washed blots were exposed to phosphorimager screens, which were read by using a Molecular Dynamics PhosphorImager. PCR primers were obtained from Integrated DNA Technologies (Coralville, Iowa), and their sequences are listed in Table 2.

TABLE 2.

Oligonucleotide primers used in this study

Primer Sequence (5′-3′) N315 locus
16S F GGTGAGTAACACGTGGATAA SArRNA01
16S R ATGTCAAGATTTGGTAAGGTT SArRNA01
RNAIII F ATGATCACACAGAGATGTGA SAS065
RNAIII R CTGAGTCCTAGGAAACTAACTC SAS065
sarA F GAGTTGTTATCAATGGTCACTTATGCTG SA0573
sarA R GTGATTCGTTTATTTACTCGACTC SA0573
hla F TTAGCCTGGCCTTCAGCC SA1007
hla R TGCCATATACCGGGTTC SA1007
saeS F GGCTTCTGAAATTACGCAACAAATG SA0660
saeS R GTTACAGTCACCGTAGTTCCCAC SA0660
rot F CTCTACTTGCAATCGCATCACTG SA1583
rot R GGGATTGTTGGGATGTTTGTTAATAC SA1583
spa F GGCACTACTGCTGACAAAATTGCTGCAG SA0107
spa R GTTCGCGACGACGTCCAGCTAATAACGCTGC SA0107
sspA F GACAACAGCGACACTTG SA0901
sspA R CTGAATTACCACCAGTTG SA0901
cap5b F GTACAGTTGTATCGAATGTAGCG SA0145
cap5b R GTGCATCAGTCACAGTATTAAC SA0145
IrgAB F GCCGGATCCGAAGTGAGCCATCTATA SA0252
IrgAB R GCCGAATTCGATAATAACAATGGCTC SA0252

Microarray analysis.

A custom DNA microarray prepared by Affymetrix for Wyeth was used. The microarray contains probe sets for all conserved, nonredundant open reading frames from six published S. aureus genome sequences, unique GenBank entries, as well as strain N315 intergenic regions. Design of the chip has been detailed by Dunman et al. (19). All microarray procedures were performed as described by Beenken et al. (3). Briefly, biotinylated cDNA was prepared and hybridized to the microarray. The arrays were read with an Affymetrix GS3000 scanner, and data were analyzed with GeneSpring software version 6.1 (Silicon Genetics, Redwood City, Calif.). All procedures were done in duplicate, and results are reported for genes with a twofold or greater expression differential (P ≤ 0.5) between the two conditions.

Exoprotein analyses.

Exoproteins were analyzed by the method of Laemmli et al. (28). Briefly, culture supernatants from equal numbers of cells were precipitated with 10% trichloroacetic acid, and the pellets were resuspended in 2% sodium dodecyl sulfate (SDS) and boiled for 3 min. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), gels were stained with Coomassie blue and scanned. β-Lactamase was assayed by the nitrocefin method adapted for use in microtiter plates (52).

Plasmid construction.

The ∼1-kb chloramphenicol resistance cassette was removed from plasmid pCN50 by digestion with ApaI and XhoI. The fragment was isolated by gel electrophoresis in Tris-acetate buffer and extracted with a QIAquick gel extraction kit (QIAGEN). Likewise, the vector backbone of pRN7044 was isolated by digestion with ApaI and XhoI followed by gel electrophoresis and gel extraction. The chloramphenicol cassette was ligated into the pRN7044 backbone with T4 DNA ligase. The ligation product was used to transform chemically competent Escherichia coli DH5α. DNA of the resulting plasmid, pRN7166, was isolated with a QIAprep spin miniprep kit, introduced into RN4220 by electroporation, and transferred to other S. aureus strains by transduction with phage φ11.

RESULTS

Effect of pH on sae transcription.

To study the effect of pH on sae expression independently of glucose, we grew cultures of strain RN6734 at pH 5.5 and 7.5 in CYGP broth without glucose and prepared Northern blots of whole-cell RNA from culture samples taken at three standard time points, T0, T3, and T6, as defined in Materials and Methods. pH did not vary by more than 0.1 to 0.2 units throughout the 6-h period. The Northern blot obtained with an saeS probe for these RNA samples (Fig. 1) demonstrates that the transition in the sae transcription pattern occurs at pH 7.5 but not at pH 5.5 and that the level of transcription is decreased at the lower pH.

FIG. 1.

FIG. 1.

Northern blot analysis of the effect of pH on saeS transcripts. Whole-cell RNA was extracted from RN6734 grown in CYGP broth without glucose and adjusted to pH 5.5 or 7.5. The RNA was separated on a denaturing gel and vacuum blotted; the blot was hybridized with a radiolabeled DNA probe complementary to saeS. The probe was detected with a PhosphorImager. Equalization was determined by subsequent blotting with a probe complementary to 16S rRNA.

Response of exoprotein gene promoters to changes in pH.

If the sae locus is involved in a global response to this pH differential, sae-regulated genes would be predicted to respond to changes in pH in this range. Accordingly, transcriptional fusions using staphylococcal β-lactamase (blaZ) as a reporter were used to test for promoter activity in response to changes in pH. The blaZ reporter, under a constitutive Pbla promoter, was insensitive to changes in pH in the range of pH 5.5 to 7.5 (data not shown). Plasmid-carried β-lactamase fusions involving Phla, Ptst, and PsspA were introduced into various strains and tested for β-lactamase activity during growth at pH 5.5 versus 7.5. The promoters for these three exoprotein genes were selected because, although postexponentially up-regulated by agr, they are regulated differentially by sar and sae (8, 36, 52; for a review, see the report of Novick 34). Results for the β-lactamase fusions are shown in Fig. 2A to C. The three promoters responded to changes in pH generally in parallel with their response to sae. Thus, at pH 7.5, Phla-blaZ shows the typical postexponential induction; however, at pH 5.5, there is a gradual increase of β-lactamase activity but no clear postexponential induction pattern. Ptst-blaZ also shows postexponential induction typical of agr-regulated exoprotein genes at pH 7.5 (34). This pattern is completely absent at pH 5.5, even though agr expression is induced at this pH. Finally, PsspA-blaZ shows a postexponential induction pattern that is similar at the two pH levels. These results imply that one or more pH-sensitive regulatory gene products may control expression from these promoters.

FIG. 2.

FIG. 2.

Transcriptional reporter analysis of the effect of pH on transcription of genes coding for several exoproteins, the effect of sarA on pH-dependent sspA transcription, and the effect of rsbU and sigB on pH-dependent hla transcription. RN6734 transduced with pRN7044 (Ptst::blaZ) (A), pRN6827 (Phla::blaZ) (B), or pRN7041 (PsspA::blaZ) (C) was assayed for β-lactamase activity during growth at pH 5.5 or pH 7.5. (D) pH-dependent β-lactamase activity of RN9388 (RN6734 ΔsarA) transduced with pRN7041, measured during growth. (E) pH-dependent β-lactamase activity of RN10029 (RN6734 rsbU+) transduced with pRN6827, measured during growth. (F) pH-dependent β-lactamase activity of RN9898 (RN6734 ΔsigB) transduced with pRN6827, measured during growth.

Effects of regulatory genes.

To test known regulatory genes for possible roles in pH-dependent regulation, we transferred null mutations in sarA, sarS, sarT, rot, arlRS, and sigB and a molecular repair of rsbU into RN6734 and tested for their effects using the β-lactamase fusion plasmids. The expectation was that a mutation affecting a required regulatory gene would abolish the pH-dependent expression differential, with respect both to expression levels and to kinetics. As shown in Fig. 2 and 3, none of the regulatory mutations tested eliminated the observed pH differential. Nevertheless, several informative results were obtained: (i) from the results shown in Fig. 2C and D, the sarA mutation dramatically increased the pH-dependent differential expression of the PsspA-blaZ reporter at pH 5.5, but had little or no effect at pH 7.5. This result may be consistent with previous experiments done in the presence of glucose, demonstrating a major increase in sspA expression in a sarA mutant background (8, 27), because these would have had a low pH at the time the samples were taken. Additionally, the sarA mutation did not eliminate the temporal up-regulation of sspA. (ii) Figure 2E and F show that hla expression was pH dependent in both wild-type and sigB mutant cells and was sharply up-regulated at pH 7.5 but not at 5.5. (iii) The mutation affecting sarA, but not those affecting sarS or sarT, profoundly decreased Ptst-blaZ expression and greatly reduced temporal induction (Fig. 3). (iv) The arl mutation appears to up-regulate the Ptst-blaZ reporter at all time points at pH 5.5 but only at the latest time point at pH 7.5. Finally, the rot mutation enhanced the temporal induction of the Ptst-blaZ reporter at pH 7.5. This result is consistent with the reported repressor activity of Rot (32), which was presumably determined at pH 7.5.

