Laboratoire de Chimie Bactérienne, UPR 9043, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, 13009 Marseille, France
Correspondence
Anne Galinier
galinier{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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Present address: Abteilung für Allgemeine Mikrobiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
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INTRODUCTION |
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This paper describes a study of the B. subtilis yvcIN operon. We characterized the promoter and showed that the gene yvcK encoded by this operon is essential for growth on intermediates of the Krebs cycle as well as on substrates of the pentose phosphate pathway (PPP), and in addition for a regular cell morphology. We propose that YvcK plays a role in carbon metabolism, presumably in gluconeogenesis.
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METHODS |
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For growth monitoring, pre-cultures were grown overnight in LB supplemented with 1 % glucose. Thereafter, the cells were washed and inoculated to an OD600 of 0·1 in the medium in which the growth test was subsequently performed. OD600 readings were periodically taken during the incubation of the cultures. For growth monitoring on solid medium, the bacteria were streaked out from pre-cultures grown in LB-glucose and kept frozen at 70 °C.
Promoter deletion analysis.
DNA fragments carrying sequences of the trxByvcI intergenic region were amplified by PCR using chromosomal DNA of strain 168 as template. The PCR reactions were carried out using the reverse primer BG17 [yvcI (+105 to +88); BamHI site within the 5' end] in combination with one of various forward primers that carried an EcoRI or MunI site at their 5' ends, respectively. The PCR fragments were digested by BamHI and EcoRI or MunI and inserted between the BamHI and EcoRI sites on plasmid pAC6 (Stülke et al., 1997). Plasmid pAC6 carries a promoterless lacZ reporter gene followed by cat downstream of the BamHI/EcoRI cloning site encompassed by the amyE-5' and amyE-3' regions, respectively. The various upstream primers used, their annealing sites and the respective assigned plasmids were as follows: primer BG18 [yvcI (419 to 403)]
plasmid pBGM9; primer BG19 [yvcI (311 to 295)]
plasmid pBGM10; primer BG20 [yvcI (205 to 189)]
plasmid pBGM11; primer BG36 [yvcI (176 to 157)]
plasmid pBGM21; primer BG37 [yvcI (153 to 135)]
plasmid pBGM22; primer BG57 [yvcI (115 to 98)]
plasmid pBGM71; primer BG39 [yvcI (80 to 62)]
plasmid pBGM25; primer BG40 [yvcI (61 to 40)]
plasmid pBGM26; primer BG41 [yvcI (40 to 22)]
plasmid pBGM27; primer BG1 [yvcI (20 to 3)]
plasmid pBGM13; primer BG22 [yvcI (+1 to +17)]
plasmid pBGM14. For the construction of plasmid pBGM23 carrying a fusion of yvcI (141 to +105) to lacZ, we used the EcoRI site occurring naturally at position 140 to 135 in front of yvcI. The 1087 bp EcoRIClaI fragment of plasmid pBGM10 was inserted between these sites on plasmid pAC6. The recombinant plasmids described above were linearized by digestion with ScaI and subsequently used to transform strain 168 by a double-crossover event with the amyE gene with selection for chloramphenicol resistance. The recombination events were verified by monitoring the absence of amylase activity, as described previously (Cutting & Vander Horn, 1990
).
Construction of gene deletions within the yvcIN operon.
For the construction of gene deletions, the generalized deletion vector pBGM35 was designed. To this end, the BamHIDraI fragment of pMUTIN2 (Vagner et al., 1998) encompassing the RBSspoVGlacZ cassette was inserted between the BamHI and SmaI sites of the previously described plasmid pBGM31 (Görke et al., 2004
). Plasmid pBGM35 therefore carries a lacZcatrrnBt1t2
t0 cassette encompassed upstream by SacII/BamHI and downstream by ApaI/KpnI cloning sites. About 500 bp of the sequences flanking the gene(s) to be deleted was amplified by PCR and successively inserted between these sites. The resulting plasmids were linearized by ScaI digestion and used to transform B. subtilis 168 by a double-crossover event with selection for chloramphenicol resistance. The resulting gene deletions, and the designation of the respective strains and of the plasmids used to construct them, were as follows:
yvcJ (SG62; pBGM59),
yvcK (SG63; pBGM61),
[yvcJK] (SG56; pBGM48),
[yvcJKL] (SG61; pBGM57),
yvcL (SG64; pBGM67),
[crh, yvcN] (SG65; pBGM69). Details of these constructions are available on request. In the resulting strains, the lacZcatrrnBt1t2
t0 cassette replaced the desired gene(s) and simultaneously a transcriptional fusion of lacZ to the gene(s) located upstream was created. The presence of the three transcriptional terminators prevented a transcriptional read-through into genes located downstream of the cassette.
