Transcriptional analysis of the acid tolerance response in Streptococcus pneumoniae

Antonio J. Martín-Galiano1,{dagger},{ddagger}, Karin Overweg2,{ddagger}, Maria J. Ferrándiz1, Mark Reuter2, Jerry M. Wells2,§ and Adela G. de la Campa1

1 Unidad de Genética Bacteriana, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain
2 Bacterial Infection and Immunity Group, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK

Correspondence
Adela G. de la Campa
agcampa{at}isciii.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptococcus pneumoniae, one of the major causes of morbidity and mortality in humans, faces a range of potentially acidic conditions in the middle and late stages of growth in vitro, in diverse human fluids during the infection process, and in biofilms present in the nasopharynx of carriers. S. pneumoniae was shown to develop a weak acid tolerance response (ATR), where cells previously exposed to sublethal pHs (5·8–6·6) showed an increased survival rate of up to one order of magnitude after challenge at the lethal pH (4·4, survival rate of 10–4). Moreover, the survival after challenge of stationary phase cells at pH 4·4 was three orders of magnitude higher than that of cells taken from the exponential phase, due to the production of lactic acid during growth and increasing acidification of the growth medium until stationary phase. Global expression analysis after short-term (5, 15 and 30 min, the adaptation phase) and long-term (the maintenance phase) acidic shock (pH 6·0) was performed by microarray experiments, and the results were validated by real-time RT-PCR. Out of a total of 126 genes responding to acidification, 59 and 37 were specific to the adaptation phase and maintenance phase, respectively, and 30 were common to both periods. In the adaptation phase, both up- and down-regulation of gene transcripts was observed (38 and 21 genes, respectively), whereas in the maintenance phase most of the affected genes were down-regulated (34 out of 37). Genes involved in protein fate (including those involved in the protection of the protein native structure) and transport (including transporters of manganese and iron) were overrepresented among the genes affected by acidification, 8·7 and 24·6 % of the acid-responsive genes compared to 2·8 % and 9·6 % of the genome complement, respectively. Cross-regulation with the response to oxidative and osmotic stress was observed. Potential regulatory motifs involved in the ATR were identified in the promoter regions of some of the regulated genes.


Abbreviations: ATR, acid tolerance response

The raw microarray data is available as supplementary material with the online version of this paper.

{dagger}Present address: Lehrstuhl für Genomorientierte Bioinformatik. Wissenschaftszentrum Weihenstephan, Am Forum 1, 85354 Freising, Germany.

§Present address: The University of Amsterdam, Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.

{ddagger}These authors contributed equally to this work.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptococcus pneumoniae is an important human pathogen and a major cause of pneumonia, meningitis, otitis media and sepsis. This broad pathogenic potential reflects its ability to adapt to different habitats within the human host. In otitis media, S. pneumoniae is suspected to be present in biofilms on the mucosal surface of the middle ear, and possibly also during asymptomatic colonization of the nasopharynx (Donlan & Costerton, 2002). Physiological changes due to the biofilm mode of growth may include a decreased pH, as in dental plaque where bacterial crowding and production of lactic acid by the closely related Streptococcus mutans and Streptococcus gordonii occurs (Cotter & Hill, 2003). In other stages of infection, S. pneumoniae must also face a mild acidification of the invaded body fluids, i.e. blood, cerebrospinal fluid and inflammatory fluids, which decrease from pH 7·3–7·4 to 6·8–7·0 as a consequence of the inflammatory response (Andersen et al., 1989; Light et al., 1980).

Some bacterial species show an increased survival rate after challenge at lethal acid pH if they are first subjected to a period of mild sublethal acidic conditions, a process known as the acid tolerance response (ATR), which has been described in Escherichia coli (Goodson & Rowbury, 1989), Salmonella typhimurium (Foster & Hall, 1990) and oral streptococci (Svensater et al., 1997). The lethal pH and the range of pH that trigger the ATR are characteristic for each species, and are indicative of their acid resistance capacity (Svensater et al., 1997). The bacterial responses to different types of physiological stresses often partially overlap, such that exposure to one stressful condition commonly gives some protection against several others (Svensater et al., 2000). Whereas the ATR has been relatively well characterized in oral streptococci, it has not been investigated in S. pneumoniae.