FIG. 3.

FIG. 3.

Transcriptional reporter analysis of the effect of pH on the transcription of genes coding for Tsst at pH 7.5 (A) and pH 5.5 (B) in various genetic backgrounds. In Emr-marked mutants, Ptst::blaZ reporter pRN7166 was used.

Identification of the MAS.

Given the apparent complexity of the response of exoprotein gene expression to changing pH, it seemed worthwhile to examine the entire MAS at the transcriptional level. For this purpose, we employed a staphylococcal DNA microarray (Affymetrix) (3, 6, 19, 20).

To perform this analysis, we prepared whole-cell RNA from two cultures of RN6734 in CYGP broth without glucose, each at three different time points. The cultures were started with an inoculum taken from an overnight GL plate (pH, ∼7.2). One culture was started at pH 5.5, the other at pH 7.5, and the two were grown as described in Materials and Methods, with samples taken at T0, T3, and T6 for RNA preparation. Comparable amounts of cDNA were hybridized to the microarrays for each experimental point. In Table 3 are shown the results for all genes whose transcript level differed by at least twofold between the two pH conditions, at any of the time points. In this presentation, to avoid any implications of regulatory mechanism, we refer to up or up-regulated and down or down-regulated in comparing transcript levels between the two conditions. In all, 424 genes were identified; approximately twice as many were up-regulated at pH 5.5 as were down-regulated. In addition to genes encoding proteins ostensibly involved in pH homeostasis, there were many encoding proteins involved in amino acid uptake or metabolism, carbohydrate uptake or metabolism, virulence (encoding either secreted or surface proteins), and transcriptional regulation and, curiously, a considerable number of prophage genes were found to be pH regulated. Among the prophage-encoded genes, a much greater number (31 of 32) were down-regulated at pH 5.5 than were up-regulated. Given that prophage genes are not generally expressed in the prophage state, this result could indicate that spontaneous prophage induction occurs more frequently at pH 7.5. Among the surface proteins, a much greater number (72 of 84) were up-regulated at pH 5.5 than were down-regulated. Of the genes encoding secreted proteins, about the same number were up- as down-regulated. As a functional category, transporters with roles in nutrient accumulation and maintenance of homeostasis were differentially expressed in a ratio that paralleled that of the stimulon as a whole, with approximately twice as many up-regulated at pH 5.5 as at pH 7.5. Some genes involved in homeostasis have roles in maintenance of intracellular pH (ure operon), others are osmoprotective (opuC operon), and some contribute to protection against both low pH and high osmolarity (kdp operon) (7, 47).

TABLE 3.