Construction of expression plasmids for the complementation analysis.
The genes yvcJ and yvcK were amplified by PCR using chromosomal DNA of B. subtilis 168 as template and the primer pairs BG53 [yvcJ (+1 to +19)] with BG54 [yvcJ (+890 to 871)] and BG55 [yvcK (42 to 24)] with BG56 [yvcK (+954 to 936)], respectively. Gene ybhK from E. coli was amplified by PCR using primers BG86 [ybhK (1 to +18)] and BG87 [ybhK (+909 to +892)] and chromosomal DNA of E. coli W3110 (Bachmann, 1972) as template. The PCR fragments were digested at the StuI sites introduced by the primers and inserted into the StuI site of the B. subtilis replicative plasmid pDG148-Stu (Joseph et al., 2001
). The resulting plasmids pBGM53, pBGM54 and pBGG1 carry the genes yvcJ, yvcK and ybhK, respectively, under control of the IPTG-inducible Pspac promoter.
-Galactosidase assays.
Enzyme assays were according to Miller (1972), as previously described (Görke et al., 2004
). Samples were assayed in triplicate and each experiment was repeated at least twice using independent cultures.
-Galactosidase activities are expressed in Miller units. Standard deviations were below 15 %.
RNA isolation and primer extension analysis.
In order to determine the transcription start of the promoter present in front of yvcI, strain SG59 was used. This strain carries a transcriptional fusion of yvcI (141 to +19) to lacZ integrated into the amyE locus. For its construction, a PCR fragment was amplified using primers BG20 [yvcI (205 to 189)] and BG58 [yvcI (+19 to +1)], and digested with EcoRI (at position 140 to 135 in front of yvcI) and BamHI (site located in primer BG58), and inserted between the corresponding sites of plasmid pAC6. The resulting plasmid pBGM52 was subsequently used to transform strain 168 by a double-crossover event with the amyE gene with selection for chloramphenicol resistance. For RNA isolation, strain SG59 was grown overnight in CE-succinate, and 2 ml of the culture was harvested. Total RNA was isolated using the High Pure RNA isolation kit (Roche) according to the supplier's protocol, but DNase treatments and column purifications were repeated in order to eliminate contamination with genomic DNA. Primer extension reactions were carried out using 50 µg total RNA, 10 pmol end-labelled primer BG25 [primes in lacZ in reverse direction (positions +71 to +48)] and 200 units of SuperScript II RNaseHreverse transcriptase (Gibco-BRL) in 20 µl 1x first-strand buffer (Gibco-BRL) containing 10 mM dithiothreitol and 0·5 mM each deoxyribonucleotide triphosphate (Amersham Pharmacia Biotech). The 5'-end labelling of the oligonucleotide was performed using T4 polynucleotide kinase (Amersham Pharmacia Biotech) and [-33P]ATP (Amersham Pharmacia Biotech). Primer extension products were analysed by electrophoresis on a 6 % polyacrylamide/6 M urea gel, alongside a sequencing ladder (lanes C, T, A and G) obtained with the same end-labelled primer and a PCR fragment encompassing the putative promoter [yvcI (141 to +19)] as template. Sequencing reactions were carried out using Sequenase purchased from United States Biochemical.
Culture observation.
Bacterial cells were observed by phase-contrast microscopy (Nikon Eclipse E800) and images were recorded using the Nikon Digital camera DXM 1200.
Construction of a chromosomal mini-Tn10 (mTn10) library and screening for suppression of the growth defect of strain SG63.