One of the systems used by Gram-positive bacteria to respond to acidic pH is the system involving the F0F1 ATPase, an enzyme that hydrolyses ATP to generate a proton gradient for a variety of transport processes, and regulates the intracellular pH via proton extrusion (Cotter & Hill, 2003; Futai et al., 1989). In the oral streptococci (Bender et al., 1986), Enterococcus hirae (Kobayashi et al., 1986), lactic acid bacteria (Koebmann et al., 2000; Kullen & Klaenhammer, 1999) and S. pneumoniae (Martín-Galiano et al., 2001), the activity of the F0F1 ATPase increases as the pH of the medium decreases. This increase in activity is regulated in S. pneumoniae at the level of transcription initiation (Martín-Galiano et al., 2001). Global changes in gene expression in response to acidification have been analysed by microarray technology in bacteria in which acid resistance is crucial for their pathogenesis, such as Helicobacter pylori (Ang et al., 2001) and Mycobacterium tuberculosis (Fisher et al., 2002). Here we have characterized the ATR in S. pneumoniae, and analysed the adaptive and maintenance phases of the response by global expression analysis.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Culture conditions, RNA extraction and microarray experiments.
To characterize the ATR, S. pneumoniae R6 was grown at 37 °C in Todd–Hewitt medium (Difco) with 0·5 % yeast extract (THYE medium), and survivors were counted after 16 h of growth by plating on THYE medium plus 1 % agar in an atmosphere containing 5 % CO2. In the short-term ATR experiment, the R6 strain was grown in THYE medium at pH 7·8 until mid-exponential phase (OD620 0·25). At this moment, the pH of the medium was reduced to pH 6·0 (the more acidic pH that allows normal growth) with 1 M HCl, and RNA was extracted from 50 ml aliquots of culture collected at 0, 5, 15 and 30 min. In the long-term ATR experiment, R6 was grown in THYE medium at pH 7·8 until OD620 0·10, the culture was diluted 50-fold in fresh THYE medium buffered at starting pH values of 7·8 and 6·0, and 50 ml samples of culture were taken when the OD620 reached 0·25, i.e. after approximately 200 min (referred as 200 min throughout this paper). Cells were then chilled on a water–ice bath, washed with cold 0·9 % NaCl and stored at –80 °C. Pellets were thawed and cells lysed for 15 min at 37 °C in 10 mM Tris, 1 mM EDTA (pH 8·0), 0·1 % sodium deoxycholate. RNA was extracted using the RNeasy midi kit (QIAGEN), including a DNase treatment according to the manufacturer's instructions, precipitated with ethanol, washed and resuspended in 40 µl RNase-free water. The concentration and purity of the RNA samples were measured using the 2100 Bioanalyser according to the manufacturer's protocols (Agilent). Details of the construction of the microarrays used in this study have been described previously (Dagkessamanskaia et al., 2004). The microarrays included probes for all strain TIGR4 annotated genes (2236) and probes for 117 R6-specific genes (i.e. those with less than 90 % similarity to any of the TIGR4 genes, as deduced by BLAST analysis). The labelling of cDNA with Cy3- or Cy5-labelled CTP, and the microarray hybridization were performed as described previously (Martín-Galiano et al., 2004). Microarrays were scanned with an Axon 4000A using GenePix Pro 3.0 software.

Microarray data normalization and analysis.
All hybridization experiments were carried out in triplicate comparing two mRNA samples from three independent replicate cultures. Hybridization datasets were normalized and analysed as described by Holmes et al. (2005). In short, normalized data from triplicate hybridization datasets were unified in one single dataset and reanalysed. However, for the data analysis described here, the mean of the fluorescence values of single genes from each hybridization dataset were unified, as single genes were represented by at least four features on the array. The boundary between genes that were equally and differentially expressed was arbitrarily chosen as about twofold greater intensity in either the Cy3 or the Cy5 fluorescence values. The genes that potentially had increased or decreased expression levels were first identified as those with mean intensities outside the boundary, and were tested statistically by two F-tests. According to these tests, genes were classified as either differentially or equally expressed in both populations. With this boundary, the error in classification on the control dataset of the short-term ATR experiment and long-term ATR experiment was 0 and 1·45 %, respectively.