Genes with a ≥2-fold change in transcript level, pH 7.5 versus 5.5a

ORF no. Gene Description Fold change at pH 5.5
Effect of regulator
Functional category
T0 T3 T6 agr sarA rot
Genes up-regulated at pH 5.5
    N315-SA0021 yycJ Conserved HP 2.0 Down
    N315-SA0091 plc 1-Phosphatidylinositol phosphodiesterase precurosr 2.4
    N315-SA0092 HP 2.3
    N315-SA0111 sirA Lipoprotein 3.8 Surface
    N315-SA0112 HP, similar to cysteine synthase 2.1
    N315-SA0121 HP 2.3
    N315-SA0131 deoD Purine nucleoside phosphorylase 2.9
    N315-SA0144 cap5A Capsular polysaccharide synthesis enzyme Cap5A 4.0 3.6 2.6 Surface
    N315-SA0145 cap5B Capsular polysaccharide synthesis enzyme Cap5B 3.3 3.3 2.1 Surface
    N315-SA0146 cap5C Capsular polysaccharide synthesis enzyme Cap8C 4.2 3.2 Surface
    N315-SA0147 cap5D Capsular polysaccharide synthesis enzyme Cap5D 3.2 3.1 Surface
    N315-SA0148 cap5E Capsular polysaccharide synthesis enzyme Cap8E 3.3 2.9 Surface
    N315-SA0149 cap5F Capsular polysaccharide synthesis enzyme Cap5F 3.3 2.9 Surface
    N315-SA0150 cap5G Capsular polysaccharide synthesis enzyme Cap5G 3.6 3.6 Surface
    N315-SA0151 cap5H Capsular polysaccharide synthesis enzyme O-acetyl transferase Cap5H 2.8 3.2 Surface
    N315-SA0152 cap5I Capsular polysaccharide synthesis enzyme Cap5I 2.7 3.5 Surface
    N315-SA0153 cap5J Capsular polysaccharide synthesis enzyme Cap5J 3.2 Up Up Surface
    N315-SA0154 cap5K Capsular polysaccharide synthesis enzyme Cap5K 2.0 Surface
    N315-SA0155 cap5L Capsular polysaccharide synthesis enzyme Cap5L 2.1 2.8 Surface
    N315-SA0156 cap5M Capsular polysaccharide synthesis enzyme Cap5M 2.9 Surface
    N315-SA0157 cap5N Capsular polysaccharide synthesis enzyme Cap5N 2.9 Surface
    N315-SA0158 cap5O Capsular polysaccharide synthesis enzyme Cap8O 2.6 Surface
    N315-SA0162 aldA Aldehyde dehydrogenase homologue 6.3 Down
    N315-SA0170 Conserved HP 3.4 Up
    N315-SA0171 fdh NAD-dependent formate dehydrogenase 2.8 4.5 4.0
    N315-SA0173 HP, similar to surfactin synthetase 2.1 Up Up Secreted
    N315-SA0184 Conserved HP 3.0 Up
    N315-SA0185 Conserved HP 3.7 Up
    N315-SA0186 HP, similar to sucrose phosphotransferase enzyme II 5.0 Down Transport
    N315-SA0187 HP, similar to transcription regulator 5.2 Up Down Regulator
    N315-SA0223 atoB Acetyl-CoA acetyltransferase homologue 3.8
    N315-SA0224 fadB HP, similar to 3-hydroxyacyl-CoA dehydrogenase 3.6
    N315-SA0225 fadD HP, similar to glutaryl-CoA dehydrogenase 3.1
    N315-SA0226 fadE HP, similar to acid-CoA ligase 2.8
    N315-SA0227 fadX Conserved HP 2.4
    N315-SA0229 HP, similar to nickel ABC transporter nickel-binding protein 9.8 6.3 3.9 Transport
    N315-SA0244 tagF HP, similar to teichoic acid biosynthesis protein F 2.1 Up Surface
    N315-SA0258 rbsK Probable ribokinase 3.3 Transport
    N315-SA0259 rbsD Ribose permease 3.3 Transport
    N315-SA0260 HP, similar to ribose transporter RbsU 3.8 Transport
    N315-SA0262 HP 3.1
    N315-SA0270 HP, similar to secretory antigen precursor SsaA 2.8 Secreted
    N315-SA0291 HP 2.1 Up
    N315-SA0304 nanA N-Acetylneuraminate lyase subunit 2.7
    N315-SA0305 HP, similar to glucokinase 2.4
    N315-SA0318 HP, similar to transport protein SgaT 4.2 2.3 Transport
    N315-SA0319 Conserved HP 3.9 2.5
    N315-SA0320 HP, similar to PTS fructose-specific enzyme IIBC component 4.0 2.6 Transport
    N315-SA0321 HP, similar to transcription antiterminator BglG family 3.3 2.9 Regulator
    N315-SA0322 HP, similar to transcription regulator 2.6 Regulator
    N315-SA0325 glpT Glycerol-3-phosphate transporter 4.1 2.3 Transport
    N315-SA0428 Conserved HP 2.6 Up
    N315-SA0432 treP PTS enzyme II, phosphoenolpyruvate dependent 3.8 Down Transport
    N315-SA0433 Alpha-glucosidase 2.2 3.7 Down
    N315-SA0510 araB Probable l-ribulokinase 2.2
    N315-SA0519 sdrC Ser-Asp rich fibrinogen-binding, bone sialoprotein-binding protein 5.7 5.6 6.9 Surface
    N315-SA0547 mvk Mevalonate kinase 2.1
    N315-SA0548 mvaD Mevalonate diphosphate decarboxylase 2.1
    N315-SA0549 mvaK2 Phosphomevalonate kinase 2.0 Down
    N315-SA0550 Conserved HP 2.5
    N315-SA0587 Lipoprotein, streptococcal adhesin PsaA homologue 2.1 Surface
    N315-SA0599 abcA ATP-binding cassette transporter A 2.1 Transport
    N315-SA0605 HP, similar to dihydroxyacetone kinase 2.8 Down
    N315-SA0606 Conserved HP 2.6
    N315-SA0607 Conserved HP 3.1
    N315-SA0612 Conserved HP 2.1
    N315-SA0620 Secretory antigen SsaA homologue, similar to autolysin 2.7 Up Secreted
    N315-SA0622 HP, similar to AraC/Xy|S family transcriptional regulator 2.1 Regulator
    N315-SA0623 HP 2.1 2.2
    N315-SA0636 Conserved HP 2.4
    N315-SA0637 Conserved HP 2.9
    N315-SA0651 HP 4.2 Up
    N315-SA0666 6-Oyruvoyl tetrahydrobiopterin synthase homologue 2.4 Up
    N315-SA0667 Conserved HP 2.4 2.7
    N315-SA0711 Conserved HP 2.5
    N315-SA0738 HP 2.7 2.0 3.7
    N315-SA0739 Conserved HP 4.2 2.2 3.6 Up
    N315-SA0820 glpQ Glycerophosphoryl diester phosphodiesterase 2.3
    N315-SA0835 clpB ClpB chaperone homologue 2.3
    N315-SA0893 Conserved HP 2.1
    N315-SA0899 sspC Cysteine protease 2.2 Up Down Down Secreted
    N315-SA0900 sspB Cysteine protease precursor 2.3 Down Down Secreted
    N315-SA0901 sspA Serine protease; V8 protease; glutamyl endopeptidase 2.4 Secreted
    N315-SA0974 Conserved HP 2.3
    N315-SA0978 LPXTG-containing conserved HP 3.2 Surface
    N315-SA0979 Conserved HP 3.2
    N315-SA0980 HP, similar to ferrichrome ABC transporter 3.1 Transport
    N315-SA0981 HP, similar to ferrichrome ABC transporter 2.6 Transport
    N315-SA0982 srtB Conserved HP 2.1 Transport
    N315-SA0983 Conserved HP 3.1
    N315-SA0996 sdhB Succinate dehydrogenase iron-sulfur protein subunit 2.3
    N315-SA1007 hla Alpha-hemolysin precursor 4.4 Up Up Down Secreted
    N315-SA1041 pyrR Pyrimidine operon repressor chainA 2.0 Up Regulator
    N315-SA1042 pyrP Uracil permease 2.9 Transport
    N315-SA1043 pyrB Aspartate transcarbamoylase chain A 2.3 2.1
    N315-SA1044 pyrC Dihydroorotase 2.2
    N315-SA1045 carA Carbamoyl-phosphate synthase small chain 2.6 2.0 Up Up
    N315-SA1046 carB Carbamoyl-phosphate synthase large chain 2.4
    N315-SA1047 pyrF Orotidine-5-phosphate decarboxylase 2.6 2.2
    N315-SA1048 pyrF Orotate phosphoribosyltransferase 2.0 2.8 2.0
    N315-SA1056 HP 2.2 Up
    N315-SA1076 rnc RNase III 2.3
    N315-SA1081 rpsP 30S ribosomal protein S16 2.1
    N315-SA1089 sucD Succinyl-CoA synthetase 2.3
    N315-SA1120 HP, similar to transcription regulator GntR family 2.2 Up Up Regulator
    N315-SA1140 glpF Glycerol uptake facilitator 2.3 Down Down Transport
    N315-SA1141 gplK Glycerol kinase 2.6 Transport
    N315-SA1149 glnR Glutamine synthetase repressor 2.1 Regulator
    N315-SA1154 Conserved HP Up 2.2
    N315-SA1162 HP 2.1
    N315-SA1176 Conserved HP 2.6
    N315-SA1211 opp-2E Oligopeptide transporter putative ATPase domain 2.5 2.2 Transport
    N315-SA1212 opp-2D Oligopeptide transport ATPase 3.7 3.0 Transport
    N315-SA1213 opp-2C Oligopeptide transporter membrane permease domain 3.9 2.8 2.0 Transport
    N315-SA1214 opp-2B Oligopeptide transporter membrane permease domain 4.5 3.6 2.7 Up Up Transport
    N315-SA1215 HP 2.4
    N315-SA1235 Conserved HP 3.2
    N315-SA1245 odhA 2-Oxoglutarate dehydrogenase E1 2.3 Up
    N315-SA1252 Conserved HP 2.2
    N315-SA1265 Conserved HP 2.2
    N315-SA1275 Conserved HP 2.2 2.9 Up Up
    N315-SA1310 ansA Probable l-asparaginase 2.1
    N315-SA1318 HP 2.1
    N315-SA1321 HP 2.0
    N315-SA1410 grpE GrpE protein 2.1
    N315-SA1434 Acetyl-CoA carboxylase (biotin carboxylase subunit) 2.1
    N315-SA1476 HP 2.6
    N315-SA1531 ald Alanine dehydrogenase 3.3
    N315-SA1551 sgtA Probable transglycosylase 2.1
    N315-SA1554 acs Acetyl-CoA synthetase 2.3
    N315-SA1601 crcB Conserved HP 2.0
    N315-SA1609 pckA Phosphoenolpyruvate carboxykinase 5.0 Up
