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1 Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, Av. Real 1, 24006 León, Spain
2 University of León, Facultad de Ciencias Biológicas y Ambientales, Campus de Vegazana s/n, 24071 León, Spain
Correspondence
Juan F. Martín
degjmm{at}unileon.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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factor of C. glutamicum, whereas the –35 and –10 boxes of P-atp2 fitted the consensus sequence for H-recognized Mycobacterium tuberculosis promoters CC/GGGA/GAC 17–22 nt C/GGTTC/G, known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0F1 operon is highly expressed at alkaline pH, probably using a H RNA polymerase.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hermann, 2003). Because of its industrial interest, the molecular genetics of C. glutamicum has received considerable attention (reviewed by Martín, 1989; Martín & Gil, 1999; Kirchner & Tauch, 2003). The genome of C. glutamicum consists of a single circular chromosome of 3·3 Mb (Correia et al., 1994; Bathe et al., 1996) and has been fully sequenced in two different strains (Kalinowski et al., 2003; Ikeda & Nakagawa, 2003). The knowledge of the genome sequence has facilitated gene organization studies and transcriptional analysis (Barreiro et al., 2001, 2004).
In the last few years considerable effort has been dedicated to understanding promoter structure in corynebacteria (Pátek et al., 1996, 2003a, b; V
sicová et al., 1999). Knowledge of heat-shock-induced or pH-stress-inducible promoters is important to understand the circuits of stress responses in these bacteria. In addition, promoters inducible by heat shock (Barreiro et al., 2004) or pH stress are useful tools to increase or modulate expression of specific genes of interest.
Each micro-organism in nature has an optimal pH and drastic changes in extracellular pH values trigger a stress response that results in overexpression of certain genes and suppression of others (Foster, 1999). The promoters of those genes respond to the pH changes (Storz & Hengge-Aronis, 2000). However, the specific mechanisms of pH control of pH-regulated promoters are poorly understood.
One known example of pH-regulated operons is that of the bacterial F0F1 operon, which encodes the F0 and F1 multiprotein complexes of the ATP synthase (frequently known as membrane ATPase) that is involved in the formation of ATP using the electrochemical force of the membrane proton gradient (Foster, 1999).
In several bacterial species, including Lactobacillus acidophilus (Kullen & Klaenhammer, 1999), Streptococcus mutans (Kuhnert et al., 2004), Streptococcus pneumoniae (Martín-Galiano et al., 2001) and Lactococcus lactis (Koebmann et al., 2000), expression of the F0F1 operon is induced by strong acid pH. This led to the belief that an increased pH gradient across the membrane, when protons were accumulated externally due to proton extrusion (by different microbial electron-transport systems), resulted in a strong induction of the F0F1 operon. However, very recently, the F0F1 operon of Escherichia coli has been shown to be induced by alkaline extracellular pH (Maurer et al., 2005). This raises the question of the molecular mechanism underlying extracellular pH sensing and transcriptional response to basic pH. Since E. coli has a different natural habitat from the acidophilic bacteria in which previous studies were done, this opens the question of the type of pH response of the F0F1 operon in different micro-organisms.
C. glutamicum has traditionally been grown at neutral pH (Kinoshita & Tanaka, 1972) for amino acid production. However, preliminary studies in our laboratory suggested that C. glutamicum is a moderately alkali-tolerant organism. Therefore, it was of great interest to analyse the stress response of the F0F1 operon in this micro-organism with the aim of increasing our understanding of pH stress control in corynebacteria.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli was grown in Luria–Bertani broth (Sambrook & Russell, 2001) at 37 °C. C. glutamicum was grown in trypticase soy broth (TSB) at 30 °C and at different pH conditions, in four identical 5 litre BIOSTAT fermenters equipped with automatic pH control. The values were maintained at ±0·1 units of the initial pH conditions (pH values 6·0 to 9·5). The pH was controlled automatically by addition of 1 M HCl or 1 M KOH. The cultures were grown aerobically in the fermenters mixed by stirring.
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DNA isolation and manipulation.
E. coli plasmid DNA was obtained by alkaline lysis. Total C. glutamicum DNA was prepared as described by Martín & Gil (1999). DNA manipulations were performed as described by Sambrook & Russell (2001). DNA fragments were isolated from agarose gels using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences). E. coli cells were transformed by standard methods (Sambrook & Russell, 2001), whereas C. glutamicum cells were transformed by electroporation (van der Rest et al., 1999).
RNA extraction.
