Department of Cell and Organism Biology, Lund University, Sölvegatan 35, SE-223 62 Lund, Sweden
Correspondence
Claes von Wachenfeldt
Claes.von_Wachenfeldt{at}cob.lu.se
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ABSTRACT |
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Text files of transcriptome data, light absorption difference spectra of membranes from strains 1A1 and LUW210 (Supplementary Fig. S1) and oligonucleotide sequences (Supplementary Table S1) are available with the online version of this paper.
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INTRODUCTION |
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In Escherichia coli, oxygen represses the anaerobic respiratory pathways and fermentation, whereas nitrate is the preferred electron acceptor under anaerobic conditions. Nitrate acts to induce nitrate reductase expression and to repress synthesis of other anaerobic pathways (Gennis & Stewart, 1996). The transcriptional regulation of the respiratory enzymes parallels the redox potentials of the corresponding electron-acceptor couples (Gennis & Stewart, 1996
). A similar hierarchical transcriptional regulation may be present in B. subtilis. The two-component regulatory system ResDE and the redox regulator Fnr are important components of the regulatory system for anaerobic adaptation. Several enzymes necessary for anaerobic growth are under the control of the ResDE two-component system (Nakano & Zuber, 2002
). ResD encodes a putative oxygen sensor kinase and ResE encodes a transcription regulator (Geng et al., 2004
). Full activation of fnr expression requires ResE (Nakano & Zuber, 2002
). Fnr is in turn an activator of the narGHJI operon, narK encoding a putative nitrite extrusion protein, and arfM encoding an anaerobic respiration and fermentation modulator (Cruz Ramos et al., 1995
; Marino et al., 2000
).
Transcription of the cydABCD operon, encoding cytochrome bd, is highly regulated and only becomes activated when oxygen is limiting (Winstedt et al., 1998). Cytochrome bd is, in analogy to the orthologous E. coli enzyme, likely to be a high-oxygen-affinity terminal oxidase. Here, we show that regulation of the cydABCD operon is independent of the resDE or fnr gene products. It has recently been shown that in the high-G+C Gram-positive bacterium Streptomyces coelicolor A3(2), expression of the cytochrome bd-encoding genes is negatively regulated by the DNA-binding Rex protein (Brekasis & Paget, 2003
). In B. subtilis, the orthologous protein is encoded by the ydiH gene. Here, we provide evidence that B. subtilis YdiH functions as a repressor not only of the cydABCD operon but also of the ldh lctP operon and of a putative formate-nitrite transporter gene, ywcJ. During the course of this work, Schau et al. (2004)
reported that YdiH is a negative regulator of the cydABCD operon.
A regulatory model is proposed in which the NADH/NAD+ ratio is key to a control YdiH activity. During the transition to oxygen-limited growth, an increased level of NADH is expected, since it is less efficiently reoxidized to NAD+ as a result of reduced respiration. This leads to the production of cytochrome bd, LDH and YwcJ.
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METHODS |
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RNA isolation.
To isolate RNA, a 12·5 ml culture sample was rapidly added to a 50 ml centrifuge tube filled with 5 g crushed ice. The sample was centrifuged at 8000 g for 10 min at 4 °C, and the pellet was suspended in 1 ml ice-cold TES buffer (50 mM Tris/HCl, pH 7·5, 5 mM EDTA, 50 mM NaCl) and transferred to a 2 ml test tube containing 0·6 ml acidic phenol, 0·12 ml chloroform and 0·75 ml zirconium-silica beads (0·1 mm diameter). The test tube was placed in a Mini-Beadbeater (Biospec Products, USA) and shaken at full speed for 80 s. The tube was then centrifuged at 5000 g for 5 min. The aqueous phase was recovered and extracted twice with 0·6 ml acidic phenol and 0·12 ml chloroform, and once with 0·7 ml chloroform. Total RNA was precipitated from the aqueous phase by adding a 1/10 volume of 3 M sodium acetate (pH 4·8) and two volumes of ice-cold 95 % (v/v) ethanol. After centrifugation and washing with ice-cold 70 % (v/v) ethanol, the pellet was suspended in 0·2 ml water pre-treated with diethyl pyrocarbonate. The RNA was then treated with 3 µl (3 U) DNase I (Invitrogen) for 30 min at 37 °C, extracted once more with 0·6 ml acidic phenol and 0·12 ml chloroform, and finally with 0·7 ml chloroform. RNA was recovered by precipitation, as described above, and suspended in 60 µl water pre-treated with diethyl pyrocarbonate. The concentration and quality of the RNA was checked by electrophoresis in a 0·8 % (w/v) agarose gel containing ethidium bromide.
cDNA labelling and microarray hybridization conditions.