    N315-SA1630 splB Serine protease SplB 2.3 Up Up Down Secreted
    N315-SA1631 splA Serine protease SplA Up Up Up Down Secreted
    N315-SA1844 agrA Accessory gene regulator A 2.8 Up Up Regulator
    N315-SA1879 kdpC Probable potassium-transporting ATPase C chain 9.2 3.0 Down Transport
    N315-SA1880 kdpB Probable potassium-transporting ATPase B chain 3.3 11.0 2.9 Transport
    N315-SA1881 kdpA Probable potassium-transporting ATPase A chain 5.7 17.6 3.4 Down Transport
    N315-SA1882 kdpD Sensor protein KdpD 12.3 Regulator
    N315-SA1883 kdpE KDP operon transcriptional regulatory protein KdpE 10.2 Regulator
    N315-SA1889 HP 2.0 Up
    N315-SA1890 Conserved HP 2.4
    N315-SA1896 thiD Phosphomethylpyrimidine kinase 2.0
    N315-SA1929 pyrG CTP synthase 2.2 2.7
    N315-SA1931 HP, similar to spermine/spermidine acetyltransferase bit 2.1 Up
    N315-SA1937 Conserved HP 2.0 Down
    N315-SA1938 pyn Pyrimidine nucleoside phosphorylase 2.1
    N315-SA1947 czrA Repressor protein 2.6 2.3 Regulator
    N315-SA1948 czrB Cation-efflux system membrane protein homolog 2.1 2.3 Transport
    N315-SA1968 arg Arginase 2.1
    N315-SA1976 Conserved hypothetical protein 2.5
    N315-SA1979 HP, similar to ferrichrome ABC transporter (binding protein) 2.7 Transport
    N315-SA2006 HP, similar to MHC class II analog 2.0 2.3 Up Up Surface
    N315-SA2007 HP, similar to alpha-acetolactate decarboxylase 4.5 2.2 Up
    N315-SA2008 budB Alpha-acetolactate synthase 7.8 3.1
    N315-SA2060 HP, similar to transcription regulator MarR family 2.1 2.5 2.1 Regulator
    N315-SA2075 fdhD FdhD protein homologue 2.1
    N315-SA2079 HP, similar to ferrichrome ABC transporter fhuD precursor 2.4 Up Up Transport
    N315-SA2081 HP, similar to urea transporter 2.3 Transport
    N315-SA2082 ureA Urease gamma subunit 3.9 10.4 6.3 Down
    N315-SA2083 ureAB Urease beta subunit 5.7 12.8 8.4 Down
    N315-SA2084 ureC Urease alpha subunit 4.5 10.2 4.8 Down
    N315-SA2085 ureE Urease accessory protein UreE 2.8 5.6 3.8 Down
    N315-SA2086 ureF Urease accessory protein UreF 2.7 6.1 4.0 Down
    N315-SA2087 ureG Urease accessory protein UreG 2.0 3.6 3.0 Down
    N315-SA2088 ureD Urease accessory protein UreD 3.6 2.7 Down
    N315-SA2093 ssaA Secretory antigen precursor SsaA homolog 2.1 2.7 Down Secreted
    N315-SA2097 HP, similar to secretory antigen precursor SsaA 3.9 Secreted
    N315-SA2101 Conserved HP 2.0
    N315-SA2120 HP, similar to amino acid amidohydrolase 2.1
    N315-SA2126 HP 2.6
    N315-SA2142 semB HP, similar to multidrug resistance protein 3.9 5.3 4.2 Transport
    N315-SA2143 Conserved HP 4.0 6.3 5.7
    N315-SA2146 tcaA TcaA protein 2.0 Regulator
    N315-SA2159 HP, similar to transcription repressor of sporulation 2.3 Regulator
    N315-SA2165 HP, similar to transcriptional regulator tetR-family 2.1 2.5 Regulator
    N315-SA2166 HP, similar to cationic transporter 2.4 2.4 Transport
    N315-SA2168 HP 2.4
    N315-SA2173 HP 2.2
    N315-SA2204 gpm Phosphoglycerate mutase 3.0
    N315-SA2208 hlgC Gamma-hemolysin component C 2.3 Up Up Down Secreted
    N315-SA2216 HP, similar to ABC transporter 2.4 Transport
    N315-SA2217 HP, similar to lipoprotein inner membrane ABC-transporter 2.2 Transport
    N315-SA2227 Truncated HP, similar to d-serine/d-alanine/glycine transporter 4.3 Transport
    N315-SA2229 Conserved HP 3.4
    N315-SA2231 HP, similar to glucose epimerase 2.2
    N315-SA2234 opuCD Probable glycine betaine/carnitine/choline ABC transporter (membrane p) opuCD 2.6 2.2 Transport
    N315-SA2235 opuCC Glycine betaine/carnitine/choline ABC transporter (osmoprotective) opuCC 2.8 2.5 Transport
    N315-SA2236 opuCB Probable glycine betaine/carnitine/choline ABC transporter (membrane p) opuCB 3.3 Transport
    N315-SA2237 opuCA Glycine betaine/carnitine/choline ABC transporter (ATP-bindin) opuCA 4.9 2.4 Transport
    N315-SA2238 Conserved HP 2.1
    N315-SA2239 HP, similar to amino acid transporter 7.7 4.6 2.4 Transport
    N315-SA2246 HP 2.2
    N315-SA2296 HP, similar to transcriptional regulator MerR family 3.0 Regulator
    N315-SA2297 HP, similar to GTP-pyrophosphokinase 3.1 2.3 Down
    N315-SA2298 Conserved HP 2.8
    N315-SA2299 Conserved HP 3.3
    N315-SA2300 HP, similar to glucarate transporter 2.2 Down Transport
    N315-SA2304 fbp Fructose-bisphosphatase 2.5
    N315-SA2311 HP, similar to NAD(P)H-flavin oxidoreductase 2.0
    N315-SA2316 srtA Sortase 2.4 Transport
    N315-SA2318 sdhA HP, similar to l-serine dehydratase 4.4 2.8 Up
    N315-SA2319 sdhB HP, similar to beta-subunit of l-serine dehydratas 5.3 2.3 Up Up
    N315-SA2320 HP, similar to regulatory protein pfoR 4.6 2.2 Regulator
    N315-SA2326 ptsG PTS system, glucose-specific IIABC component 2.1 Down Transport
    N315-SA2328 cidB Conserved HP 2.4 2.6
    N315-SA2329 cidA Conserved HP 5.3 3.1
    N315-SA2330 cidR HP, similar to transcription regulator 2.4 Regulator
    N315-SA2332 HP, similar to secretory antigen precursor SsaA 2.8 Secreted
    N315-SA2338 HP 3.1 2.6
    N315-SA2341 rocA 1-Pyrroline-5-carboxylate dehydrogenase 3.0 Up Down
    N315-SA2343 HP 2.7 Up Up
    N315-SA2355 Conserved HP 2.4 4.1
    N315-SA2356 isaA Immunodominant antigen A 2.5 Surface
    N315-SA2405 betA Choline dehydrogenase 2.5
    N315-SA2406 gbsA Glycine betaine aldehyde dehydrogenase gbsA 3.6
    N315-SA2408 cudT Choline transporter 2.5 Transport
    N315-SA2411 citM HP, similar to magnesium citrate secondary transporter 2.1
    N315-SA2413 cysJ Sulfite reductase (NADPH) flavoprotein 2.0
    N315-SA2414 gpxA HP, similar to glutathione peroxidase 3.2 2.6 2.3 Down
    N315-SA2423 clfB Clumping factor B 2.8 Surface
    N315-SA2428 arcA Arginine deiminase 2.1 2.6 Up
    N315-SA2430 aur Zinc metalloproteinase aureolysin 2.0 Up Down Secreted
    N315-SA2434 Fructose phosphotransferase system enzyme fruA homolog 2.3 Down
    N315-SA2443 HP 3.1
    N315-SA2444 HP 2.5 3.0
    N315-SA2445 HP 3.1 3.2
    N315-SA2446 secY HP, similar to preprotein translocase secY 3.1 3.4 Transport
    N315-SA2447 HP, similar to streptococcal hemagglutinin protein 5.8 2.2 Up Up Surface
    N315-SA2459 icaA Intercellular adhesion protein A 3.0 Surface
    N315-SA2460 icaD Intercellular adhesion protein D 2.2 Surface
    N315-SA2463 lip Triacylglycerol lipase precursor 4.1 2.0 Up Down Secreted
    N315-SA2480 drp35 Drp35 2.8
    N315-SA2481 Conserved hypotehtical protein 2.1
    N315-SA2494 cspB Cold shock protein cspB 2.6
    N315-SAS014 HP Up
    N315-SAS017 HP 2.3
    N315-SAS039 HP 2.0
    N315-SAS064 HP (bacteriophage phiN315) 2.2
agrC 3.6 Regulator
LPXTG-motif cell wall achor domain protein, similar to ClfA 3.6 Surface
Similar to surface protein Pls Up Surface
Similar to Staphylococcus lugdunensis gene Fbl Up 3.0
    COL-SA0109 HP 2.1
    COL-SA0484 HP 2.1
    COL-SA0492 HP Up
    COL-SA0794 HP 2.9
    COL-SA0850 HP 2.9 2.1 3.9
    COL-SA0852 HP 5.6 7.8
    COL-SA0933 HP 2.7
    COL-SA1042 HP 2.4
    COL-SA1043 Glycosyl transferase group 1 2.5
    COL-SA1170 HP 2.3
    COL-SA1177 HP 2.3
    COL-SA1186 Antibacterial protein 23.5 Secreted
    COL-SA1187 Antibacterial protein 28.7 Secreted
    COL-SA1332 HP 2.4
    COL-SA1339 HP 2.0
    COL-SA1343 HP 3.4
    COL-SA1347 HP 2.3
    COL-SA1528 HP 2.2
    COL-SA1529 HP 2.0
    COL-SA1865 splE Serine protease SplE 2.3 Secreted
    COL-SA1877 epiB Lantibiotic epidermin biosynthesis protein EpiB 2.2 Secreted
    COL-SA2027 HP 3.4
    COL-SA2065 HP 9.5 2.8
    COL-SA2069 HP 13.9 3.2
    COL-SA2629 HP 3.1
    COL-SA2637 HP 2.2
    COL-SA2676 LPXTG-motif cell wall anchor domain protein 5.2 2.3 Surface
    COL-SA2693 HP 3.9 3.7
    Mu50-SAV0850 HP 2.2
    Mu50-SAV0866 HP Up
    Mu50-SAV0888 HP 3.1
    Mu50-SAV1991 HP 2.2
    Mu50-SAV1996 HP 2.2
    MW1600 2.4
    MW1681 Up
    MW1896 HP 2.3
    MW1926 HP 2.4
    MW2575 Ser/Thr repeat surface, similar to hemagglutinin 3.1 Surface
Genes downregulated at pH 5.5
    N315-SA0100 Conserved HP 5.7 Up