Total RNA from corynebacteria grown to OD600 3·5–4·0 at different pHs was extracted essentially as described by Eikmanns et al. (1994), except that the cell pellet obtained after centrifugation was frozen with liquid nitrogen and kept at –70 °C until RNA extraction (Barreiro et al., 2001). The RNA concentration was determined spectrophotometrically by the absorbance at 260 nm.
Northern hybridizations.
Denaturing RNA electrophoresis was performed in 0·9 % agarose gels in MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA pH 7·0) with 17 % (v/v) formaldehyde. RNA (30–40 µg) was dissolved in denaturing buffer [50 % formamide, 20 % formaldehyde, 20 % MOPS (5x) with 10 % DYE (Sambrook & Russell, 2001) and 1 % ethidium bromide]. RNA probes for atpI, atpB and atpD were labelled with digoxigenin and Northern hybridizations were performed following the instructions of the DIG Northern Starter Kit (Roche). The hybridization temperature was 68 °C.
Promoter fusion to the cat reporter gene.
DNA fragments of 240 bp (P-atp1) and 355 bp (P-atp2) were amplified from total DNA of C. glutamicum with Platinum pfx DNA polymerase, using the PxB-U1/PxB-D3 and PxB-U/PxB-D2 pairs of primers, respectively (Table 2; see also Fig. 4a). Both fragments, digested with BamHI/SacI, were cloned in plasmid pET2 digested with the same enzymes. These two putative promoter regions of the F0F1 ATPase operon were sequenced with the PETH and CM4 primers. Both constructions with the promoter regions of the F0F1 operon (named pET2-atp1 and pET2-atp2) were transformed into E. coli DH5
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sicová et al., 1998). The cultures were grown at 30 °C under different pH conditions (pH 6·0, 7·0 and 9·0). Primer extension was performed essentially as described by Pátek et al. (2003a). RNA (100–150 µg) was hybridized with 3 pmol fluorescein-labelled CM4 primer or with the CM5 primer (Table 2
) complementary to the cat (chloramphenicol acetyltransferase) sequence in pET2. The precipitated RNA with the hybridized primer was dissolved in 19 µl of the following reaction mixture: 4 µl 5x First-Strand Buffer (Invitrogen), 5 µl dNTP (2 mM), 1 µl RNasin (40 U) (Promega), 2 µl actinomycin D (500 µg ml–1) (Sigma), 2 µl DTT (0·1 M) (Sigma) and 5 µl H2O. After precipitation, the sample was dissolved in 6 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 7·6) and 6 µl formamide loading buffer (stop buffer) from the Thermo Sequenase Primer Cycle Sequencing kit (Amersham). A 6 µl sample was heat-denatured, loaded onto 6 % polyacrylamide sequencing gel next to the sequencing ladder and run in the automatic DNA sequencer (ALF, Pharmacia). The product of a sequencing reaction performed with the same promoter fragment cloned in pET2 and the CM4 or CM5 primers was used as sequencing ladder. The results were evaluated using the Fragment Manager V1.1 program (Pharmacia Biotech).
Chloramphenicol resistance assay.
The minimal inhibitory concentration of chloramphenicol was determined on LB or TSB plates (at pH values 6·0, 7·0 and 9·0) by the method of Ozaki et al. (1984).
RT-PCR and quantification of the RT-PCR products.
The RT-PCRs for the atpI and atpB genes were performed with the SuperScript One-Step RT-PCR system with Platinum Taq (Invitrogen), using five sets of primers (Table 2) for the corresponding genes (ATPI-U/ATPI-D, ATPB-U/ATPB-D, ATPI-U/ATPB2, ATPI-U/ATPI-4 and ATPI-U/ATPB-D), and another set of primers for the 16S rRNA (16S-3/16S-5) as a control of total RNA. The reactions were performed with 0·5 µg RNA. The intensity of the bands was analysed with the Quantity One program (The Discovery Series, 1-D Analysis Software, Bio-Rad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Confirmatory experiments were performed in four identical Braun Biostat fermenters at fixed pH values of 6·0–9·5 to avoid the possible effect of the buffering substances on the growth rate. The results of the fermenter studies showed that the growth rate of C. glutamicum was optimum at pH values 7·0–9·0 (Fig. 1). The highest biomass was obtained at pH 9·0. The growth rate was clearly lower at pH 6·0 or pH 9·5. These results indicate that C. glutamicum is an alkali-tolerant bacterium.
