Coordinated patterns of cytochrome bd and lactate dehydrogenase expression in Bacillus subtilis

Jonas T. Larsson, Annika Rogstam and Claes von Wachenfeldt

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A variety of pathways for electron and carbon flow in the soil bacterium Bacillus subtilis are differentially expressed depending on whether oxygen is present in the cell environment. This study characterizes the regulation of the respiratory oxidase cytochrome bd and the NADH-linked fermentative lactate dehydrogenase (LDH). Transcription of the cydABCD operon, encoding cytochrome bd, is highly regulated and only becomes activated at low oxygen availability. This induction is not dependent on the gene encoding the redox regulator Fnr or the genes encoding the ResDE two-component regulatory system. The DNA-binding protein YdiH was found to be a principal regulator that controls cydABCD expression. Transcription from the cyd promoter is stimulated 15-fold by a region located upstream of the core promoter. The upstream region may constitute a binding site for an unidentified transcription activator that is likely to influence the level of transcription but not its timing, which is negatively controlled by YdiH. This report provides evidence that YdiH also functions as a repressor of the ldh gene encoding LDH and of a gene, ywcJ, which encodes a putative formate-nitrite transporter. Based on the similarity between YdiH and the Rex protein of Streptomyces coelicolor, it is proposed that YdiH serves as a redox sensor, the activity of which is regulated by cellular differences in the free levels of NAD+ and NADH. It is suggested that ydiH be renamed as rex.


Abbreviations: CcpA, carbon catabolite protein A; LDH, lactate dehydrogenase; MUG, 4-methylumbelliferyl-{beta}-D-galactoside

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aerobic and facultative aerobic bacteria can respond to changes in the environment by synthesizing different respiratory pathways (Poole & Cook, 2000; von Wachenfeldt & Hederstedt, 2002). During aerobic growth the final step in respiration, the four-electron reduction of dioxygen to two water molecules, is catalysed by a group of membrane-bound enzymes called terminal oxidase. The free energy released in this process is used to establish and maintain a charge and pH gradient across the cytoplasmic membrane. This electrochemical gradient drives the synthesis of ATP and other energy-requiring reactions in the cell. Most prokaryotes have the capacity to produce multiple terminal oxidases: they have branched pathways for electron transfer from different low-redox-potential electron donors to oxygen. The spore-forming, low-G+C Gram-positive bacterium Bacillus subtilis has genes that encode three distinct terminal oxidases: cytochrome caa3, cytochrome aa3 and cytochrome bd (von Wachenfeldt & Hederstedt, 2002). In addition, the presence of a fourth terminal oxidase of bd type, YthAB, can be predicted from the genome sequence (Winstedt et al., 1998; von Wachenfeldt & Hederstedt, 2002). No single terminal oxidase is essential, but one of the quinol oxidases, cytochrome aa3 or cytochrome bd, is required for aerobic growth of B. subtilis (Winstedt & von Wachenfeldt, 2000). By changing the levels of the terminal oxidases and other respiratory enzymes, bacteria can conserve energy under widely varying environmental conditions. B. subtilis is also able to use nitrate (NO3) or nitrite (NO2) as electron acceptors for respiration or to grow fermentatively (Nakano & Zuber, 2002). In B. subtilis, the respiratory nitrate reductase is encoded by the narGHI genes of the narGHIJ gene cluster (Cruz Ramos et al., 1995). Nitrate reductase is a membrane-bound complex, which probably contributes to the proton-motive force by a redox loop in which oxidation of menaquinol and release of two protons takes place at the positive side (outer side) of the membrane. Nitrite reduction is catalysed by a soluble NADH-dependent nitrite reductase that is encoded by nasD and nasE (Nakano & Zuber, 2002). Under fermentative growth conditions, sugars are converted to reduced organic compounds, such as ethanol, lactate, acetoin and 2,3-butanediol. Lactate dehydrogenase (LDH), encoded by ldh, is a cytoplasmic NADH-linked enzyme that converts pyruvate to lactate. During fermentation, LDH is the key enzyme involved in reoxidation of the NADH formed by glycolysis (Cruz Ramos et al., 2000).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. B. subtilis strains were grown at 37 °C in nutrient sporulation medium with phosphate (NSMP) and in NSMP supplemented with 0·5 % (w/v) glucose (NSMPG) (Winstedt et al., 1998). Tryptose blood agar base medium (TBAB) was used for growth of bacteria on plates. L broth or L agar was used for the growth of E. coli strains (Sambrook & Russel, 2001). The following antibiotics were used when required: chloramphenicol (5 mg ml–1), tetracycline (15 µg ml–1), spectinomycin (150 µg ml–1), and a combination of erythromycin (0·5 µg ml–1) and lincomycin (12·5 µg ml–1) for B. subtilis strains, and ampicillin (100 µg ml–1) for E. coli strains.