For transposon mutagenesis, the mTn10 delivery vector pIC333 was introduced into strain SG63; transformants were selected at 28 °C on LB-glucose containing erythromycin. Ten independent colonies were inoculated into LB-glucose containing spectinomycin and grown overnight at 28 °C. These cultures were subsequently diluted 100-fold in 2 ml of the same medium and grown for 4 h at 28 °C. After shifting the temperature to 37 °C, the cultures were grown further for 5 h. Subsequently, the cells were washed with CE-medium lacking any carbon source and plated on DSM as well as on CE-gluconate containing spectinomycin and incubated at 37 °C. On both types of plates mutants appeared which resumed growth, and a representative number of clones were isolated. In order to verify that suppression of the growth defect was linked to the respective transposon insertion, chromosomal DNA was prepared from these suppressor mutants and retransformed into the original mutant strain SG63. Transformants were selected on LB-glucose containing spectinomycin and subsequently replica-plated onto the medium on which the respective transposon insertion was initially isolated. Those transformants for which 100 % of the colonies were able to grow on both types of plate were kept for a further characterization. In addition, it was verified that the transformants were sensitive to erythromycin, in other words that the pIC333 vector had been lost.
Identification of mTn10 insertion sites by ST-PCR.
The mTn10 insertion sites in the chromosomes of the various suppressing mutants were identified by the semi-random, two-step PCR (ST-PCR) protocol, which was originally developed for yeast (Chun et al., 1997). The PCR conditions were as described by Chun et al. (1997)
, but the primer sequences were different. For the amplification of the sequences next to the left-hand end of the inserted mTn10, a first PCR was performed using the primers BG80 (5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNGATC) and BG84 [5'-AAGAGCGCCCAATACGCAAACCGCC; mTn10 (145121)]. One microlitre of this PCR product was subsequently used as a template for a second PCR using the primer BG81 (5'-GCTCTAGAGGCCACGCGTCGACTAGTAC), which carries the sequence identical to the defined sequence preceding the ten random bases of primer BG80, and primer BG85, which anneals downstream of primer BG84 within the transposon [mTn10 (11285)]. Similarly for amplification of the sequences next to the right-hand end of the transposon, the first PCR reaction was performed by using the primers BG80 and BG82 [5'-TTTGCATGCTTCAAAGCCTGTCGGAATTGG; mTn10 (21892218)]. The second PCR reaction was carried out using the primers BG81 and BG83 [5'-GCTCTAGAATTCACGGTTTACCCACTTATAAACAAAAGATCGG; mTn10 (22412275)], which anneals downstream of primer BG82. The various PCR products were subsequently sequenced by using the primers BG83 or BG85 for the sequences located to the right and the left-hand end of the transposon, respectively.
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RESULTS AND DISCUSSION |
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YvcK is required for growth on Krebs cycle intermediates and on PPP substrates
LB and DSM broth are both complex media based on enzymic digests of protein combined with yeast extract (LB) or beef extract (DSM). According to a typical analysis, LB broth contains 1·14 % (w/v) amino acids and 0·17 % carbohydrates, whereas DSM contains 0·48 % amino acids and only 0·03 % carbohydrates (Difco Manual, 11th edition, 1998, Difco Laboratories, Sparks, MD 21152, USA). Therefore DSM is a poor source of carbohydrates. When glucose was added to DSM or LB medium, the growth impairments of the strains lacking YvcK were completely cured (Fig. 2B; data not shown). These observations suggested that the yvcK mutants require the presence of certain carbohydrates, such as glucose, for growth. In order to determine these requirements in more detail, the various strains were examined in the chemically defined CE-minimal medium supplemented with different carbon sources, on plates as well as in liquid medium (Fig. 3
). All the strains grew normally on PTS substrates (glucose, sucrose, salicin, maltose, fructose, mannitol) and on glucitol, glycerol and inositol (Fig. 3A
; examples are shown for glucose and glucitol in Fig. 3B
). All these substrates are catabolized via the EmbdenMeyerhoffParnas (EMP) pathway. However, no growth of the strains lacking YvcK could be observed on plates containing arabinose, ribose, gluconate, citrate or succinate, and growth was significantly impaired on fumarate and malate. In liquid medium supplemented with gluconate, fumarate (Fig. 3B
) or malate (data not shown), the yvcK deletion strain SG63 started to grow and reached a certain cell density before a decline of the OD600 could be observed. In the presence of citrate or succinate (Fig. 3B
), strain SG63 grew slower than the wild-type and stopped growth when an OD600 of about 0·5 was reached, whereas the wild-type strain grew to an OD600 of 1·6 or 1·0, respectively. For the other strains lacking YvcK, comparable growth properties were observed (data not shown). Interestingly, growth of the yvcK mutants was impaired if gluconeogenesis was required, i.e. on carbon sources which are substrates of the Krebs cycle or of the pentose phosphate pathway (PPP).
