Unité de Régulation de lExpression Génétique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Neurobiologie et Diversité Cellulaire, CNRS UMR 7637, Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris, 10 rue Vauquelin, 75005 Paris, France2
INRA, Domaine de Vilvert, 78352 Jouy en Josas, France3
Author for correspondence: Isabelle Martin-Verstraete. Tel: +33 1 45 68 72 95. Fax: +33 1 45 68 89 48. e-mail: iverstra{at}pasteur.fr
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ABSTRACT |
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Keywords: sulfur metabolism, proteome analysis, regulation
Abbreviations: 2D, two-dimensional; ABC, ATP-binding cassette; DIG, digoxigenin; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight
a Present address: Genopole, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
c Present address: HKU Pasteur Research Center, Dexter HC Man Building, 8 Sassoon Road, Pokfulam, Hong Kong.
b Present address: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
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INTRODUCTION |
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Under sulfate-starvation conditions, E. coli synthesizes a set of proteins which are absent during growth in the presence of sulfate. These proteins are involved in the biosynthesis of cysteine from sulfide and in the utilization of alternative sulfur sources such as sulfonates (Uria-Nickelsen et al., 1993 ). Random lacZ fusions and 2D gel electrophoresis revealed a specific increase in the synthesis of several proteins, such as oxygenases (TauD, SsuD), high-affinity uptake systems (ATP-binding cassette ABC transport systems, sulfate- and cystine-binding proteins), alkyl hydroperoxide reductase (AhpC), O-acetylserine lyase A (CysK), and of several unidentified proteins (Kertesz et al., 1993
; Quadroni et al., 1996
; van der Ploeg et al., 1996
, 1999
). Several proteins induced under sulfate-starvation conditions have also been identified in Pseudomonas aeruginosa (Hummerjohann et al., 1998
; Kertesz et al., 1999
; Quadroni et al., 1999
).
In Bacillus subtilis, sulfate assimilation and cysteine biosynthesis may proceed via an E. coli-like pathway. The enzymes leading to the conversion of sulfate into sulfide, and to its incorporation into cysteine, are present in B. subtilis (Kunst et al., 1997 ; Pasternak et al., 1965
). The expression of the cysH gene, which encodes 3'- phosphoadenosine 5'-phosphosulfate sulfotransferase, was found to be repressed by both cysteine and sulfide (Mansilla & de Mendoza, 1997
). The cysH gene is the first gene of an operon which encodes a sulfate permease (CysP) and enzymes catalysing the first steps of the sulfate assimilation pathway (Mansilla & de Mendoza, 2000
). Little is known about the regulation of sulfur metabolism. In Gram-positive bacteria, a highly conserved sequence, the S-box, is located in the leader region of several operons including genes involved in the biosynthesis of methionine or cysteine. These genes are proposed to form a new regulon controlled by a global transcription-termination control system (Grundy & Henkin, 1998
).
Only a few B. subtilis genes necessary for the assimilation of sulfur from sulfonates have been identified (van der Ploeg et al., 1998 ). Three of them encode an ABC transport system (ssuBAC) and another one encodes a monooxygenase (ssuD).
To get insight into the global regulation of sulfur-related gene expression in B. subtilis, we associated the recent knowledge of the whole genome sequence (Kunst et al., 1997 ), the highly sensitive technique of 2D gel electrophoresis (OFarrell, 1975
) and mass spectrometry. 2D gel electrophoresis now allows the separation of more than 1000 proteins (Bernhardt et al., 1999
) and many can be identified by MALDI TOF spectrometry (Shevchenko et al., 1996
). We took advantage of this technique to identify proteins whose synthesis is regulated by sulfur availability in B. subtilis. The protein synthesis patterns of a wild-type strain grown with either sulfate or glutathione as a sulfur source were compared. Synthesis of 15 proteins was up- or down-regulated. These proteins were identified unambiguously by MALDI TOF spectrometry. For most of them, the alteration in expression of the corresponding genes under these two conditions was confirmed by slot-blot analyses or lacZ fusions.
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METHODS |
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MALDI-TOF spectrometry identification.
Protein spots were cut off and digested in gel slices with trypsin (Roche) as described previously (Shevchenko et al., 1996 ). The matrix used for the desalted digestions was a saturated solution of 2,5-dihydroxybenzoic acid in 0·1% trifluoroacetic acid. MALDI-TOF spectra of the peptides were obtained with a Voyager-DE STR Biospectrometry Workstation mass spectrometer (PE Biosystems). The analysis was performed in positive ion reflector mode. The trypsin autoproteolysis products were used as internal calibrants. Data mining was performed using ProFound and MS-FIT software against non- redundant databases. A mass deviation of 0·10·3 Da was allowed in the database searches.