The normalized and unified control experiments were also used to estimate the boundaries between genes that were equally and differentially expressed in the two samples (Holmes et al., 2005). This boundary would allow the detection of changes equivalent to about 1·5-fold and 1·9-fold greater intensity in one of the fluorescence channels for the short-term ATR experiment and long-term ATR experiment, respectively. Differentially expressed genes identified from this analysis are shown in Table 2 if they are part of an operon, and at least one of the genes of the operon was found to have at least twofold changes in expression level.


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Table 2. Genes with altered transcript levels in ATR, listed by role and subrole

 
Real-time RT-PCR experiments.
Analysis of selected genes was carried out with 20 µl reverse transcription reactions containing 1 µg RNA, 0·5 mM each dNTP, 0·2 µM each gene-specific primer (Table 1), 40 U RNaseOUT ribonuclease inhibitor and 200 U SuperScript III RNase H reverse transcriptase (Invitrogen), in the buffer recommended by the manufacturer. The reactions were incubated at 55 °C for 60 min, terminated by 15 min incubation at 70 °C, and the template RNA was removed by 20 min incubation at 37 °C in the presence of 20 U RNase H (Amersham Biosciences). The cDNA obtained was stored at –20 °C. The LightCycler FastStart DNA Master SYBR Green I system (Roche) was used for real-time PCR (LightCycler 2.0 instrument) in a 20 µl reaction containing 2 µl diluted cDNA, 0·4 µM each primer, a variable amount of MgCl2 (2–3 mM) and 2 µl SYBR Green mix. PCR amplifications were carried out as follows: 1 initial cycle of denaturation (10 min at 95 °C), and 42 cycles of amplification [95 °C, 0 s; test annealing temperature, 5 s; 72 °C, 8 s elongation and signal acquisition (single mode)]. To check the purity of the amplification product, 1 cycle of melting (95 °C, 0 s; test annealing temperature, 10 s; 95 °C, 0 s at the step acquisition mode) was performed. For relative quantification of the fluorescence values, a calibration curve of serial dilutions of cDNA samples of each amplicon was made. To normalize the three independent cDNA replicate samples, values were divided by those obtained for the amplification of spr1863, a gene not showing variation in the microarrays. The mean of the relative values of the three replicates (coefficient of variation<=25 %) was used.


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Table 1. Validation of microarray data by real-time RT-PCR

 
Searching for regulatory motifs in the upstream regions of transcriptionally altered genes.
The 100 nt region located upstream of the start codon of genes was analysed. To assign genes to operons, the gene organization on The Institute for Genomic Research (TIGR) web page (http://www.tigr.org) was analysed, based on the method published by Ermolaeva et al. (2001), and with STRING (von Mering et al., 2005). Two contiguous genes were considered as part of the same operon if they had a confidence value over 75 % according to the TIGR web page and/or a STRING score higher than 0·5. When genes were predicted to be part of the same operon, or located next to each other with similar transcriptional profiles, only the upstream region of the first gene of the operon was considered. The P value was calculated by comparing the potential motif with its random appearance considering the mean GC content of S. pneumoniae (40 mol% G+C). All sequences presented in at least two promoter regions of similar controlled genes with P<=2x10–3 were analysed as potential acid-dependent regulatory motifs.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Acid tolerance response of S. pneumoniae
The lethal pH, defined by a survival rate of 10–4 after an exposure of 2 h, was estimated in order to undertake the subsequent ATR experiments, and corresponded to pH 4·4 (Fig. 1a). Exposure of bacteria to mild sublethal acid conditions (pH 5·8–6·6) for 2 h prior to lethal pH challenge, increased the cellular survival rate threefold to tenfold (Fig. 1b). Moreover, the survival rate was notably increased, by up to three orders of magnitude, when cells at stationary phase were compared to cells at exponential phase, without any additional period at sublethal pH (Fig. 1c) due to the gradual acidification of the medium caused by the production of lactic acid. The pH values at which the ATR is triggered in the optical density experiment are concordant with those in the mild acid shock assay (compare Figs 1b, c).