For cDNA synthesis, 20 µl of total RNA (1 µg ml1) was mixed with 1 µl (0·1 pmol) of a mixture of coding-sequence-specific primers (Eurogentec), 8 µl of Superscript II buffer (Invitrogen), 3 µl of a 20 mM mixture of dATP, dGTP and dTTP (containing equimolar amounts of the nucleotides), 1 µl of 2·5 mM dCTP, 1 µl of 1 mM Cy3- or Cy5-labelled dCTP analogues (Amersham Biosciences) and 4 µl of 0·1 M DTT. The mixture was incubated at 65 °C for 5 min and subsequently at 42 °C for 5 min. Then 1 µl (40 U) of RNasin (Promega) and 2 µl (400 U) of SuperScript II reverse transcriptase (Invitrogen) were added and the mixture was incubated at 42 °C for 1 h. To stop the reaction and hydrolyse RNA, 5 µl of 50 mM Na/EDTA (pH 8·0) and 2 µl of 10 M NaOH were added, and the sample was incubated at 65 °C for 20 min. Unincorporated dye-labelled dCTP was removed and cDNA was concentrated to 15 µl by using a microconcentrator (Microcon YM-30, Millipore). DNA microarrays consisting of duplicated spots of PCR products representing 3925 of the 4100 predicted ORFs of the B. subtilis 168 genome (Eurogentec) were prehybridized using 47·5 µl DIG Easy Hyb solution (Roche) and 2·5 µl yeast RNA (10 mg ml1 in DIG Easy Hyb solution) at 42 °C for 2 h. Solutions were applied to the microarrays by capillary action under a coverslip. The coverslip was removed by dipping the slides into 0·1x SSC. The slides were dried by centrifugation for 30 s. For hybridization with cDNA, 42·5 µl DIG Easy Hyb solution was mixed with 2·5 µl yeast RNA and 5 µl dye-labelled cDNA. The slides were incubated with this mixture for 16 h at 42 °C. Microarray slides were washed twice for 5 min each at room temperature with 0·1x SSC, 0·1 % (w/v) SDS and again for 1 min in 0·1x SSC. Slides were dried by centrifugation and were immediately scanned with a confocal laser scanner with excitation at 635 and 532 nm (Axon GenePix 4000B; Axon Instruments, Inc.). Synthesis of cDNA from RNA isolated from untreated (aerobic) and argon-exposed cells was made in the presence of Cy5 and Cy3, respectively. Each experiment, except with LUW219, was performed at least two times, with RNA isolated from independent bacterial cultures.
Microarray data analysis.
Images were analysed using GenePix Pro 4.1 (Axon Instruments). The images were inspected for artifacts. Spots affected by, for example, smears were excluded from further analysis. The results from the image analysis, including background and median spot intensities, were transferred into the software BASE 1.2 (BioArray Software Environment) (Saal et al., 2002). The data were normalized with a LOWESS (locally weighted scatterplot smoothing) algorithm [0·33 window size (fraction of points), 0·1 minimum log (intensity) step, 4 iterations]. For detection of differentially expressed genes, a standard deviation analysis obtaining a Z score representing the change in expression for each gene was performed (Saal et al., 2002
; Yang et al., 2003
). Briefly, the Z scores were calculated as follows. From within the assay, n spots were selected using a sliding window across, where Ch1 and Ch2 were the background-corrected intensities from the scanning at 635 and 532 nm, respectively. The sliding window was set to 400 spots.