    N315-SA0107 spa Immunoglobulin G-binding protein A precursor Down Down Down Up Surface
    N315-SA0129 LPXTG-containing HP 2.1 Surface
    N315-SA0131 deoD Purine nucleoside phosphorylase 2.1
    N315-SA0162 aldA Aldehyde dehydrogenase homologue 2.3 Down
    N315-SA0206 msmX multiple sugar-binding transport ATP-binding protein 2.5 Transport
    N315-SA0207 HP, similar to maltose/maltodextrin-binding protein 3.6 Transport
    N315-SA0208 Maltose/maltodextrin transport permease homologue 5.2 Transport
    N315-SA0209 Maltose/maltodextrin transport permease homologue 5.9 Transport
    N315-SA0210 HP, similar to NADH-dependent dehydrogenase 5.0
    N315-SA0211 HP, similar to NADH-dependent dehydrogenase 4.4
    N315-SA0212 Conserved HP 3.9 Down Down
    N315-SA0214 uhpT Hexose phosphate transport protein 4.1 Transport
    N315-SA0218 pflB Formate acetyltransferase 2.6 2.9
    N315-SA0219 pflA Formate acetyltransferase activating enzyme 2.0 2.2
    N315-SA0232 ldh l-Lactate dehydrogenase 2.2
    N315-SA0233 PTS enzyme (EC 2.7.1.69), factor II homologue, maltose and glucose specific 2.3 Transport
    N315-SA0252 lrgA Holin-like protein LrgA 5.2 16.9 7.3 Surface
    N315-SA0253 lrgB Holin-like protein LrgB 5.7 20.5 10.2 Down Surface
    N315-SA0265 lytM Peptidoglycan hydrolase 2.7 Secreted
    N315-SA0303 HP, similar to sodium-coupled permease 2.7 Transport
    N315-SA0304 nanA N-Acetylneuraminate lyase subunit 2.9
    N315-SA0357 HP, similar to exotoxin 2 6.1 2.6 Secreted
    N315-SA0388 set12 Exotoxin 12 2.2 Secreted
    N315-SA0395 HP (pathogenicity island SaPIn2) 10.5
    N315-SA0478 Conserved HP 2.2
    N315-SA0562 adh Alcohol dehydrogenase I 3.7
    N315-SA0639 HP, similar to ABC transporter required for expression of cytochrome bd 2.2 Transport
    N315-SA0640 HP, similar to ABC transporter required for expression of cytochrome bd 3.0 Transport
    N315-SA0650 norA Quinolone resistance protein 2.7 Transport
    N315-SA0660 saeS Histidine protein kinase 2.1 2.3 Regulator
    N315-SA0661 saeR Response regulator 2.3 2.3 Regulator
    N315-SA0662 HP 5.2 3.0
    N315-SA0663 HP 9.0 3.4 Down
    N315-SA0691 sstD Lipoprotein, similar to ferrichrome ABC transporter 2.9 Transport
    N315-SA0744 ssp Extracellular ECM and plasma binding protein 2.6 Surface
    N315-SA0746 nuc Staphylococcal nuclease 2.2 Down Secreted
    N315-SA0754 HP, similar to lactococcal prophage ps3 protein 05 3.0 Up
    N315-SA0773 Conserved HP 3.4
    N315-SA0807 mnhG Na+/H+ antiporter subunit 2.0 Transport
    N315-SA0808 mnhF Na+/H+ antiporter subunit 2.3 Down Transport
    N315-SA0810 mnhD Na+/H+ antiporter subunit 2.0 Transport
    N315-SA0820 glpQ Glycerophosphoryl diester phosphodiesterase 2.4
    N315-SA0889 HP 31.1 17.6 10.3
    N315-SA0890 Conserved HP 6.2 32.8 9.5
    N315-SA0937 Cytochrome d ubiquinol oxidase subunit I homologue 2.6
    N315-SA0938 Cytochrome d ubiquinol oxidase subunit II homologue 3.0
    N315-SA0963 pyc Pyruvate carboxylase 2.0
    N315-SA1000 HP, similar to fibrinogen-binding protein 4.1 3.9 Surface
    N315-SA1001 HP 4.6
    N315-SA1003 12.2 4.1
    N315-SA1004 fib HP, similar to fibrinogen-binding protein 2.4 13.1 4.7 Surface
    N315-SA1270 HP, similar to amino acid pearmease 3.5 Down Transport
    N315-SA1272 ald Alanine dehydrogenase 4.0 Down
    N315-SA1490 yhjN Conserved HP 2.2
    N315-SA1583 rot Repressor of toxins Rot 2.7 3.0 4.0 Up Regulator
    N315-SA1591 arsR Arsenical resistance operon repressor homologue 2.0 Regulator
    N315-SA1628 splD Serine protease SplD 2.5 Up Up Down Secreted
    N315-SA1629 splC Serine protease SplC 2.7 Down Secreted
    N315-SA1630 splB Serine protease SplB 2.2 Up Up Down Secreted
    N315-SA1631 splA Serine protease SplA Up Up Down Secreted
    N315-SA1637 lukD Leukotoxin, LukD (pathogenicity island SaPIn3) 16.1 9.9 Secreted
    N315-SA1638 lukS Leukotoxin LukE 16.6 9.5 Secreted
    N315-SA1750 mapW_1 Truncated map-w protein 9.7 7.6 Surface
    N315-SA1751 mapW_2 Truncated map-w protein 7.8 6.1 Surface
    N315-SA1753 HP (bacteriophage phiN315) 2.4
    N315-SA1755 HP (bacteriophage phiN315) 27.2 11.8
    N315-SA1758 sak Staphylokinase precursor 2.4 Secreted
    N315-SA1759 Lytic enzyme 2.5 Secreted
    N315-SA1784 HP (bacteriophage phiN315) 2.2
    N315-SA1789 HP (bacteriophage phiN315) 2.0
    N315-SA1790 HP (bacteriophage phiN315) 2.8
    N315-SA1792 Single-strand DNA-binding protein 3.1
    N315-SA1795 HP (Bacteriophage phiN315) 2.6
    N315-SA1796 HP (Bacteriophage phiN315) 2.8
    N315-SA1812 lukE HP, similar to synergohymenotropic toxin precursor 12.3 5.9 Down Secreted
    N315-SA1813 lukM HP, similar to leukocidin chain lukM precursor 11.7 5.2 Secreted
    N315-SA2095 HP, similar to d-octopine dehydrogenase 2.3
    N315-SA2108 HP, similar to transcription regulator RpiR family 2.1 3.3 Regulator
    N315-SA2114 glvC PTS system, arbutin-like IIBC component 2.3 Transport
    N315-SA2135 gltS HP, similar to sodium/glutamate symporter 3.9 Up Transport
    N315-SA2147 tcaR TcaR transcription regulator 2.1 2.5 Regulator
    N315-SA2156 l-Lactate permease lctP homologue 3.2 Transport
    N315-SA2194 HP, similar to Zn-binding lipoprotein adcA 3.6 Surface
    N315-SA2206 sbi IgG-binding protein SBI 6.0 4.2 Surface
    N315-SA2207 hlgA Gamma-hemolysin chain II precursor 3.1 16.2 Secreted
    N315-SA2208 hlgC Gamma-hemolysin component C 6.7 2.7 Up Up Down Secreted
    N315-SA2209 hlgB Gamma-hemolysin component B 7.4 2.8 Up Up Down Secreted
    N315-SA2210 bioX HP, similar to BioX protein 5.9
    N315-SA2211 bioW HP, similar to 6-carboxyhexanoate-CoA ligase 3.6
    N315-SA2251 opp-1F Oligopeptide transporter putative ATPase domain 2.1 Up Transport
    N315-SA2253 opp-1C Oligopeptide transporter putative membrane permease domain 2.1 Transport
    N315-SA2254 opp-1B Oligopeptide transporter putative membrane permease domain 2.2 Transport
    N315-SA2255 opp-1A Oligopeptide transporter putative substrate binding domain 2.1 Transport
    N315-SA2300 HP, similar to glucarate transporter 2.7 Down Transport
    N315-SA2302 stpC HP, similar to ABC transporter 5.8 5.1 3.0 Transport
    N315-SA2302 stpC HP, similar to ABC transporter 5.8 5.1 3.0 Transport
    N315-SA2321 HP 2.9 2.3 Up Up
    N315-SA2430 aur Zinc metalloproteinase aureolysin 2.5 Up Down Secreted
    N315-SAS025 HP 2.6
    N315-SAS035 HP 2.2 2.4
    N315-SAS051 HP 2.3
    N315-SAS058 HP (bacteriophage phiN315) 2.4
splF 4.6 Secreted
    COL-SA0256 HP Down
    COL-SA0334 Bacteriophage L54a, conserved HP 2.4
    COL-SA0472 exotoxin 2 2.3 Secreted
    COL-SA0478 exotoxin 3 Exotoxin 3 3.4 Secreted
    COL-SA0906 Terminase small subunit 2.2
    COL-SA1165 HP 2.1
    COL-SA1477 ilvA Threonine dehydratase catabolic 2.7 4.1
    COL-SA1865 splE Serine protease SplE 2.4 Secreted
    COL-SA1870 HP 2.2
    COL-SA2014 Terminase small subunit 2.1
    COL-SA2420 HP, RpiR family 2.1 10.4 Regulator
    COL-SA2480 HP 2.8
    COL-SA2511 fnbA Fibronectin-binding protein A 2.2 Surface
    COL-SA2568 HP 2.1
    Mu50-SAV0861 HP, bacteriophage? Down
    Mu50-SAV0883 HP, bacteriophage? Down
    Mu50-SAV0888 HP, bacteriophage? 2.4
    Mu50-SAV0889 HP, bacteriophage? 2.6
    Mu50-SAV0893 HP, bacteriophage? 2.6
    Mu50-SAV0894 HP, bacteriophage? 2.8
    Mu50-SAV0895 HP, bacteriophage? 2.0
    Mu50-SAV0898 HP, bacteriophage? 2.2
    Mu50-SAV0899 HP, bacteriophage? 2.2
    Mu50-SAV0900 HP, bacteriophage? 2.6
    Mu50-SAV0902 HP, bacteriophage? 2.6
    Mu50-SAV0903 HP, bacteriophage? 2.3
    Mu50-SAV0905 HP, bacteriophage? 2.3
    Mu50-SAV0906 HP, bacteriophage? 2.6
    Mu50-SAV0907 HP, bacteriophage? 2.7
    Mu50-SAV0909 HP, bacteriophage? 2.5
    Mu50-SAV0910 HP, bacteriophage? 2.7
    Mu50-SAV0911 HP, bacteriophage? 2.9
    Mu50-SAV1985 HP, bacteriophage? 2.8
    Mu50-SAV1988 HP, bacteriophage? 3.8
    MW1382 HP 3.3
    MW1441 HP 2.4
    MW1920 HP 2.2
    MW1927 HP 3.1
    MWP018 Down
a