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulations and transformation.
Methods for plasmid isolation, agarose gel electrophoresis, use of restriction and DNA-modification enzymes, DNA ligation, PCR and electroporation of E. coli cells were performed according to standard protocols (Sambrook & Russel, 2001). Isolation of chromosomal DNA and transformation of B. subtilis cells with chromosomal or plasmid DNA were done as described by Hoch (1991).

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 ml–1) 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 1–5 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 ml–1 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:

{3323equ1}
where M(w) is the mean log2 ratio of the spots in the window surrounding i:

{3323equ2}
SD(w) is the corresponding standard deviation.

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 7·3x10–6 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.

{beta}-Galactosidase activity measurements.
{beta}-Galactosidase activity was assayed using MUG (4-methylumbelliferyl-{beta}-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 0·8–1·0 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 {beta}-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 promoter–lacZ 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 p{Delta}yjbIH1. This plasmid was used to transform strain 1A1 to spectinomycin resistance. The deletion–insertion 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 650–626 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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Role of the cyd leader
The expression of cytochrome bd in B. subtilis is induced in cells grown in broth media containing glucose and reaches its maximum when the cells are entering the early stationary growth phase (Winstedt et al., 1998). B. subtilis LUW95 that carries a transcriptional cydlacZ fusion was grown in a broth medium with (NSMPG) or without glucose (NSMP), and {beta}-galactosidase activities were recorded. High expression of the lacZ reporter gene was only detected in NSMPG-grown cells harvested in the stationary phase (Fig. 1A). To study if the leader alone is responsible for the observed expression pattern, the LUW241 strain was constructed. This strain carries a cydlacZ fusion lacking most of the DNA corresponding to the untranslated cyd leader region. Removing the leader region drastically increased expression of cydlacZ in cells grown in NSMP (Fig. 1B). In NSMPG, an increased expression in exponentially growing cells was observed (Fig. 1A). When the cyd untranslated leader region followed by lacZ was placed downstream of the constitutive {sigma}A-type glpD promoter (Holmberg & Rutberg, 1992) (strain LUW217), a similar expression pattern as seen for LUW95 was observed (Fig. 1A, B).



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Fig. 1. Time-courses of {beta}-galactosidase activity of various cydlacZ fusions. Cells were grown at 37 °C on a rotary shaker (100 r.p.m.) in NSMPG (A, C–F) or NSMP (B). Samples were collected and assayed as described in Methods. t0 marks the end of exponential growth. Data were averaged from at least three independent trials. Error bars represent the standard deviation of the means. 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). Strains: (A, B, C, E, F) LUW95 (cyd region –270 to +199) ({bullet}), (A, B) LUW241 (cyd region –270 to +10) ({circ}), (A, B) LUW217 (cyd region +1 to +199) ({blacktriangledown}), (C) ccpA : : spc mutant strain LUW214 ({circ}), (D) PglpD-cyd-lacZ strain LUW217 ({bullet}), ccpA : : spc mutant strain LUW277 ({circ}), (E) fnr : : spc mutant strain LUW287 ({circ}), (F) resDE : : tet mutant strain LUW269 ({circ}).

 
Role of the upstream region of the cyd promoter
To define the minimal upstream sequences required for transcription of the cydABCD operon, a series of 5' deletion variants of the cyd promoter were used to drive expression of the lacZ reporter gene (Fig. 2). Upstream of the cydA transcription start site, a potential housekeeping {sigma}A-type –10 (TAAAGT) sequence is present. It is preceded by a poor –35 signal (TTTATT), suggesting the involvement of a transcription activator. The deletion analysis shows that transcription from the cyd promoter is stimulated approximately 15-fold by a cis-acting DNA sequence located between –79 and –74 upstream of the transcription start point (Fig. 2). Deletion of the upstream region affected the maximum expression level but not the timing of expression of the cydlacZ fusions (data not shown).