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Organization of the C. glutamicum F0F1 operon
The genetic organization of the C. glutamicum F0F1 ATP synthase operon maintains the canonical order of the eight structural genes, atpBEFHAGDC (Fig. 2), as in most prokaryotes (Table 3).
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), although the putative protein encoded by this gene does not appear to form part of the F0F1 ATPase complex (see Discussion). A comparative search of ORFs located immediately upstream of the F0F1 operon of other Gram-positive bacteria revealed that genes similar to atpI occur in Streptomyces coelicolor (28·9 % identical amino acids) (Hensel et al., 1995), Clostridium thermaceticum (Amaresh & Ljungdahl, 1997), Bacillus subtilis (32·2 % identity) (Santana et al., 1994), and also in the Gram-negative E. coli (28·8 % identity).
Transcriptional analysis of the F0F1 operon shows strong induction of a 7·5 kb polycistronic transcript and a 1·2 kb monocistronic transcript at alkaline pH
The expression of the F0F1 operon was tested in exponential-phase cells (OD600 3·5–4·0) using probes named B, B1, B2 and D (Fig. 2) of 750, 275, 350 and 850 bp, respectively, internal to the atpB and atpD genes.
As shown in Fig. 2, two transcripts, of 7·5 kb and 1·2 kb, were observed when the total RNA was hybridized with the B or B2 atpB probes. The 7·5 kb band appears to correspond to a transcript of the full atpBEFHAGDC operon. This was confirmed by hybridization with the D probe (internal to the atpD gene located in the distal part of the operon), which highlighted the same 7·5 kb transcript. The 1·2 kb band observed with the B probe appears to correspond to a different transcript specific for the atpB gene.
Formation of both transcripts was clearly induced at alkaline pH (9·0). The 7·5 kb transcript was probed with either the atpB or atpD probe. Similar results were obtained with the B2 probe (Fig. 2); the signals obtained with the B1 probe were very weak, probably due to the interference of the secondary structures of the mRNA in this region.
RT-PCR analysis confirms the alkaline pH induction of the F0F1 operon
The increased expression of the atpB gene at alkaline pH was confirmed by RT-PCR using several primer pairs as indicated in Fig. 3(a). As shown in this figure, the atpB gene showed a clearly higher expression at pH values 7·0 and 9·0 than at pH 6·0, whereas no differences were observed in the levels of the 16S rRNA control. Controls without reverse transcriptase or without RNA were run to confirm that PCR amplification of possible DNA contaminations was not occurring. The PCR reactions (with Taq polymerase without RT) gave no amplification at all.
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, RT-PCR analysis using primers ATPI-U and ATPI-D, corresponding to the atpI gene, proved that this gene has a low level of expression, as compared to the 16S rRNA or the atpB gene. Interestingly, this low expression increased at pH 9·0, suggesting that the promoter of the atpI gene is also regulated by pH.
The lower expression of the atpI gene suggests that it is expressed from a promoter different from that of the polycistronic mRNA, although we could not exclude a priori that the atpI transcript might be formed by processing of the 7·5 kb polycistronic transcript. To clarify this point, RT-PCR studies of the atpI transcript were performed using the same upstream primer (ATPI-U) and three different downstream primers ATPB2, ATPI-4 and ATPB-D (Fig. 3b). The results unequivocally showed that amplification of the atpI transcript was only observed with the first downstream primer (ATPB2). This result, together with the finding of two separate promoters (see below), indicates that the atpI and atpB genes are expressed from separate promoters.
Analysis of the P-atp1 and P-atp2 promoters by coupling to the cat reporter gene
Two putative promoter regions located upstream of the atpI gene (named P-atp1) and upstream of the atpB (named P-atp2), respectively, were studied by coupling these regions to a chloramphenicol acetyltransferase (cat) gene in the promoter-probe vector pET2 (Vsicová et al., 1998) (Fig. 4a). Both promoters showed transcription-initiation ability in C. glutamicum and in E. coli, a common feature of several C. glutamicum promoters that are recognized by the E. coli RNA polymerase (see Discussion).
The P-atp1 promoter expressed cat, conferring resistance to 80 µg chloramphenicol ml–1 in C. glutamicum. In this regard, this promoter is similar to several other C. glutamicum promoters characterized in our laboratory (Fig. 4b). The P-atp2 promoter expressed cat producing resistance to 80 µg chloramphenicol ml–1 in C. glutamicum.