“Fold change” indicates increase or decrease for up-regulated and down-regulated genes, respectively. Values in bold indicate low expression under one condition introduced uncertainty in the fold difference value. “Down” denotes saturation under this condition or absence under other condition such that no fold difference could be calculated; “Up” denotes saturation under this condition or absence under other condition such that no fold difference could be calculated.

We have analyzed the microarray data on the basis of individual genes. However, the MAS includes several clusters of adjacent genes that may represent single transcription units. The individual genes in each cluster are oriented in the same direction and have the same temporal patterns and pH responses. Thus, the conclusions would have been the same if the analysis had been based on transcription units rather than individual genes.

Northern blot hybridization analysis.

Northern blot hybridization was used to validate the microarray data for several genes, and it provided an important comparison with gene fusion data. In addition to several extensively studied genes, including hla, spa, sspA, sarA, and rnaIII, we analyzed three loci, rot, cap5b, and lrgAB, whose pH-dependent expression was first revealed by the microarray data. Northern blot hybridization results are presented in Fig. 4, from which the following conclusions are apparent. (i) The blotting data for cap5b and lrgAB were consistent in direction if not magnitude with the profiling data. The cap5b transcript level was increased at pH 5.5, whereas lrgAB was increased at pH 7.5. Two or three transcripts are visible in the cap blot and seem to behave somewhat differently. It is planned to determine their sizes and investigate their differential abundance. (ii) hla data from the two techniques could not be compared because expression of hla reached the GeneChip upper-threshold values at T3 and T6 and was so low at T0 that the hla transcript could not be detected by blotting. Comparison of the hla Northern blotting data with the gene fusion results revealed that transcript levels and promoter activity were similar in direction if not magnitude. (iii) With the exception of the T6 time point, the Northern blotting data for sspA were relatively consistent with both the profiling data and with the gene fusion data. Although at T3 the sspA transcript appeared higher at pH 5.5 and at T6 it appeared higher at pH 7.5, these differences were less than twofold. One possibility for the differences observed between gene fusion and Northern blot data could be that the gene fusion construct used may not have included all 5′ regulatory elements (Fig. 2 and 4). At T0, the transcription level was too low to be evaluated at either pH. (iv) The spa data could not be confirmed by Northern blotting of RN6734 RNA because the level of expression was too low at most time points in this agr+ strain. Therefore, we prepared blots with RNA from the isogenic agr-null strain, RN7206. Although the blots from one strain cannot be directly compared with the microarray data from another strain, spa expression in RN7206 was greater at pH 7.5 throughout growth and, in RN6734, it was greater at pH 7.5 only at the earliest time point. (v) Although the pH regulation of rot seen in the microarray was confirmed by Northern blot hybridization, rot transcription increased postexponentially at both pHs rather than being constitutive as has been reported elsewhere (32). (vi) The Northern blots for agr-rnaIII and sae were consistent with the microarray results.

FIG. 4.

FIG. 4.

Northern blot analysis of the effect of pH on the transcript levels of genes coding for several exoproteins and their regulators. (A and B) Blots of whole-cell RNA from RN6734 grown in CYGP broth without glucose and adjusted to pH 5.5 or 7.5. (B) Blots of transcripts identified by microarray analysis as pH dependent. The 16S rRNA blot above panels A and B shows equal loading. (C) pH-dependent expression of spa in RN7206 (RN6734 Δagr); equal loading is shown by the adjacent 16S rRNA blot.

Although sarA does not belong to the MAS based on analysis of RN6734, interpretation of sarA results is problematic; sarA is transcribed from three separate promoters (12), one of which, P3, uses σB and is not active in RN6734 and other derivatives of NCTC8325, owing to the rsbU defect in these strains. In an rsbU+ strain this promoter is much more active at pH 5.5 than at pH 7.5 (unpublished data). Conversely, the P1 promoter appeared to be considerably less active at pH 5.5 than at pH 7.5 (the level of the P2 transcript was too low to be relevant). Thus, the role of pH in the overall production of SarA protein, which may be highly strain specific, remains to be determined.

Effect of pH on overall exoprotein patterns.

Given that the MAS seems large and complex and that it includes several exoprotein genes, including those encoding known virulence factors, we have begun to analyze the effects of pH on the extracellular proteome. For this purpose, supernatants were collected from cultures of RN6734 grown at pH 5.5 versus pH 7.5 in the absence of glucose at the T6 time point. Supernatant exoprotein profiles, as determined by SDS-PAGE, are shown in Fig. 5. Several exoproteins were observed to be more abundant at pH 7.5 but were considerably reduced or absent at pH 5.5, and vice versa; additionally, there was a considerable asymmetry in the size distributions of the bands corresponding to the two pH conditions, with higher-molecular-weight species predominant at pH 5.5 and lower-weight species at pH 7.5. To test for the possibility that this asymmetry could represent differential proteolytic activity, we mixed equal quantities of the two supernatants, adjusted the mixtures to either pH 5.5 or 7.5, incubated them for 30 min at 37°C, and analyzed them by SDS-PAGE. The resulting exoprotein patterns, shown in the two rightmost lanes in Fig. 5, were indistinguishable with the exception of a high-molecular-weight band, which we have identified as geh lipase (unpublished data). This band is somewhat weaker after incubation of the mixture at pH 7.5 than at 5.5, presumably owing to differential proteolysis (30, 42, 48). Overall, these results are consistent with transcription-level regulation of exoprotein production, and it is clear that proteolysis is not responsible for most of the pH-dependent differences in the exoprotein profile.