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Fig. 2. Deletion analysis of the cyd promoter. B. subtilis strains carrying cyd promoter variants were grown at 37 °C on a rotary shaker (100 r.p.m.) in NSMPG, and the promoter activity was detected at t+1. The promoter activity of LUW95 was used for the calculation of the relative promoter activity. The 100 % activity corresponds to 26·8±2·2 MUG units according to the mean and standard deviation from three independent experiments. The other promoter activities are calculated as the mean percentage expression of the full-length promoter (–270) from three independent experiments. The promoter activity for each construct is plotted versus the 5'-deletion end-point relative to the cydA transcriptional start point. The strains used were: LUW95 (–270), LUW96 (–116), LUW243 (–92), LUW270 (–89), LUW271 (–79), LUW244 (–74), LUW245 (–60), LUW107 (–37) and LUW108 (–7).

 
Carbon catabolite protein A (CcpA) indirectly affects expression of the cydABCD operon
The expression pattern of the cydABCD genes (Fig. 1A, B) suggests that they are subject to glucose activation. Data from transcriptome analysis indicates that the glucose activation is mediated by the transcription factor CcpA (Moreno et al., 2001). A mutation in the ccpA gene was found to suppress the maximal transcription of the cydlacZ fusion approximately 2·5-fold without affecting the timing of the transcription (Fig. 1C). The reduced level of expression was confirmed by an approximately threefold reduction of cytochrome bd enzyme produced (see Supplementary Fig. S1). The cyd untranslated leader region was next placed downstream of the constitutive glpD promoter. In this construct also, the ccpA mutation reduced transcription (approx. 3·5-fold), indicating that CcpA activation does not involve the cydABCD core promoter region or upstream regions (Fig. 1D).

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 ResD–ResE 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. 3A). 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. 4A). 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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Fig. 3. (A) Time-courses of {beta}-galactosidase activity of a cydlacZ fusion in wild-type or in a yjbIH mutant. Cells were grown at 37 °C on a rotary shaker (100 r.p.m.) in NSMPG. Sampling was done as described in the legend to Fig. 1. Strains: parental strain LUW95 ({bullet}), yjbIH : : spc mutant strain LUW219 ({circ}). (B) Growth and (C) dissolved oxygen concentrations in cultures of LUW95 ({bullet}) and LUW219 ({circ}) in NSMPG. In (C), the horizontal line (10 mbar of O2) indicates the approximate limit of microaerobic growth conditions (Arras et al., 1998).

 


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Fig. 4. Changes in gene expression as determined by microarray analysis of exponentially, aerobically growing cells and cells challenged with 5 min of oxygen limitation. B. subtilis strains were grown aerobically in NSMPG until reaching OD600 0·6. At this point, the cells were left untreated or exposed to thorough bubbling with argon. This treatment rapidly removes oxygen from the culture medium. (A) wild-type, (B) LUW219 ({Delta}yjbIH), (C) LUW273 (ydiH insertion mutant). The horizontal line marks the Z score threshold value that is considered significant. The intensity-dependent Z score measures the number of standard deviations of a particular data point from the mean (for details see Methods). Data points above this line represent genes which are significantly upregulated in oxygen-depleted cells. In (C), circled dots represent genes that are transcribed from {sigma}B-dependent promoters, and the boxed dots show genes that were found to be upregulated in wild-type cells (A). It should be noted that the microarrays used do not contain functional probes for cydC and cydD. The transcriptome datasets can be found in supplementary data text files 1, 2, 3, 4 and 5.

 
YdiH regulates cyd, ldh and ywcJ
DNase I footprinting experiments indicate that YdiH in vitro binds to three regions located in the 5'-untranslated region preceding cydA. The reported YdiH DNA-binding consensus sequence is TTTGTGAADTABTGAKCAAWDT (Schau et al., 2004). By searching the B. subtilis genome with the consensus sequence, highly related sequences are found in the ldh and ywcJ promoter regions (Fig. 5), indicating that YdiH also regulates these genes. In addition, a related but reversed sequence is present upstream of yjlC that encodes a protein of unknown function. A related sequence is also found to overlap the –10 region in the alsSD promoter. As described above, ldh and ywcJ, together with the cyd genes, are induced when an exponentially growing aerobic culture is rapidly challenged with oxygen limitation (Fig. 4A). When the same experiment was done with a YdiH mutant, no induction of cydAB, ldh, lctP or ywcJ transcription could be detected (Fig. 4C). The relatively high signal intensities observed for cydAB, ldh, lctP and ywcJ indicate that these genes were constitutively expressed at high levels in the mutant (Fig. 4C). In the YdiH mutant, oxygen limitation induced transcription of at least 103 genes, of which 54 are known to be under the control of the general stress sigma factor {sigma}B (Petersohn et al., 2001; Price et al., 2001) (Fig. 4C and supplementary data text files 3, 4 and 5).