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Fig. 1. Characterization of the ATR of S. pneumoniae R6: lethal pH determination (a), ATR (b) and optical density dependence of the ATR (c). (a) Cells grown to OD620 0·1 at pH 7·8 were diluted 1/50 and incubated for 2 h in media with increasingly acidic pHs. (b) Cells grown to OD620 0·1 were diluted 1/50 in medium with the indicated pHs. Then, a volume corresponding to 106 cells (as measured by OD620 determination) was inoculated into 5 ml medium at pH 4·4 and incubated for 2 h. (c) Fractions of a culture corresponding to 106 cells grown in medium at pH 7·8 were incubated for 2 h at pH 4·4. Survival rates (grey bars) are the mean±SEM of three independent experiments. {blacktriangleup}, pH.

 
Global transcription analysis
The short-term (5, 15 and 30 min) and long-term (200 min) transcriptome response to low pH was analysed in exponentially growing cells by labelling cDNA from the control and test samples with different fluorescent dyes (Cy3 and Cy5), followed by subsequent co-hybridization on a single microarray slide. For these experiments S. pneumoniae R6 cultures were grown in triplicate as described in Methods. Cells diluted at pH 6·0 suffered a transition stage of around 30 min as deduced from the serious delaying effect on the growth rate. After that period, the cells recovered displaying equivalent duplication times (34 min) to the parallel control culture at pH 7·8 (38 min). Although the stationary-phase ATR was the predominant form of acid adaptation, the exponential phase of growth was chosen to compare the transcriptional status of cells. This decision was made on the basis of discerning among acid adaptation and other characteristics dependent of the phase of growth, such as a lack of primary sources of carbon and nitrogen, accumulation of metabolic by-products, alterations in the duplication rate or potential activation of quorum-sensing processes. Triplicate hybridization datasets were normalized and analysed as described in Methods. Most genes remained unaltered, showing comparable expression levels between the samples at pH 7·8 and pH 6·0, whereas 6·0 % of genes (126 out of 2084 R6 strain genes) had a significantly different signal intensity. A similar number of genes were affected in experiments comparing S. pneumoniae gene expression in different body fluids (Orihuela et al., 2004). We have detected variations of up to 6·1-fold for genes with increased expression levels, and up to 7·8-fold for genes with decreased expression levels. The magnitude of these changes were small compared to the 10-fold to 100-fold changes detected in the levels of certain proteins during the ATR of S. mutans (Len et al., 2004a, b), Streptococcus sobrinus (Nascimento et al., 2004) and Streptococcus oralis (Wilkins et al., 2001). Of the 126 genes with altered transcript levels, 59, 30 and 37 were associated with short-term, both short- and long-term, or long-term ATRs, respectively (Table 2). The patterns of expression were very consistent, with only two exceptions. Genes spr1667 and spr0456 (dnaJ) showed increased expression at 5 and 30 min, but not at 15 min. In these two cases, the increases at 15 min were of 1·4-fold, just below the threshold limit. Whilst the number of genes showing up- or down-regulation was nearly equivalent in the short-term acid response (i.e. 64·4 % were up-regulated), the majority of genes (91·9 %) specifically altered in the long-term acid response were down-regulated. An intermediate situation was observed for genes with altered expression in both periods (66·7 % were down-regulated). The number of genes with an altered transcription, and their function and general type of regulation (the balance of up-/down-regulated genes) strongly support the inference of a different type of regulation in short-term adaptation compared to long-term maintenance responses to acidification.

Genes encoding products predicted to have a role in protein fate were overrepresented among those affected by acidification, i.e. they made up 8·7 % of the acid responsive genes compared to 2·8 % in the genome complement. Similarly, 24·6 % of the acid responsive genes were predicted to have a role in transport, whereas only 9·6 % of the genome set have this putative function (Table 2).

Around half of the genes (59 out of 126) with an altered expression could be grouped in 23 putative operons comprising 2–4 genes (Table 2), as deduced by parallel expression patterns and contiguous localization in the chromosome of their genes. Almost all of these operons (87 %) were also computationally predicted by the algorithm utilized by TIGR (http://www.tigr.org) and/or the STRING database procedure.