For each spot i, the Z score was calculated as:
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An arithmetic mean of the Z scores was calculated from all replicates in each assay. Genes present in less than n (total replicates)1 were excluded from the results. The Z score cut-off was chosen as ±3SD. This corresponds to a confidence level of 99·73 %, or a P value of 7·3x106 for a gene to be differentially expressed for two slides (outside the Z score cut-off for both slides). With 3925 data points, this results in 0·03 errors in this experiment.
-Galactosidase activity measurements.
-Galactosidase activity was assayed using MUG (4-methylumbelliferyl-
-D-galactoside) as substrate. Liquid culture measurements were done essentially as described by Youngman (1990)
. The bacteria were grown in 25 ml cultures in 250 ml Erlenmeyer flasks with indentations. The cultures were first inoculated to an OD600 of 0·1, grown to an OD600 of 0·81·0 and then diluted to an OD600 of 0·1. At various times during growth, 1 ml samples were removed and frozen in liquid nitrogen. Samples were stored at 80 °C until assayed for
-galactosidase activity. One unit of activity is defined as 1 nmole of MUG hydrolysed per millilitre of culture sample per minute, normalized for culture cell density (OD600).
Construction of B. subtilis strains carrying cydlacZ or ywcJlacZ fusions.
Transcriptional lacZ fusion plasmids were constructed in vector pDG1661. Strains LUW95, LUW96, LUW107, LUW108, LUW217, LUW243, LUW244, LUW245, LUW270, LUW271, LUW241 and LUW275 were constructed by using pDG1661 and primer pairs (see Supplementary Table S1): CYDA6/CYDA10, CYDA6/CYDA9, CYDA6/CYDA11, CYDA6/CYDA12, glpDcyd/CYDA6, CYDA6/CYDA22, CYDA6/CYDA20, CYDA6/CYDA21, CYDA6/CYDA24, CYDA6/CYDA25, CYDA6/CYDA30, CYDA6/CYDA31 and YWCJ1/YWCJ2, respectively. The various DNA fragments were digested with EcoRI and BamHI and ligated into pDG1661 digested with the same enzymes, and then transformed into E. coli XL-1 Blue selecting for ampicillin resistance. Plasmids extracted from E. coli were used to transform B. subtilis 1A1 to chloramphenicol resistance. In the resulting transformants, the promoterlacZ fusion had integrated at the amyE locus by double-crossover recombination. All constructs generated from PCR-amplified fragments were subjected to sequence analysis to verify the fidelity of amplification.
Construction of a yjbIH null mutant.
The upstream and downstream regions flanking the yjbIH genes were amplified by PCR from B. subtilis 1A1 chromosomal DNA using primer pairs YJBI6/YJBI7 and YJBI8/YJBI9, respectively (see Supplementary Table S1). The YJBI6/YJBI7 PCR fragment was cut with NdeI and EcoRI and ligated into pDG1726 digested with the same enzymes and transformed into E. coli TOP10 selecting for ampicillin resistance. The resulting plasmid was named pYjbI1. The YJBI8/YJBI9 PCR fragment was cut with SphI and ligated into pYjbI1 cut with SphI to give plasmid pyjbIH1. This plasmid was used to transform strain 1A1 to spectinomycin resistance. The deletioninsertion within the chromosomal yjbIH locus arising from a double crossover recombination event was confirmed by sizing the PCR fragments generated from this chromosomal region (data not shown).
Inactivation of ydiH.
Plasmid pYdiH1, carrying an internal fragment of ydiH, was integrated into the chromosome of B. subtilis strain 1A1 via a single-crossover recombination event. Integration of pYdiH1 in the ydiH gene was confirmed by sizing of PCR fragments generated from this chromosomal region (data not shown). The integration disrupts the ORF of ydiH. To obtain plasmid pYdiH1, a 663 bp DNA fragment was amplified using PCR with B. subtilis 1A1 chromosomal DNA as template and the primers YDIH1 and YDIH2. The DNA product was cut with DraI and EcoRI and ligated into HindII/EcoRI-cut pDG1727.
Miscellaneous methods.