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Fig. 5. (A) Putative YdiH-binding sites and location in different B. subtilis promoter regions. Grey rectangles show potential –10 and –35 {sigma}A-type promoter recognition sites and black rectangles indicate the position of YdiH-binding sites. The arrows indicate transcription start sites. The transcription start site of the cydABCD operon (Winstedt et al., 1998) and of the ldh lctP operon was obtained from primer extension analysis (Cruz Ramos et al., 2000). The location of the promoter and of the transcription start site of ywcJ was predicted (Jarmer et al., 2001). The predicted ywcJ promoter sequences are: –10 (TATACT) and –35 (GTGAAA). The upstream region of ywcJ was sequenced and found to be identical to the published genomic sequence (Glaser et al., 1993), with the exception of a single change of C to T corresponding to the most-5' nucleotide of the –10 sequence. This change has been confirmed by directly sequencing a PCR product originating from this region of the chromosome. (B) Sequence logo of the YdiH-binding sites identified in the genome sequences of: Bacillus anthracis str. Ames, Bacillus cereus ATCC 10987, B. cereus ATCC 1457, B. cereus E33, Bacillus halodurans C-125, Bacillus licheniformis ATCC 14580, B. subtilis 168, Bacillus thuringiensis serovar konkukian str. 97-27, Bacillus clausii KSM-K16, Geobacillus kaustophilus HTA426, Listeria innocua Clip11262, Listeria monocytogenes str. 4b F2365, L. monocytogenes EGD-e and Oceanobacillus iheyensis HTE831. The Shannon information content is shown on the y axis; Shannon's unit of non-randomness is the bit (short for ‘binary digit’) (Crooks et al., 2004). The proposed 18 bp YdiH-binding consensus sequence is shown above the sequence logo. It is composed of a tandem inverted repeat of two weakly conserved 7 bp half-sites, as indicated by the arrows. B. subtilis Fnr- (Makita et al., 2004) and ResD- (Nakano et al., 2000) binding consensus sequences are also shown.

 
Activation of {sigma}B is achieved through a complex phosphorylation/dephosphorylation cascade in response to various cellular stimuli. Two independent signalling pathways regulate {sigma}B activity. The energy pathway requires the RsbP phosphatase, and the environmental pathway depends on the RsbU phosphatase (Hecker & Völker, 2001). To identify the pathway by which {sigma}B is activated in the YdiH mutant in response to oxygen starvation, an RsbP/YdiH and an RsbU/YdiH mutant strain were tested under these conditions (Fig. 6). In the RsbU/YdiH mutant, the {sigma}B regulon was activated. In contrast, in the RsbP/YdiH mutant or in an RsbP/RsbU/YdiH mutant strain, no activation of the {sigma}B regulon could be detected, indicating that the activation is RsbP dependent.



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Fig. 6. An RsbP-dependent signal transduction cascade controls induction of {sigma}B-dependent promoters in a YdiH mutant under oxygen starvation. The isogenic set of B. subtilis strains (A) LUW292, YdiH; (B) LUW289, YdiH RsbU; (C) LUW295, YdiH RsbP; (D) LUW293, YdiH RsbU RsbP; was grown and analysed as described in the legend to Fig. 7. Black dots represent genes that are transcribed from {sigma}B-dependent promoters. Circles and arrows indicate the positions of signals corresponding to sigB and gsiB transcripts, respectively. The gsiB gene is often used as a reporter for activation of the {sigma}B regulon as it is transcribed exclusively from a {sigma}B-dependent promoter (Brigulla et al., 2003). The presented data are from a single experiment. The horizontal line marks Z score=3, corresponding to a confidence level of 95 %. The intensity-dependent Z score measures the number of standard deviations of a particular data point from the mean. Data points above this line represent genes which are significantly upregulated in oxygen-depleted cells. The transcriptome datasets are available in supplementary data text file 6.