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Deletion of yvcK leads to aberrant and strikingly different cell shapes in media in which growth is impaired
The courses of the growth curves of the yvcK mutants in the various non-permissive media indicated that the cells were able to use the respective carbon sources at least for some generations before the cells stopped growth (Fig. 2). This suggested that either a toxic compound accumulated in the cells or that compound(s) or protein(s) required for growth could not be synthesized any more and were subsequently diluted by cell division. In CE minimal liquid media containing gluconate, malate or fumarate as carbon source, the OD600 of the YvcK-lacking strains declined after an initial phase of growth (Fig. 2
), suggesting that the bacteria lysed. To check this observation, we grew the yvcK deletion strain SG63 and the wild-type strain 168 as a reference in various media. Samples were taken during the incubation and the bacteria were examined by microscopy. When grown in minimal medium containing glucose, strain SG63 exhibited a normal cell morphology, similar to that of the wild-type strain 168 (data not shown). In gluconate-containing minimal medium, the cells of strain SG63 adopted bubble-like shapes, starting soon after inoculation of the medium, and resembled the L-form of B. subtilis, which has been shown to lack a cell wall (Gilpin et al., 1973
). This abnormality exhibited its most drastic extent 46 h after inoculation, shortly before the OD600 declined (Fig. 5
A). At that time, cell debris became visible, confirming that the bacteria had lysed. When the OD600 of the culture rose again, bacteria exhibiting normal cell shapes started to appear, indicating that the compensatory mutation(s), which suppress the growth defect, also suppress the abnormal morphology.
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High concentrations of Mg2+ restore growth and wild-type cell morphology of the yvcK mutant
Cell wall deficiency can be the result of degradation by cellular enzymes, a process known as autolysis, or due to a failure in biosynthesis or of the biosynthesis of precursors. Autolysis occurs in B. subtilis under any condition that causes the dissipation of protonic potential (Jolliffe et al., 1981; Kemper et al., 1993
). An inhibited cell envelope synthesis has been observed in B. subtilis mutants defective in the pathway that converts glucose 6-phosphate into teichoic acids, which are major constituents of the cell wall (Prasad & Freese, 1974
; Lazarevic et al., 2005
). A gtaC mutant deficient in the
-phosphoglucomutase catalysing the first step of this pathway, i.e. the interconversion of glucose 6-phosphate to
-glucose 1-phosphate, is morphologically similar to a yvcK mutant grown on gluconate (Fig. 5A
; Lazarevic et al., 2005
). It has been reported that abnormal cell morphologies due to cell wall deficiency caused by mutations in the pathway leading to teichoic acids or due to a mreB null mutation can be cured by the addition of magnesium (Lazarevic et al., 2005
; Formstone & Errington, 2005
). To check the idea that cell wall synthesis might be affected in the yvcK mutants when cultivated on the non-permissive media, the yvcK deletion strain SG63 and, as a reference, the wild-type strain 168 were grown in CE-gluconate minimal liquid medium in the presence or absence of an additional concentration of 25 mM MgCl2. The addition of magnesium ions perfectly restored normal growth rates (Fig. 6
A) and the wild-type cell morphology of strain SG63 (Fig. 6B
). This result suggested that cell wall biosynthesis is affected in the yvcK mutant.
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Identification of suppressor mutations by transposon mutagenesis
In order to gain insight into the reason for the growth impairments and for the cell wall deficiency exhibited by the yvcK mutant, we aimed to identify mutations able to suppress the growth defects. Therefore a transposon mutagenesis of the yvcK deletion strain SG63 was carried out using a mTn10 delivery system harboured on a temperature-sensitive plasmid. Briefly, several independent libraries of strain SG63 carrying random mTn10 insertions in the genome were generated and screened in parallel on DSM and on CE-gluconate plates for a resumption of growth. Several independent transposon mutants were isolated from both types of plate. Control experiments verified that suppression of the growth defect was linked to the respective transposon insertion and was not due to a second mutation which could have occurred spontaneously during the procedure (see Methods). The sites of the mTn10 insertions in the chromosome were identified by ST-PCR. Finally, the mTn10 insertion sites of 13 mutants could be identified, of which seven were isolated on DSM plates and six on CE-gluconate plates (Fig. 7A).
