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
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ABSTRACT |
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The raw microarray data is available as supplementary material with the online version of this paper.
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.
These authors contributed equally to this work.
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INTRODUCTION |
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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.
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METHODS |
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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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RESULTS |
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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 24 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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DISCUSSION |
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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 hrcAgrpEdnaKdnaJ 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 pyrBcarAcarB and pyrKpyrDb 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:
-amylase; acetyl xylan esterase, dextran glucosidase and
-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 2540 % 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.
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ACKNOWLEDGEMENTS |
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Received 30 May 2005;
revised 14 September 2005;
accepted 16 September 2005.
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