FIG. 5.

FIG. 5.

Exoprotein profiles of cultures grown at pH 5.5 or 7.5, and the effect of pH-dependent protease activity on exoprotein profiles. Exoprotein was prepared from 6-h cultures grown in CYGP broth adjusted to either pH 5.5 or pH 7.5. Supernatants from equal numbers of cells were precipitated with trichloroacetic acid and subjected to SDS-PAGE. The right two lanes show exoprotein from both of the left lanes combined, adjusted to each pH, and incubated at 37°C for 30 min to investigate the effect of differential protease activity.

Identification of differentially abundant exoproteins.

We identified several of the differentially abundant exoproteins by mass spectrometry. Due to differences in loading and resolution between the gel from which the bands were excised and the gel shown in Fig. 5, these proteins are not labeled in Fig. 5. In addition to the cleaved form of lipase, these included an immunodominant surface antigen, IsaA (more abundant at pH 5.5), the precursor form of autolysin Atl (more abundant at pH 5.5), and the serine protease-like SplF (more abundant at pH 7.5). Comparing these and the microarray results, some of the differential abundances of these proteins are a reflection of pH-dependent differences in transcription (IsaA and SplF); others are likely the result of posttranscriptional mechanisms (Geh and Atl).

DISCUSSION

This study began with the observation that the glucose-induced down-regulation of certain staphylococcal virulence genes is largely a result of the pH reduction resulting from glucose fermentation. The end point of this pH reduction is generally around pH 5.5 in standard glucose-supplemented broth cultures of S. aureus; in cultures grown at this pH the classical agr-dependent postexponential induction of virulence genes such as hla and tst, seen at pH 7.5, is eliminated. Microarray analysis revealed that the transcript levels of over 400 S. aureus genes were affected, and the overall pattern is here defined as the MAS. More than twice as many genes are up-regulated at pH 5.5 than are down-regulated, relative to effects at pH 7.5.

It is well known that bacteria have elaborate mechanisms by which they maintain their internal pH within tight limits. Major, potentially lethal reductions in external pH activate an acid stress response, which mobilizes a variety of resources in an attempt to defend the cytoplasmic pH (14, 37, 38). At the same time, bacteria such as staphylococci can grow and divide normally over a considerable pH range—at least 5 to 9. In the present work we have analyzed the lower half of this range and found that there is a large set of genes that are differentially regulated between pH 5.5 and 7.5—the MAS. We suggest that this modulation of gene expression is likely to represent an adaptation to pH-variable environments, rather than a global acid stress response. In other words, pH could be one of the factors that determine locale-specific gene expression patterns, since certain body sites are characterized by variations in pH from the homeostatic 7.4 (Fig. 6) (5, 10, 16, 21, 22, 31, 41, 53). Given the breadth of the MAS, plus the fact that the intracellular pH is maintained within very narrow limits, the pH differential must be sensed by one or more surface receptors and transduced to the interior of the cell. As there is a complex regulatory network that coordinates the expression of accessory genes (34), the MAS must interact with one or more regulatory genes involved in this network. For any regulatory genes that belong to the MAS, it is predicted that their regulons will follow suit, insofar as the regulation is direct. That is, if transcription of a regulatory gene is reduced at one pH, the genes that it up-regulates will also be reduced at that pH, and vice versa. Genes that do not follow this rule must be regulated indirectly. For those regulatory genes that do not belong to the MAS, any target genes that do belong must also be regulated indirectly. Therefore, examination of stimulons and their overlaps with regulons is expected to aid greatly in the understanding of the overall regulatory network. Results presented here may be viewed as the beginning of this analysis.

FIG. 6.

FIG. 6.

pH ranges of various niches S. aureus can colonize in the human host. Blood, pH 7.4 (Robinson 1975); vagina, 4.2 to 6.6 (Wagner and Ottesen 1982); abscess, 6.2 to 7.3 (Bessman, Page et al. 1989); urinary tract (UT), 4.6 to 7 (McClatchey 1994); lung, 6.8 to 7.6 (Cheng, Rodriguez et al. 1998); mouth, 5 to 7 (Dong, Pearce et al. 1999); nose, 6.5 to 7 (England, Homer et al. 1999); skin, 4.2 to 5.9 (Ehlers, Ivens et al. 2001).

A number of tantalizing relationships between the MAS and certain other stimulons and regulons have been revealed; these relationships are noted here and are clearly in need of further study.

One example is the stimulon defined by the response to subinhibitory concentrations of cell wall-active antibiotics (bacitracin, d-cycloserine, and oxacillin) (50). The MAS appears to overlap significantly with the cell wall-stress stimulon. Twenty-five of the 158 genes in this stimulon are also in the MAS, and for these genes the two stimulons are concordant, raising the question of whether there is some common adaptivity between the two, such as the involvement of any of these 25 genes in transducing the pH stimulus. Seventeen of these 25 genes were both up-regulated by cell wall-active antibiotics and were up-regulated at pH 5.5. Seven of the remaining eight genes were down-regulated by antibiotics and were also down-regulated at pH 5.5. The only exception was a hypothetical gene that could correspond to an unidentified regulator (a repressor) of the MarR type. Because the microarray used in defining the cell wall-stress stimulon was less comprehensive than the one used in the present study, it is possible that the relationship between the two stimulons is even greater than observed here.

Another stimulon that was recently defined is the set of genes that are differentially expressed during growth in a mature flow cell-resident biofilm (3). The biofilm state has been shown to be relevant to several different infection niches for growth, persistence, and resistance to antibiotic treatment (17). Although the strain used by Beenken et al. is not of the NCTC8325 lineage and the cultures were grown with glucose, genes they identified as differentially expressed between exponential-phase planktonic cultures, in which the pH may have not yet dropped below pH 6.0, and mature biofilms may be relevant for comparison to the MAS. Of the 95 genes that are reported up-regulated in a biofilm, well over half (55) are also up-regulated under the mild acid condition. The operons up-regulated under both conditions include those coding for capsular polysaccharide biosynthesis, pyrimidine biosynthesis, the potassium-proton antiporter system kdp, the urease system, and the ssp operon. Among the 17 genes down-regulated under both conditions are rot, spa, nuc, pycA, and the opp1 and spl operons. None of the mild-acid-down-regulated genes were up-regulated in the biofilm, and only 17 of the 278 biofilm down-regulated genes were up-regulated in mild acid. The results presented here support the suggestion of Beenken et al. that a mature biofilm is an acidic environment. The incomplete concordance of the biofilm and mild acid stimulons may reveal sets of genes specific to biofilm or planktonic lifestyles. In sum, the mild acid-biofilm correlation provides an example of an important niche where pH-dependent gene expression may be critical for growth and persistence.

A third example involves the conditionally essential two-component signal transduction system (TCS), YycG/YycF. Five of 12 genes recently reported to be controlled by YycG/YycF were identified in the MAS, although the TCS itself was not. YycF recognizes a pair of directly repeated hexamers within the promoter region of target genes (18). Two of the target genes with their direct repeats oriented in one direction were down-regulated at pH 5.5; three with their repeats in the opposite direction were up-regulated at pH 5.5. This difference in the orientation of cis regulatory elements in the promoters of genes belonging to the YycG/YycF TCS regulon correlates with the differential pH effects on these genes, suggesting an interesting possible relationship between this TCS and transduction of the pH signal.