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In the group of suppressor mutants isolated from the CE-gluconate plates, five different types of insertion were identified. One insertion was also found in zwf in the same position as in the zwf mutants isolated on DSM (see above). This was the only mutation that was found on both types of media used in the screen. The other insertions were located in genes yqfF, secDF, mfd and mrpB. The function of yqfF is unknown, and the transposon insertion might also affect the expression of genes located downstream. The gene secDF encodes a membrane protein required for the efficient secretion of proteins via the Sec-dependent pathway in B. subtilis (Bolhuis et al., 1998). A transcriptional terminator is present downstream of secDF. Interruption of secDF in B. subtilis impairs the high-level production of secretory proteins (Bolhuis et al., 1998
), and might also affect the secretion of YfnI. In E. coli, SecDF couples protein translocation to the proton-motive force (pmf) (Duong & Wickner, 1997
). The gene mfd encodes the transcription repair coupling factor, which displaces RNA polymerase (RNAP) stalled on the DNA by nucleotide lesions in the DNA template or by proteins that block elongation downstream of a promoter (Ayora et al., 1996
; Roberts & Park, 2004
). Finally, one insertion mapped in the gene mrpB, which is the second of seven genes within the mrp operon, whose products are all required for the activity of the Mrp antiporter that extrudes Na+ or K+ in exchange for protons (Krulwich et al., 2001
). The Mrp antiporter has been shown to play a central role in pH homeostasis and is required for cytoplasmic pH regulation in the presence of NaCl concentrations similar to those found in LB (Ito et al., 1999
, 2001
). Accordingly, the mrpB : : mTn10 mutant was not viable on LB (Fig. 7
; Kobayashi et al., 2003
).
It is noteworthy that not all these mTn10 insertion mutations restored growth of the yvcK mutant simultaneously in DSM and in gluconate minimal medium (Fig. 7
). Indeed, the insertions in yfnI, zwf, yqfF and mfd restored growth in both media, whereas the remaining insertions suppressed the growth defect either exclusively in DSM (insertion in cggR) or in CE-gluconate (insertions in secDF and mrpB).
Effects of defined mutations in zwf, gapA and cggR on growth of the yvcK mutant
Most of the transposon insertions obtained in the screen were not very informative, since they resided in genes of unknown function (yfnI and yqfF) or presumably yielded pleiotropic effects (insertions in secDF, mfd and mrpB). However, the insertions in zwf and cggR affected central enzymes of carbon metabolism and caused growth impairments on substrates of the EMP pathway, such as glucose or glycerol (Fig. 7B). In order to explore in more detail the effects of these two transposon insertions, defined knock-out mutations were introduced into the
yvcK deletion strain SG63.
First we constructed a conditional zwf knockout mutation in which expression of zwf is inducible by IPTG. The resulting double-mutant strain yvcK, zwf-5' : : pMUTIN (SG94) was able to grow in both media, CE-gluconate as well as DSM, but exclusively in the absence of IPTG. Induction of zwf expression by the addition of IPTG resulted in the same growth defects observed for the
yvcK single mutant (data not shown). This result clearly establishes that the mTn10 insertion eliminated Zwf activity in the cell and that this abolition leads to suppression of the
yvcK phenotype.
To clarify the effect of the transposon insertion in cggR, we tested the suppressing effect of gapA and cggR mutations. A conditional gapA knockout mutation in which gapA is expressed exclusively in the presence of IPTG (Fillinger et al., 2000) was introduced into the yvcK mutant SG63. In the absence of IPTG, the resulting double-mutant strain
yvcK, gapA-5' : : pMUTIN2 (SG92) was unable to grow in DSM, in CE-gluconate as well as in CE-glucose (data not shown). This indicated that a switch-off of gapA expression does not lead to suppression of the growth defects. Next, we crossed into strain SG63 (
yvcK) a cggR-frameshift mutation which leads to constitutive expression of gapA at a high level (Ludwig et al., 2001
). The resulting double mutant
yvcK,
cggR (strain SG91) grew in all of the above-mentioned media, demonstrating that the
cggR allele completely suppressed the growth impairments of the yvcK mutant (data not shown). Thus, neither the gapA-knockout mutation nor the
cggR mutation resulted in the same growth properties caused by the cggR : : mTn10 insertion (Fig. 7B
). How can these differences be explained? It has been shown that a
cggR mutation also results in a higher expression level of the genes pgK, tpiA, pgm and eno, which are encoded downstream of gapA. Although these genes are transcribed from their own promoter located in the gapApgk intergenic region (see Fig. 7A
), they can additionally be co-transcribed together with gapA from the promoter located in front of cggR (Ludwig et al., 2001
). The cggR : : mTn10 mutant isolated here was unable to grow on glucose (Fig. 7B
), which is indicative of a gapA-knockout mutation (Fillinger et al., 2000
; and see above). Thus, it may be concluded that the cggR : : mTn10 mutation abolished gapA expression, perhaps by interference with the mRNA-processing event taking place at the 3' end of the cggR message (Meinken et al., 2003
), but led at the same time to an elevated expression of the genes downstream of gapA. This would mean that a higher expression of the genes downstream of gapA rather than of gapA itself might be responsible for suppression of the growth impairments. These genes encode enzymes of the lower part of the EMP pathway and are required for both glycolysis and gluconeogenesis.