Verification of transcriptome data by real-time RT-PCR
The reverse transcription products of seven genes (i.e. six acid responsive genes and one non-varying gene, spr1863, as an internal standard) were measured by real-time RT-PCR at 5, 15, 30 and 200 min after acid shock. Representative acid-responsive genes were selected on the basis of their different temporal patterns of expression as revealed by transcriptome analysis. One of these genes (dnaK) showed an elevated transcription level at 5 min. Gene spr1680, encoding a transcription regulator of the MarR family, was selected as this was up-regulated at 5, 15, 30 and 200 min. Four genes showing decreased transcript levels were analysed. Two of them were down-regulated at 5, 15 and 30 min (glnA and zwf). The other two were down-regulated at 15, 30 and 200 min (spr1538, coding xylan esterase I and spr1625, coding a general stress protein). Additionally, relative expression levels of spr1514 (atpC), the first gene of an operon that was increased in response to low pH (Martín-Galiano et al., 2001), encoding the c subunit of the F0F1 ATPase, was also included. Ratios of microarray and real-time RT-PCR expression measurements showed a good correlation, with a slope of 1·08 (Fig. 2).



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Fig. 2. Correlation of the measured change in RNA transcript levels (at pH 7·8 versus pH 6·0) using data from microarray hybridizations and real-time quantitative RT-PCR, r2=0·74.

 
Gene clustering and promoter analysis
The 100 nt regions located upstream of the predicted start codon for the genes with altered expression were analysed for the presence of potential regulatory motifs, as described in Methods. For genes belonging to operons, only the region upstream of the first gene of the operon was analysed. In this way, 29 regions of 51 up-regulated genes, and 59 regions of 75 down-regulated genes were analysed. Almost one-third (35 out of 110, 31·8 %) of the genes had typical promoters consisting of two conserved hexameric boxes (matching the TTGACA-17±1 nt-TATAAT consensus). Four of the nineteen genes up-regulated at 5 min had specific regulatory sequences: a CIRCE element was found in the promoter region of the hrcA gene, the first gene of the hrcA–grpE–dnaK operon (Zuber & Schumann, 1994), and a CtsR-repressor-binding sequence is located upstream of clpL, as described by Kwon et al. (2003) (Fig. 3). Sequence comparisons revealed a number of putative regulatory sequences. The GTTRTWWTTWTWTCARAAATT sequence (where R is A or G, and W is A or T), was found 3 nt upstream of the –35 box of the putative promoters of the manganese (psaB–psaC–psaA) and iron (fatD–fatC–fecE–fatB) ABC transporter operons, which are up-regulated at 15 and 30 min, with very similar fold changes. Two up-regulated ABC transporters (spr1203 and spr1382/aliB) share a GAGGTTTT sequence located 10–22 bp upstream of the putative –35 region. The putative promoters of the first genes of two down-regulated operons, the one encoding Gln synthetase (glnR) and the five-gene operon encoding a phosphate ABC transporter (pstS), have the sequence TTAGACTATAA overlapping their potential –10 box.



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Fig. 3. Common sequences found in the 5' upstream regions of genes with altered expression. The –35 and –10 boxes are underlined twice, those bases matching the consensus sequence are shown in bold. Sequences with P values of 2·0x10–3 or lower are shown, this value was calculated as the random possibility of finding the sequence considering a genome with a content of 40 mol% G+C. Cases indicates the number of sequences expected in the genome of R6. The real number found in R6 and TIGR4, respectively, are shown in parentheses.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
S. pneumoniae has been shown to be an acid-sensitive lactic acid bacterium according to the optimum pH (7·5) of its F0F1 ATPase (Martín-Galiano et al., 2001) and, as shown here, also by the characteristics of its ATR (high lethal pH, high pH values triggering the response). All the pH values are noticeably higher than those estimated for S. mutans, an aciduric oral streptococcus (Hamilton & Svensater, 1998). This weak ATR of S. pneumoniae was further enhanced by the growth of bacteria to stationary phase, where the pH steadily decreases due to the production of lactic acid as an end product of fermentation, as also observed in S. mutans (Li et al., 2001). Then, the predominant ATR in this organism seems to occur at stationary phase. Additionally, it is worth mentioning that the ATR assays were performed on the R6 laboratory strain, and it cannot be completely ruled out that other strains would display slightly different behaviours in response to acid.