For membrane preparations, liquid cultures of B. subtilis were grown in 1·5 l batches in 5 l Erlenmeyer flasks with indentations. The cultures were incubated at 37 °C on a rotary shaker (200 r.p.m.). Membranes were isolated as described by Hederstedt (1986) and suspended in 20 mM MOPS buffer (pH 7·4). Absorption spectra were recorded as described previously (Schiött et al., 1997
). The absorption difference at 650626 nm was used to evaluate the relative concentration of cytochrome bd in the membranes. Protein concentrations were estimated using the bicinchoninic acid (BCA) method (Pierce) with BSA as standard. Dissolved oxygen measurements were done with a Mettler Toledo InPro 6000 series oxygen sensor. The RSAT tools were used to search genomic sequences for putative YdiH binding sites (van Helden et al., 2000
). The WebLogo was prepared according to Crooks et al. (2004)
.
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RESULTS |
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Role of Fnr, ResDE and YjbIH in regulation of cydABCD
Cytochrome bd shows an apparent oxygen-dependent expression pattern and is maximally expressed at low oxygen tension (Winstedt et al., 1998). Several of the O2-responsive gene regulators of bacteria are members of the Fnr protein family of transcriptional regulators (Korner et al., 2003
). However, mutations in fnr had no apparent effect on cydABCD expression in NSMP, but may influence the maximum level of expression in NSMPG (Fig. 1E
). ResD of the ResDResE two-component signal transduction system has been suggested to directly sense a decreased oxygen tension leading to activation of the ResDE regulon (Geng et al., 2004
). Deletion of resDE had little effect on the expression of a cydlacZ fusion (Fig. 1F
).
The yjbI gene of the putative yjbIH operon encodes a truncated haemoglobin (Giangiacomo et al., 2005). The physiological roles of truncated haemoglobins are largely unknown. However, experimental data suggest a variety of roles for these proteins related to the binding of oxygen for storage, transfer or sensing (Wittenberg et al., 2002
). A yjbIH mutation drastically decreased cydlacZ expression (Fig. 3
A). It was noted that the yjbIH mutation also affected bacterial growth and oxygen consumption in NSMPG (Fig. 3B, C
). To investigate if YjbIH is a direct oxygen sensor, we used transcriptome analysis to see which genes are differentially expressed when an aerobic culture is rapidly challenged with oxygen limitation. Wild-type and yjbIH mutant cells grown aerobically to exponential phase in NSMPG were left untreated or exposed to argon to rapidly deplete oxygen from the culture medium. After 5 min, total RNA was isolated from the untreated and argon-exposed cultures. The cydAB (encoding cytochrome bd), ldh lctP (encoding LDH and lactate permease, respectively) and ywcJ (encoding a putative formate-nitrite transporter) transcripts showed a significant accumulation in the wild-type samples exposed to argon (Fig. 4
A). Transcriptome analysis of the yjbIH mutant showed significant induction of cydA and ldh expression (Fig. 4B
). The results indicate that YjbIH is not directly involved in the regulation of these genes.
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DISCUSSION |
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The ResDE two-component system and the redox regulator Fnr play important roles in B. subtilis during transition from aerobic to anaerobic growth (Nakano & Zuber, 2002). Our work demonstrates that ResDE does not appear to influence transcription of the cydABCD genes encoding the cytochrome bd terminal oxidase. In an Fnr mutant, the maximum level of
-galactosidase activity from the cydlacZ reporter was slightly reduced, but the timing of expression was not influenced. The recently described regulator YdiH seems to be a principal regulator of cydABCD expression in B. subtilis. Three putative binding sites for YdiH are located downstream from the transcription start site (Schau et al., 2004
), suggesting that repression of the cyd operon occurs via a transcriptional roadblock mechanism that inhibits transcript elongation. However, it is possible that additional factors control expression of the cyd genes. Our observation that the sequence upstream (up to position 79) of the 35 hexamer of the core cyd promoter significantly stimulates promoter activity suggests the involvement of an as-yet-unidentified transcription activator. Transcription activation by the putative regulator is likely to influence the level of transcription but not its timing, which is negatively controlled by YdiH.