The B. subtilis YvcK protein can be functionally replaced with its E. coli homologue YbhK
Homologues of YvcK are present in many Gram-positive as well as Gram-negative bacteria, suggesting that they accomplish an important function for the bacterial cell. The corresponding homologous protein YbhK from E. coli exhibits 34 % identity and 52 % similarity to YvcK at the amino acid sequence level. In order to test whether or not YbhK could substitute for YvcK in B. subtilis, ybhK was cloned under the control of an IPTG-inducible promoter on a plasmid able to replicate in B. subtilis. The resulting plasmid pBGG1 was used to transform the yvcK deletion strain SG63, and growth of the resulting transformant was tested in DSM in the presence of IPTG for the induction of ybhK expression. The presence of ybhK restored the growth of the yvcK mutant, although not to the level that was observed when the yvcK expression plasmid pBGM54 was present in this strain (Fig. 4
, compare filled triangles and open diamonds). This result indicates that YbhK from E. coli can at least partially substitute for YvcK in B. subtilis and suggests that these proteins share some similar functions and interaction partners.
A recent global study of the protein-interaction network in E. coli proposes that several proteins interact with YbhK (Butland et al., 2005). This is in agreement with our data indicating that YvcK may have several roles in the cell, and that its inactivation may yield pleiotropic effects. Among the putative interaction partners of YbhK, Butland et al. (2005)
identified SecA, belonging to the Sec protein secretion machinery, and the essential cell division protein FtsK, involved in septum formation. This supports our finding that a secDF mutation partly suppresses the growth defect of the yvcK mutant (Fig. 7
) and that the yvcK mutants exhibit a filamentous cell shape under certain growth conditions (Fig. 5
). Furthermore, AceE and LpdA, two subunits of the pyruvate-dehydrogenase complex, were identified as YbhK interaction partners. The pyruvate-dehydrogenase complex converts pyruvate into acetyl-CoA, a key reaction in the lower part of the EMP pathway. This observation, together with our finding that mutations in zwf and cggR suppress the growth defect of the yvcK mutants, is intriguing.
In conclusion, we assume that an enzyme involved in the conversion of D-glyceraldehyde 3-phosphate into acetyl-CoA within the lower part of the EMP pathway, e.g. the pyruvate-dehydrogenase complex, may exhibit an abnormally high activity in the yvcK mutant and may redirect the carbon flux almost completely towards the production of acetyl-CoA. Alternatively, the activity of an enzyme required for gluconeogenesis, i.e. the conversion of oxaloacetate into glucose 6-phosphate, could be lowered or abolished in the yvcK mutant. This would slow down the synthesis of cell wall precursor molecules from carbon sources that do not directly enter the EMP pathway. It would explain the growth impairment and abnormal cell shape of the yvcK mutants on substrates of the PPP and of the Krebs cycle. This hypothesis is currently under investigation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Asai, K., Baik, S. H., Kasahara, Y., Moriya, S. & Ogasawara, N. (2000). Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis. Microbiology 146, 263271.[Medline]
Ayora, S., Rojo, F., Ogasawara, N., Nakai, S. & Alonso, J. C. (1996). The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination. J Mol Biol 256, 301318.[CrossRef][Medline]
Bachmann, B. J. (1972). Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36, 525557.[Medline]
Bagyan, I., Hobot, J. & Cutting, S. (1996). A compartmentalized regulator of developmental gene expression in Bacillus subtilis. J Bacteriol 178, 45004507.