Our analysis of the global transcription response of S. pneumoniae to low pH is, as far as we know, the first reported study on global expression of the ATR for any member of the genus Streptococcus. A total of 126 genes were shown to be involved in ATR. Based on their temporal pattern of expression, we classified acid tolerance responsive genes as those involved in an adaptation response (with altered expression at 5, 15 and 30 min after the acid shock) and those involved in the maintenance of the response (altered in the long term). Six of the nineteen genes with elevated expression levels at 5 min are involved in protein fate. Four are part of the hrcA–grpE–dnaK–dnaJ operon, encoding GrpE, DnaK and DnaJ chaperones, the HrcA repressor and two proteases (clpL and prtA). Early activation of clpL might play a role in the degradation of misfolded proteins due to acidification, and also as a chaperone to assist the refolding of proteins (Kwon et al., 2003). DnaK and ClpL proteins has been shown to increase in S. mutans at acidic pH (Len et al., 2004b), and an increase in the transcription of clpL has been observed in response to heat and cold shock in Lactococcus lactis (Skinner & Trempy, 2001). Proteins that cannot be folded by molecular chaperones may be degraded, in order to recycle amino acids for de novo protein synthesis. This might explain the early up-regulation of the prtA and ptrB protease genes, with prtA having the highest increase in expression of all up-regulated genes at 30 min. The elevated proteolytic activity would generate assimilated nitrogen sources, such as ammonia, thus possibly explaining why expression of the two enzymes involved in the synthesis of glutamine from 2-oxoglutarate and ammonium, NADP-glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA), showed a reduction in expression. In accordance with this idea, gdhA is down-regulated in S. mutans at low pH (Wilkins et al., 2002), and in Bacillus subtilis the glnRA operon is autoregulated such that expression is lowered when the cellular nitrogen content is high (Wray et al., 1996). On the other hand, the decreased expression of the proteases encoded by spr1282 and spr2045, and the up- and down-regulation of various amino acid transporters, suggests that low pH might induce changes in the amino acid metabolism as an adaption to the new demands for protein synthesis.

A change in nucleotide metabolism was also observed after 5 min, involving elevated expression of the pyrB–carA–carB and pyrK–pyrDb operons involved in the first four steps of the de novo biosynthesis of pyrimidine ribonucleotides. In addition, reduced activity of the oxidative branch of the pentose phosphate pathway was predicted, due to the down-regulation of the zwf gene, which encodes glucose-6-phosphate dehydrogenase, the first and key enzyme of the pathway. Conversely, most genes of the non-oxidative route of the pentose phosphate pathway were up-regulated, suggesting that the pathway was redirected to the production of pentose phosphate instead of NADPH, possibly to support the biosynthesis of new nucleic acids (Kamada et al., 2003).

Maintenance of the new cellular state should involve energy-demanding mechanisms, which would require a cellular energetic shift. Glycolysis appeared to be enhanced since the gene encoding its rate-limiting enzyme, 1-phosphofructokinase, was transcribed at higher levels. Additionally, the incorporation of galactose intermediates into glycolysis through the tagatose pathway (involving the lacC and lacD genes) was also enhanced. For efficient utilization of galactose in S. mutans, a combined function of both tagatose and Leloir pathways is required (Abranches et al., 2004). In this respect, the gene encoding the galactose-1-phosphate uridyl transferase (spr1667) of the Leloir pathway was up-regulated in the pneumococcal ATR. However, transcript levels of several genes encoding enzymes that degrade carbohydrate polymers like maltodextrines, xylan, dextran and glycogen were decreased: {alpha}-amylase; acetyl xylan esterase, dextran glucosidase and {beta}-galactosidase. Interestingly, dextran glucosidase was also down-regulated in the ATR of S. mutans (Wilkins et al., 2002).