Among the genes belonging to the MAS are 26 known or putative regulatory genes. The response of target gene subsets should, to a first approximation, follow the response of the controlling regulatory gene. The sae regulon appears to fit this scheme, at least with respect to the few target genes that have been individually analyzed—for example, sae up-regulated leukotoxin and gamma hemolysin are down-regulated at pH 5.5, like sae. The generality of this effect will be reported elsewhere in connection with an ongoing analysis of the sae regulon. The transcript levels of 19 other known or putative regulatory genes are increased at pH 5.5, and those of six are decreased. In Table 3 are compiled data on known regulatory interactions involving two of the better studied of these regulators, agr and rot, whose regulons have been determined (20, 43). We note that the RNA samples used for determination of the agr and rot regulons were all prepared from bacteria grown in the presence of glucose, and the rot regulon was determined with an agr-null strain. Additionally, they were determined with an earlier version of the microarray lacking many important genes. Nevertheless, most of the agr-regulated and rot-regulated genes adhere to the principle that target genes follow the response of the controlling regulator. For genes that do not, the simplest possibility is that the regulatory connection is indirect. Thus, agr is up-regulated 3.6-fold at pH 5.5 and T0 (but not at the other time points). At this time point, when agr is a member of the MAS, the agr-regulated members of the MAS follow the regulation of agr, whereas at other time points there is no consistency to the response of agr-regulated genes. Interestingly, although agr belongs to the MAS, no known regulators of agr, including svrA, sarA, sarU, ssrAB, and arlRS, belong to the stimulon. Of course, any of these regulators could be affected at the posttranscriptional level.

rot was identified as a member of the MAS and was shown to be down-regulated at pH 5.5 at all three time points. Thirty-nine genes (9.3% of the stimulon) are rot regulated, of which 28 demonstrated higher transcript titers at pH 5.5. This is largely consistent with the reported role of rot as a repressor (32). Genes encoding surface proteins, such as spa, which is up-regulated by rot, are down-regulated at pH 5.5. rot regulates spa via SarS (43), which is not listed in the MAS. However, the stimulon list was generated by using an arbitrary cutoff value of a twofold change in transcription. Further analysis indicated that although it is not decreased twofold, sarS is indeed moderately down-regulated at the lower pH tested. Only one gene, SA0173, a hypothetical gene similar to surfactin synthetase of Bacillus subtilis, is regulated in the same direction by agr and rot (up).

Microarray data for the sarA regulon have also been published (20) and are included in Table 3, although sarA does not appear to belong to the MAS in strains of the NCTC8325 lineage, such as RN6734. Forty-six genes in the stimulon are SarA regulated. Most of these have increased transcript levels at pH 5.5, and most SarA-regulated genes with pH-sensitive expression are SarA up-regulated. These genes are regulated predominantly through agr.

It has been suggested that SarA is important in the transduction of the pH signal and, superficially, the data on the relation of SarA to sspA shown in Fig. 2C and D would seem to bear this out. SarA seems to be a repressor of sspA only at pH 5.5. However, a closer look at these data suggests that it is not SarA but a separate positive regulator that may be responsible. We note that at pH 7.5, deletion of sarA has no effect on sspA expression in the NCTC8325-derived background tested here; i.e., sarA cannot be a repressor of sspA at pH 7.5. If it were a repressor only at pH 5.5, then the level of sspA at pH 7.5 should match the higher expression level at pH 5.5, rather than the lower. The simplest interpretation here is that SarA is not a pH-dependent repressor, but rather that there is a pH-dependent activator that functions at pH 5.5, only in the absence of SarA.

It has also been suggested that σB may have a role in transducing the pH signal (9, 39); on the basis of data presented here, it is suggested that there may be two pH-sensing pathways, one independent of σB and the other possibly σB dependent. The microarray analysis was performed with RN6734 because of the enormous experience and wide use of this and other NCTC8325 derivatives, despite their rsbU defect (23, 26). As these strains have only weak residual σB activity, the studies reported here will have defined a pH-sensing pathway that is essentially independent of σB. This has been confirmed by a recently published DNA microarray analysis of the σB regulon (6). Of the 198 genes found to be σB up-regulated by transcriptional profiling of three different rsbU+ strains, 25 were also up-regulated by mild acid. None of these 198 genes was down-regulated by mild acid. Eighteen of the 53 σB down-regulated genes were also down-regulated by mild acid; however, 19 of these 53 were up-regulated by mild acid. These results are consistent with the absence of sigB or members of the sigB operon in the MAS and the pH-insensitive transcription pattern of sigB in RN6734 (data not shown). However, sarA has a σB-dependent promoter that is also pH dependent (unpublished data), so that in an rsbU+ strain, genes regulated by SarA may define a pH-dependent pathway that is σB dependent. This interpretation is highly tentative since, as noted above, it is not known whether the interplay of the three sarA promoters results in σB-dependent differences in SarA protein levels.

In conclusion, our results have pointed to a number of approaches that could help to identify the pathway(s) by which external pH is sensed by the organism. Additionally, they address but do not solve the basic question of the role of pH-dependent gene expression in pathogenesis. On the one hand, as shown here, it is clear that mild acid modulates the expression of a large set of staphylococcal genes, including virulence genes; on the other hand, it is well known that certain sites in an animal host are characterized by pHs different from the homeostatic 7.4. The basic question, then, is whether there is a significant connection between these two separate findings. In other words, is pH-dependent modulation of virulence gene expression adaptive for the organism with respect to different sites in the animal host that are maintained at particular pHs (different from 7.4)? And, if so, would this be an example of bacteria modulating the expression of particular genes for adaptation to particular tissue sites?

The available studies of pH-dependent and host-niche-specific staphylococcal virulence gene expression reveal little that suggests such a connection (1, 2, 4, 9, 11, 24, 25, 29, 33, 36, 40, 44-46, 49, 51, 54, 55). A potentially informative study, however, is that of Coulter et al. (1998), which identified genes required for growth and persistence in various animal models of staphylococcal infection. Mutations in opp2, sspA, odhA, and epiB were shown to cause attenuation in a systemic infection model but not in abscess or burn wound models, while those in opp1, pycA, and pflB caused attenuation in an abscess model but not in systemic infection or burn wound models (15). The genes associated with systemic infections were up-regulated, while those associated with abscesses were down-regulated at pH 5.5. A homologous oligopeptide permease (opp) system in B. subtilis is required for the uptake of signaling peptides necessary for pH-dependent expression of srf, which encodes a surfactin synthetase (13). A surfactin synthetase homologue is also part of the staphylococcal MAS and is up-regulated at pH 5.5, as is opp2. This could represent a conserved adaptation to the mild acid condition.

The interesting correlation between tissue-site specificity and pH modulation of these genes raises the possibility that tissue-specific pH variation could impact on pathogenesis. It seems doubtful, however, that the pHs at the sites in question prior to infection are consistent with the observed pH-dependent modulation in bacterial gene expression, because these sites are typically maintained at pH 7.4 (Fig. 6). Perhaps a local pH change as a result of the infection requires an adaptive response, prohibiting bacteria incapable of such a response from maintaining an infection.

A quite different and rather dramatic example of how pH-dependent virulence gene expression could impact human health, but in a manner that is also not consistent with the concept of pH-dependent tissue site adaptation, is menstrual toxic shock. The human vagina is maintained at a pH of <5, a pH at which toxic shock syndrome toxin 1 is not produced (45) because tst, the gene encoding the toxin, is essentially silent (Fig. 2). However, during menses the pH rises above 7, a pH at which tst is strongly expressed—which could very well account for the well-known relation between toxic shock syndrome and menstruation (4). However, whether pH-dependent expression of tst has any adaptive role for the organism or is simply coincidental is far from clear. Indeed, it is not obvious whether causing toxic shock syndrome is in any way advantageous for the organism; instead, maintaining the vaginal pH of <5 could represent a host defense against toxic shock, a defense that the bacteria have clearly not countered by pH-dependent gene expression but one that is inadvertently breached by the host through menstrual bleeding.

The in vivo relevance of the pH-dependent accessory gene expression we report here will require more accurate measurement of the pH of the host environment during infection and testing of knockouts of the members of the signal transduction network in relevant animal models. Regardless of the role of differential expression in an infection, elucidation of the MAS will forward attempts to map the S. aureus accessory gene regulatory network. Regulons have great value in establishing epistasis and suggesting functional roles for the corresponding regulator, but their usefulness is limited because regulators often act in concert and effects may be absent or even contradictory in single mutants. Stimulons allow us to perturb genetically complete organisms but require analysis of the relevant mutants to dissect cause and effect. The combination of these methods should in the future allow construction of a comprehensive model of the S. aureus accessory gene regulatory network.

Acknowledgments

We thank Hope F. Ross for discussion and critical reading of the manuscript.

This work was supported by National Institutes of Health grant R01 AI030138-14 to Richard P. Novick. Brian Weinrick was supported in part by an NIH training grant to the Department of Microbiology, NYU School of Medicine (T32 AI007180-21).

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