Boël, G., Mijakovic, I., Maze, A. & 7 other authors (2003). Transcription regulators potentially controlled by HPr kinase/phosphorylase in Gram-negative bacteria. J Mol Microbiol Biotechnol 5, 206215.[CrossRef][Medline]
Bolhuis, A., Broekhuizen, C. P., Sorokin, A., van Roosmalen, M. L., Venema, G., Bron, S., Quax, W. J. & van Dijl, J. M. (1998). SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins. J Biol Chem 273, 2121721224.
Butland, G., Peregrin-Alvarez, J. M., Li, J. & 11 other authors (2005). Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 433, 531537.[CrossRef][Medline]
Chun, K. T., Edenberg, H. J., Kelley, M. R. & Goebl, M. G. (1997). Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast 13, 233240.[CrossRef][Medline]
Cutting, S. M. & Vander Horn, P. B. (1990). Genetic analysis. In Molecular Biological Methods for Bacillus. Edited by C. R. Harwood & S. M. Cutting. Chichester: Wiley.
Deutscher, J., Galinier, A. & Martin-Verstraete, I. (2002). Carbohydrate uptake and metabolism. In Bacillus subtilis and its Closest Relatives, pp. 129150. Edited by A. L. Sonnenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
Duong, F. & Wickner, W. (1997). The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling. EMBO J 16, 48714879.
Fillinger, S., Boschi-Muller, S., Azza, S., Dervyn, E., Branlant, G. & Aymerich, S. (2000). Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem 275, 1403114037.
Formstone, A. & Errington, J. (2005). A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis. Mol Microbiol 55, 16461657.[CrossRef][Medline]
Galinier, A., Haiech, J., Kilhoffer, M. C., Jaquinod, M., Stülke, J., Deutscher, J. & Martin-Verstraete, I. (1997). The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression. Proc Natl Acad Sci U S A 94, 84398444.
Gilpin, R. W., Young, F. E. & Chatterjee, A. N. (1973). Characterization of a stable L-form of Bacillus subtilis 168. J Bacteriol 113, 486499.[Medline]
Görke, B., Fraysse, L. & Galinier, A. (2004). Drastic differences in Crh and HPr synthesis levels reflect their different impacts on catabolite repression in Bacillus subtilis. J Bacteriol 186, 29922995.
Graupner, M., Xu, H. & White, R. H. (2002). Characterization of the 2-phospho-L-lactate transferase enzyme involved in coenzyme F(420) biosynthesis in Methanococcus jannaschii. Biochemistry 41, 37543761.[CrossRef][Medline]
Helmann, J. D. & Moran, C. P. (2002). RNA polymerase and sigma factors. In Bacillus subtilis and its Closest Relatives, pp. 289312. Edited by A. L. Sonnenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
Hilden, I., Krath, B. N. & Hove-Jensen, B. (1995). Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis. J Bacteriol 177, 72807284.
Hirose, I., Sano, K., Shioda, I., Kumano, M., Nakamura, K. & Yamane, K. (2000). Proteome analysis of Bacillus subtilis extracellular proteins: a two-dimensional protein electrophoretic study. Microbiology 146, 6575.[Medline]
Ito, M., Guffanti, A. A., Oudega, B. & Krulwich, T. A. (1999). mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J Bacteriol 181, 23942402.
Ito, M., Guffanti, A. A. & Krulwich, T. A. (2001). Mrp-dependent Na(+)/H(+) antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett 496, 117120.[CrossRef][Medline]
Jolliffe, L. K., Doyle, R. J. & Streips, U. N. (1981). The energized membrane and cellular autolysis in Bacillus subtilis. Cell 25, 753763.[CrossRef][Medline]
Joseph, P., Fantino, J. R., Herbaud, M. L. & Denizot, F. (2001). Rapid orientated cloning in a shuttle vector allowing modulated gene expression in Bacillus subtilis. FEMS Microbiol Lett 205, 9197.[CrossRef][Medline]
Kemper, M. A., Urrutia, M. M., Beveridge, T. J., Koch, A. L. & Doyle, R. J. (1993). Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis. J Bacteriol 175, 56905696.[Abstract]
Kobayashi, K., Ehrlich, S. D., Albertini, A. & 96 other authors (2003). Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100, 46784683.