A link between ATR and other kinds of stress responses, such as oxidative and osmotic stress responses, was found. Acid stress induced increased transcript levels of the gor gene encoding glutathione reductase, an enzyme that regenerates glutathione to avoid oxidation of many cellular molecules, including proteins and fatty acids. Accordingly, several genes (ribD, ribE, ribH) of the operon that is involved in riboflavin biosynthesis appeared to be up-regulated. As S. pneumoniae has an anaerobic metabolism and lacks a respiratory chain, we speculate that proteins containing riboflavin as a cofactor (flavoproteins) might be involved in redox reactions that are relevant to the oxidative branch of the ATR. Additionally, the genes encoding the three subunits of the transporter for manganese, a cofactor required for superoxide dismutase function, were up-regulated. This enzyme plays a role in anti-oxidative defences and is important for virulence (Tseng et al., 2002). The 33 kDa chaperone encoded by the gene hslO was up-regulated in the maintenance phase of the ATR. It is well documented that this chaperone is subject to regulation under different environmental conditions (e.g. oxidative stress) compared to other chaperones, such as DnaK (Winter et al., 2005), which were up-regulated within a few minutes of acidification in our experiments.

A possible link between the ATR and osmotic stress was also indicated by the increased expression of the operon encoding a choline transporter (genes proV and proWX). These genes are homologues of the permease and ATPase subunits of the B. subtilis opuC ABC transporter, which transports glycine betaine (Kappes et al., 1996). This kind of transporter is able to concentrate osmoprotectants like proline and glycine betaine, which can retain water under adverse osmotic circumstances (Baliarda et al., 2003; Kappes et al., 1999; Lucht & Bremer, 1994; Proctor et al., 1997). In accordance with this, the S. mutans response to acid partially protects against osmotic stress and also oxidative stress, and 25–40 % of the proteins expressed in response to these stresses are common between them (Svensater et al., 2000).

Especially interesting are the manganese and iron transporters. The acquisition of both elements is crucial for bacterial pathogens since they are in limiting amounts inside the host body. Manganese metabolism is important during biofilm formation in S. gordonii (Loo et al., 2003) and essential in the pneumococcal colonization of the nasopharynx (Tseng et al., 2002). Iron is an essential cofactor for the function of several enzymes, for example alcohol dehydrogenases, that take part in alternative energetic pathways, and possibly also play a role in the anti-oxidative response (Echave et al., 2003).

In other bacteria, the ATR involves alternative sigma factors. The alternative sigma B factor of Listeria monocytogenes is involved in acid response and osmotic stress (Wemekamp-Kamphuis et al., 2004). RpoS, the alternative Escherichia coli sigma factor, is regulated under acid shock at the translational level (Audia & Foster, 2003). In S. pneumoniae, different patterns of expression in response to low pH, and the absence of common sequences in the promoters, would indicate multiple regulatory mechanisms. A similar expression profile could occur in vivo during the infection or in different stages of the carrier and transmission process of S. pneumoniae.

Although expression of the operon encoding the F0F1 ATPase did not show a significant (>=2-fold) increase in microarray experiments, the 1·5-fold increase detected for the first gene of the operon (atpC) was in agreement with other quantitative measurements using primer extension (1·6-fold), Northern blot (1·4-fold) and dot blot (1·5-fold) (Martín-Galiano et al., 2001). This indicates that the inherent variability of microarray data may lead to some biologically important changes in gene expression being statistically excluded from the analysis.


   ACKNOWLEDGEMENTS
 
A. J. M.-G gratefully acknowledges receipt of a fellowship from Comunidad Autónoma de Madrid, Spain. This work was supported by grant BIO2002-01398 from Ministerio de Ciencia y Tecnología. M. R., K. O. and J. M. W. acknowledge financial support for the microarray construction from EC contract QLK2-CT-2000-00543.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Abranches, J., Chen, Y. Y. & Burne, R. A. (2004). Galactose metabolism by Streptococcus mutans. Appl Environ Microbiol 70, 6047–6052.[Abstract/Free Full Text]

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Received 30 May 2005; revised 14 September 2005; accepted 16 September 2005.



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