The ldh lctP genes, which encode LDH and lactate permease, respectively, and the ywcJ gene, which encodes a predicted formate-nitrite transporter, show a pattern of expression that is very similar to that of the cyd genes. The present work demonstrates that YdiH acts as a negative regulator that coordinates the expression of these genes during the transition from aerobic to microaerophilic and finally to anaerobic growth. When aerobically and exponentially growing bacteria encounter depletion of oxygen in the medium, NADH reoxidation to NAD+ by the respiratory chain becomes less efficient. The oxygen limitation leads to induction of the cyd genes and production of cytochrome bd, an enzyme that is likely to have a higher affinity for O2 but a lower energetic efficiency with respect to proton translocation compared to the cytochrome aa3 haem-copper type oxidase (Jünemann, 1997). To further increase NADH reoxidation, expression of the genes encoding the fermentative NADH-linked LDH is induced. By the action of cytochrome bd and LDH, the NADH/NAD+ ratio is decreased, leading to repression of the expression of the corresponding genes by YdiH. From the available data and the sequence similarity between Rex and YdiH, we suggest that ydiH be renamed rex.
Our results show that a rapid shift to oxygen-starved conditions activates the B-dependent general stress response in a strain lacking ydiH. Moreover, activation of
B under these circumstances is primarily dependent on the energy-stress sensor RsbP. Induction of
B by energy stress correlates with a drop in the intracellular levels of ATP. It is not known if ATP itself is the signal that communicates energy stress to RsbP (Hecker & Völker, 2001
). However, sequence analysis suggests that the protein is composed of two domains, a PAS domain and a catalytic protein phosphatase 2C (PP2C)-like serine phosphatase domain. PAS domains are involved in several signalling proteins, where they are used as cytosolic signalling modules. It has been suggested that the PAS domain of RsbP could directly sense a change in the redox potential of the cytoplasm (Vijay et al., 2000
). Further studies are needed to understand why RsbP is activated in the YdiH (Rex) mutant strain.
Many factors and growth conditions contribute to changes in the ratio of NADH to NAD+. For instance, the observed reduction of cyd expression in the mutant lacking the truncated haemoglobin (YjbIH mutant) is most likely an indirect effect due to altered growth properties of the YjbIH mutant. During growth of the YjbIH mutant, the levels of dissolved oxygen in the medium did not decrease below 10 mbar of O2. In contrast, growth of the wild-type resulted in very low O2 tensions in the stationary growth phase. In a similar way, the involvement of the transcription regulator CcpA in cyd expression is most likely mediated via YdiH (Rex). Glucose repression of the ctaCDEF genes encoding the cytochrome caa3 terminal oxidase is reported to be dependent on CcpA (Yoshida et al., 2001). Analysis of membranes from a ccpA mutant (see Supplementary Fig. S1) indicates the presence of cytochrome caa3. Increased levels of cytochrome caa3 may contribute to NADH reoxidation, leading to YdiH (Rex)-mediated repression of the cyd genes.
Nitrate can be used as an electron acceptor for anaerobic growth of B. subtilis. It has previously been shown that nitrate represses transcription of ldh and lctP (Cruz Ramos et al., 2000). The mechanism for this regulation has not been reported. However, it has been shown that expression of the respiratory nitrate reductase is needed to allow nitrate regulation (Cruz Ramos et al., 2000
). Nitrate respiration contributes to the oxidation of NADH. This strongly indicates that the observed nitrate repression of ldh lctP is mediated via YdiH (Rex) due to the nitrate-respiration-dependent NADH oxidation.
The role of Fnr in the regulation of ldh lctP expression is less clear. It is interesting to note that the potential binding site for Fnr, and also to some extent ResD, overlaps with the proposed YdiH (Rex)-binding sequence (Fig. 5B). Putative Fnr-binding sites are found in the ywcJ and the ldh lctP promoter regions, and a ResD-binding site overlaps with the middle YdiH (Rex) box in the cyd promoter (Fig. 5A
). It remains to be investigated if Fnr and ResD can function as transcriptional repressors. The interplay between multiple regulatory systems could help to ensure the appropriate expression of cytochrome bd and LDH in response to changes in the oxygen tension and redox state of the cells.
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ACKNOWLEDGEMENTS |
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Received 15 April 2005;
revised 14 July 2005;
accepted 15 July 2005.
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