Krulwich, T. A., Ito, M. & Guffanti, A. A. (2001). The Na(+)-dependence of alkaliphily in Bacillus. Biochim Biophys Acta 1505, 158168.[Medline]
Kunst, F. & Rapoport, G. (1995). Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol 177, 24032407.
Lazarevic, V., Soldo, B., Medico, N., Pooley, H., Bron, S. & Karamata, D. (2005). Bacillus subtilis alpha-phosphoglucomutase is required for normal cell morphology and biofilm formation. Appl Environ Microbiol 71, 3945.
Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F. M., Hecker, M. & Stülke, J. (2001). Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon. Mol Microbiol 41, 409422.[CrossRef][Medline]
Martin-Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. (1990). Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol 214, 657671.[CrossRef][Medline]
Meinken, C., Blencke, H. M., Ludwig, H. & Stülke, J. (2003). Expression of the glycolytic gapA operon in Bacillus subtilis: differential syntheses of proteins encoded by the operon. Microbiology 149, 751761.[CrossRef][Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Popham, D. L. & Stragier, P. (1991). Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis. J Bacteriol 173, 79427949.[Medline]
Prasad, C. & Freese, E. (1974). Cell lysis of Bacillus subtilis caused by intracellular accumulation of glucose-1-phosphate. J Bacteriol 118, 11111122.[Medline]
Rasko, D. A., Ravel, J., Økstad, O. A. & 12 other authors (2004). The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32, 977988.
Roberts, J. & Park, J. S. (2004). Mfd, the bacterial transcription repair coupling factor: translocation, repair and termination. Curr Opin Microbiol 7, 120125.[CrossRef][Medline]
Saier, M. H., Jr, Goldman, S. R., Maile, R. R., Moreno, M. S., Weyler, W., Yang, N. & Paulsen, I. T. (2002). Transport capabilities encoded within the Bacillus subtilis genome. J Mol Microbiol Biotechnol 4, 3767.[Medline]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory.
Schaeffer, P., Millet, J. & Aubert, J. P. (1965). Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54, 704711.
Song, B. H. & Neuhard, J. (1989). Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase. Mol Gen Genet 216, 462468.[CrossRef][Medline]
Stülke, J., Martin-Verstraete, I., Zagorec, M., Rose, M., Klier, A. & Rapoport, G. (1997). Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol 25, 6578.[CrossRef][Medline]
Tanaka, K., Kobayashi, K. & Ogasawara, N. (2003). The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium. Microbiology 149, 23172329.[CrossRef][Medline]
Vagner, V., Dervyn, E. & Ehrlich, S. D. (1998). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 30973104.[Medline]
Veith, B., Herzberg, C., Steckel, S. & 9 other authors (2004). The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7, 204211.[CrossRef][Medline]
Wacker, I., Ludwig, H., Reif, I., Blencke, H. M., Detsch, C. & Stülke, J. (2003). The regulatory link between carbon and nitrogen metabolism in Bacillus subtilis: regulation of the gltAB operon by the catabolite control protein CcpA. Microbiology 149, 30013009.[CrossRef][Medline]
Warner, J. B. & Lolkema, J. S. (2002). Growth of Bacillus subtilis on citrate and isocitrate is supported by the Mg2+citrate transporter CitM. Microbiology 148, 34053412.[Medline]
Warner, J. B. & Lolkema, J. S. (2003). CcpA-dependent carbon catabolite repression in bacteria. Microbiol Mol Biol Rev 67, 475490.
Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z. & Graham, M. W. (1989). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 34693478.[Abstract]
Zalieckas, J. M., Wray, L. V., Jr, Ferson, A. E. & Fisher, S. H. (1998). Transcription-repair coupling factor is involved in carbon catabolite repression of the Bacillus subtilis hut and gnt operons. Mol Microbiol 27, 10311038.[CrossRef][Medline]
Zamboni, N., Fischer, E., Laudert, D., Aymerich, S., Hohmann, H. P. & Sauer, U. (2004). The Bacillus subtilis yqjI gene encodes the NADP+-dependent 6-P-gluconate dehydrogenase in the pentose phosphate pathway. J Bacteriol 186, 45284534.
Received 4 May 2005;
revised 20 July 2005;
accepted 21 July 2005.
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