Using the reporter constructs, the pH regulation of both P-atp1 and P-atp2 was analysed. The results (Fig. 4b) showed that there is a clear induction of reporter expression at pH 9·0 for both promoters. By contrast, the P-K promoter corresponding to the ribosomal protein operon rplK-rplA, used as control (Barreiro et al., 2001), was not affected by changes in pH from 6·0 to 9·0 (Fig. 4b).
The lack of effect of pH changes on expression of the cat reporter from the control P-K promoter validates the use of this gene as reporter in C. glutamicum promoter studies.
Transcription start points in the P-atp1 and P-atp2 promoters
To characterize in more detail the two promoter regions upstream of the atpI and atpB genes, the transcription start points of both promoters were identified by primer extension studies. The results (Fig. 5) showed that there was a transcription start point in the P-atp1 region. A single peak in the primer extension reaction product was identified, corresponding to an adenine located 45 bp upstream of the translation start codon of the atpI gene (GTG) (Fig. 5c). Identification of the transcription start point at this position allowed us to define the –10 (TAGTCG) and –35 (TTAGGT) regions of the P-atp1 promoter.
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) (see Discussion).
The F0F1 operon is flanked by transcriptional terminators
An analysis of inverted repeat (IR) sequences that may form stem and loop structures in the F0F1 operon revealed that there is a 15 nt transcriptional terminator of the gene located upstream of atpI encoding an unidentified putative protein (Fig. 2). Downstream of atpC (the last gene of the operon) there was another long IR sequence (25 nt) that is likely to act as transcriptional terminator of the F0F1 operon.
In addition, two IRs of 16 and 19 nt were found downstream of the atpI gene inside the coding frame of atpB (Fig. 3b). These two IR sequences may act as terminators of the atpI gene, but they also may serve as modulators (attenuators) of expression of the 7·5 kb polycistronic transcript.
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DISCUSSION |
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; Martín & Gil, 1999). We observed that C. glutamicum ATCC 13032 is a moderate alkaliphile which grows optimally at pH 7·0–9·0. Therefore, pH 6·0 and 9·0 were used for comparative gene expression studies. Similar pH values have been used to study pH-stress conditions in E. coli K-12 (Maurer et al., 2005), Bacillus pseudofirmus (Gilmour et al., 2000) and Shigella flexneri (Small et al., 1994). An adaptation phenomenon was observed when cultures grown at pH 9·0 were reinoculated at pH 10·0. The adaptative ability to grow at pH 10·0 supports the alkaliphilic nature of C. glutamicum.
We report here the transcriptional pattern of the F0F1 operon in C. glutamicum as established by Northern blot studies. A 7·5 kb transcript was found that includes all the structural genes of the atpBEFHAGDC operon, which was clearly induced under basic conditions. In contrast, several studies showed that this operon is induced by acid pH in other bacteria (Kuhnert et al., 2004; Martín-Galiano et al., 2001; Kullen & Klaenhammer, 1999). The recent finding that the E. coli F0F1 operon is induced by alkaline pH (Maurer et al., 2005) modifies the previous belief that this operon was induced by a high extracellular proton concentration. An intriguing question is why expression of this operon is elicited by opposite pH in different bacteria. Our results showed that in C. glutamicum the F0F1 operon is induced at alkaline pHs, particularly at pH 9·0; at this pH, growth of C. glutamicum is optimal. Probably the same occurs with other alkaliphilic micro-organisms (Saito & Kobayashi, 2003).
The 7·5 kb polycistronic transcript might be cleaved endonucleolytically to form the 1·2 kb transcript, a mechanism that has been demonstrated to regulate unequal expression of genes of other operons in E. coli (Newbury et al., 1987; Patel & Dunn, 1995). The coordinated expression of the eight genes ensures the availability of equimolecular amounts of the different proteins to form the ATPase complex. A small 1·2 kb transcript, which is also induced by pH, corresponds to a specific expression of the atpB gene.
The F0F1 operon of C. glutamicum is preceded by the atpI gene, as in E. coli (Walker et al., 1984), Bacillus subtilis (Santana et al., 1994), Bacillus pseudofirmus O4 (Ivey & Krulwich, 1991), and Streptomyces lividans (Hensel et al., 1995). The role of the small atpI gene in the F0F1 operon is intriguing. The atpI gene has not been reported to be present in all sequenced F0F1 operons and the AtpI protein from different organisms exhibits poor sequence conservation, which could indicate a dispensable function of this gene. We found a weak expression of the atpI gene in comparison with that of the atpB gene, as occurs in E. coli and Ilyobacter tartaticus (Schneppe et al., 1991; Meier et al., 2003). Poor or null atpI gene expression is a common feature among bacteria (Amaresh & Ljungdahl, 1997; Walker et al., 1984; Hicks et al., 2003).