 
To further investigate experimentally if YdiH is a regulator of ldh and ywcJ, ldhlacZ and ywcJlacZ expression in wild-type and YdiH mutant cells was compared. Expression of the ldhlacZ and ywcJlacZ constructs was induced in cells grown in media containing glucose (NSMPG). The expression reached its maximum levels when the cells entered the early stationary growth phase, similar to that seen for the cydlacZ fusion (Fig. 7). When the cultures were grown in NSMP or NSMPG at high aeration, expression of cydlacZ, ldhlacZ and ywcJlacZ was essentially undetectable. In contrast, in a YdiH mutant, expression of cydlacZ, ldhlacZ and ywcJlacZ was relatively high, both in NSMP and in the highly aerated NSMPG-grown cells (Fig. 7). Taken together, these data suggest that YdiH is a direct regulator not only of the cydABCD operon but also of ywcJ and the ldh lctP operon.



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Fig. 7. Time-courses of {beta}-galactosidase activity of cydlacZ, ldhlacZ and ywcJlacZ fusions. Cells were grown at 37 °C at different aeration levels by setting the rotary shaker to 100 (A–C, J–K, G–I, P–R) or 400 r.p.m. (D–F, M–O). The growth medium was NSMPG (A–F, J–O) or NSMP (G–I, P–R). Sampling was as described in the legend to Fig. 1. Strains: LUW95 (A, D, G), LUW276 (B, E, H), LUW275 (C, F, I), LUW274 (J, M, P), LUW281 (K, N, Q) and LUW280 (L, O, R).

 
Refining the YdiH recognition sequence by a bioinformatics approach
B. subtilis YdiH is highly similar to the corresponding proteins predicted to be present in several low-G+C-content Gram-positive bacteria. We selected YdiH homologues that have identical amino acid sequences in the presumed DNA-binding region. It is likely that these proteins bind to a common DNA sequence. Thus we scanned the selected genome sequences with a YdiH DNA-binding consensus sequence derived from the five predicted binding sites in B. subtilis (TGTGAADTRBTKMDCAA) and found related sequences in the promoter regions of genes homologous to cydABCD, ldh lctP and ywcJ. The YdiH DNA-binding regions were used to create a weight matrix graphically represented as a sequence logo in Fig. 5(B).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
For many bacteria, changes in oxygen tension serve as an important environmental cue to trigger adaptive changes in metabolism (Green & Paget, 2004; Poole & Cook, 2000). Bacteria have evolved different mechanisms to directly and indirectly sense oxygen levels. Here we show that in B. subtilis, three transcriptional units, cydABCD, ldh lctP and ywcJ, previously known to be induced under microaerobic or anaerobic growth conditions, are directly regulated by YdiH. In a recent study, YdiH has also been shown to function as a negative regulator of cytochrome bd (cydABCD) gene expression in B. subtilis by binding to the DNA corresponding to the 5'-untranslated region of the cydABCD transcript (Schau et al., 2004). The predicted ydiH translation product shares significant similarity with Rex from S. coelicolor A3(2), a regulator suggested to modulate transcription in response to changes in the cellular NADH/NAD+ ratio. Rex binds both the oxidized and reduced form of NAD(H). Binding of NADH results in the disruption of a Rex–DNA complex (Brekasis & Paget, 2003). Database searches indicate that Rex-related proteins are present in many Gram-positive bacteria, particularly in the low-G+C group. The structure of a Rex family repressor from Thermus aquaticus in complex with NADH has been determined (Sickmier et al., 2005). The protein has an N-terminal DNA-binding domain with a winged helix–turn–helix fold and a C-terminal NAD(H)-binding domain. A local sequence alignment between S. coelicolor Rex and B. subtilis YdiH shows that about 40 % of the amino acid residues are invariant. Comparative structural modelling (data not shown) suggests that the overall structure of YdiH is similar to that of T. aquaticus Rex. Based on analogy with the T. aquaticus and S. coelicolor Rex proteins, YdiH is likely to serve as a redox sensor that is regulated by cellular differences in the free levels of NAD+ and NADH.

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 {beta}-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 {sigma}B-dependent general stress response in a strain lacking ydiH. Moreover, activation of {sigma}B under these circumstances is primarily dependent on the energy-stress sensor RsbP. Induction of {sigma}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.


   ACKNOWLEDGEMENTS
 
We thank Lars Rutberg for valuable comments on the manuscript, Isabelle Martin-Verstraete, Dieter Jahn, Jörg Stülke, Kazuo Kobayashi, Yoshito Sadaie, Philippe Glaser and Marion Hulett for the gift of B. subtilis strains, and Markus Ringnér for advice on statistical analysis of microarray data. This work was supported by grants from Carl Tryggers Stiftelse and Emil och Wera Cornells Stiftelse.


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Received 15 April 2005; revised 14 July 2005; accepted 15 July 2005.



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