The results of the RT-PCR experiments correlate well with those observed in the Northern blot analysis, supporting the conclusions obtained with this second technique. These experiments also confirmed the expression of the atpI gene (expected size band of 250 nt) and also proved that this gene is not transcribed as part of the large 7·5 kb F0F1-ATP synthase mRNA, as shown by the different intensity of the atpI and the atpB amplified bands.
The separate expression of atpI from the F0F1 operon might be related to its physiological function. The atpI gene of B. pseudofirmus OF4 (Hicks et al., 2003) has been reported to encode a hypothetical Mg2+ or Ca2+ transporter. The nucleotide substrates for the F0F1 ATP synthase are usually complexed with Mg2+, and the cation has been suggested to play a role in establishing the asymmetry of the catalytic sites of the F0F1 complex (Frasch, 2000). In C. glutamicum the atpI gene is induced at basic pH, in contrast to what occurs in E. coli, in which there is no effect on atpI expression when the pH of the medium is changed (Kasimoglu et al., 1996). The alkaline induction of atpI in C. glutamicum suggests that its function is related to that of the F0F1 operon, which is also induced under the same conditions.
Promoter activity was found in the upstream regions of the atpI and atpB genes. Both regions showed high transcription initiation ability when they were tested in C. glutamicum or as heterologous sequences in E. coli. The transcriptional initiation ability in both micro-organisms indicates that these two promoter regions are recognized by the RNA polymerases of E. coli and C. glutamicum. These promoters belong to the type I corynebacteria promoters (Martín et al., 1990; Cadenas et al., 1991), also known as CEP (‘Corynebacteria-E. coli promoters’), described in several genes of C. glutamicum (Pátek et al., 2003a; Barreiro et al., 2004).
The transcription start point of the atpI and atpB promoters was determined by primer extension analysis; this allowed the identification of their –10 boxes. There is considerable variation in the conservation of nucleotides in the –10 region of corynebacterial promoters. The atpI and atpB –10 boxes are compared in Fig. 6(a) with the consensus 12 nt extended sequence tgngnTA(c/t)aaTgg (a high percentage of conservation is shown by capital letters) of C. glutamicum (Pátek et al., 2003b) recognized by the vegetative subunit of the RNA polymerase (Oguiza et al., 1996). Two of the three most conserved positions (6th and 7th) and the G in position 12 are present in the P-atp1 promoter. Downstream of the –10 region of P-atp1 there is a CATTA sequence that has four identical nucleotides (indicated in bold) to the CATGA pentamer found at this position in seven promoters of C. glutamicum downstream of the –10 boxes (Pátek et al., 2003b). The –10 box for the P-atp2 promoter is clearly less conserved with respect to promoters recognized by the vegetative subunit. However, it agrees with the –10 box of the consensus promoters recognized by H in Mycobacterium tuberculosis or R in Streptomyces coelicolor (Fig. 6b), which was also found in the C. glutamicum dnaK promoter (Barreiro et al., 2004).
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sicová et al., 1999). In the case of the P-atp2 promoter, the –35 box TGGAT has four identical positions (in bold) with the consensus H sequence (Fig. 6b), supporting the involvement of this sigma factor in the alkaline stress response. In summary, we have established that the abundance of the mRNA of the F0F1 operon of C. glutamicum is increased at basic extracellular pH, which indicates the existence of regulation at the level of transcription. On the other hand, the upstream atpI gene is transcribed independently from the operon but is also induced at basic pH. The P-atp2 promoter may be activated by the alternative sigma factor of the RNA polymerase, whose synthesis would be activated when the bacteria are growing at basic pH. A change in external pH might trigger a transient change in internal pH, which may subsequently serve as an intracellular signal to induce expression of the F0F1 operon, thus allowing a higher rate of ATP synthesis and increased growth at its optimal alkaline pH.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 July 2005;
revised 4 October 2005;
accepted 18 October 2005.
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), pH 7·0 (
), pH 9·0 (
) and pH 9·5 (*). The same inoculum culture was used to seed the four fermenters. (a) Optical density versus time; (b) dry weight versus time. Vertical bars indicate standard deviations of the mean values of three determinations (some of the bars are small and overlap the symbols).