Slot-blot analyses.
Digoxigenin (DIG)-labelled probes corresponding to ytmI, dppE, pyrF, serA, purQ, glyA, aroF and upp were obtained using the PCR DIG probe synthesis kit from Roche. Total RNA was extracted from exponentially growing cells using the High Pure RNA Isolation Kit (Roche). Increasing amounts (50, 100, 200 and 400 ng) of total RNA were transferred to a positively charged nylon membrane and hybridized with the DIG-labelled probes. Chemiluminescent detection was performed with anti-DIG-AP and CDP-Star (Roche). The chemiluminescent signal was detected on X-ray film. Slot-blots were analysed and quantified using the Image Master 1D software from Pharmacia.
Plasmid constructs.
A 1055 bp DNA fragment containing part of the ytlI gene from position -209 to +846 (numbering is relative to the translational start site) was amplified by PCR using chromosomal DNA as template and two specific primers containing an EcoRI or a BamHI restriction site. The EcoRIBamHI fragment was cloned into the pJH101 vector (Ferrari et al., 1983 ) to give pDIA5576. A 1·5 kb ClaI DNA fragment corresponding to the kanamycin-resistance gene aphA3 was inserted at the unique ClaI site of pDIA5576. The resulting plasmid pDIA5577 linearized with ScaI was used to transform B. subtilis 168, leading to the disruption of the ytlI gene by insertion of a kanamycin cassette (Table 1
).
A 336 bp and a 474 bp DNA fragment corresponding to the serA promoter (from position -270 to +66 relative to the translational start site) and to the cysK promoter (from position -210 to +264 relative to the translational start site) was amplified by PCR. Oligonucleotides were used to create an EcoRI restriction site at the 5' end and a BamHI restriction site at the 3' end of the fragments. After digestion of the PCR products with BamHI and EcoRI, they were inserted into plasmid pAC6 (Stülke et al., 1997 ). The resulting serAlacZ and cysKlacZ fusions were subsequently integrated at the amyE locus (Table 1
).
Within the framework of a European project on the functional analysis of the genome of B. subtilis, more than 1100 genes have been disrupted by fusion with the lacZ reporter gene (see http://locus.jouy.inra.fr/cgi-bin/genmic/madbase/progs/madbase.operl). An internal fragment of each gene was amplified by PCR using primers creating HindIII and BamHI restriction sites at the 5' and 3' ends of the PCR product, respectively. After digestion with HindIII and BamHI, these PCR products were cloned into the pMUTIN4 vector (Vagner et al., 1998 ). The pMUTIN derivatives were then integrated into the B. subtilis chromosome by homologous recombination at the target locus. This strategy was used to inactivate yurL, ssuA and six genes of the ytmI locus while creating a lacZ fusion (see Table 1
).
ß-Galactosidase assays.
Cells were grown in minimal medium in the presence of different sulfur sources. Samples of the cultures were taken during exponential growth and harvested by centrifugation. The ß-galactosidase activity was measured using the Miller assay (Miller, 1972 ) with cell extracts obtained by lysozyme treatment. All the assays were repeated at least twice. One unit (U) of ß-galactosidase is defined as the amount of enzyme which produces 1 nmol o-nitrophenol min-1 at 28 °C.
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RESULTS |
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To create a 2D reference map, the experimental conditions optimizing gel resolution and MALDI TOF spectrometry identification rates were established (See Methods). We identified 47 spots which were used as markers allowing the comparison of our gels with other B. subtilis 2D gels available on the internet (http://microbio2.biologie.uni-greifswald.de:8880/). Furthermore, these markers facilitate the prediction of the molecular mass and the isoelectric point of a spot according to its position on the gel. These reference spots represent proteins involved mainly in the metabolism of carbohydrates, lipids or C1 units, in the biosynthesis of nucleotides, or in response to stress and in oxidative phosphorylations (data not shown).
We then compared the protein synthesis patterns (proteome) of the wild-type strain grown on sulfate or glutathione. For both sulfur sources, two independent protein preparations were made and for each preparation at least two gels were run, silver-stained and analysed. This allowed us to identify 24 proteins whose synthesis was up- or down-regulated (by at least a factor of 2) in the presence of one of these sulfur sources (Fig. 1). The synthesis of 10 proteins was increased in the presence of glutathione while 14 others were more abundant in the presence of sulfate (Fig. 1
). These 24 spots were cut out of the gel, digested with trypsin and submitted to MALDI TOF spectrometry. In these conditions, 15 spots were unambigously identified (Table 2
) while 9 others remained unidentified. In the presence of glutathione, a significant increase in the synthesis of uptake systems (DppE, SsuA), of an oxygenase (SsuD), of two proteins of unknown function (YtmI, YurL) and of cysteine synthase (CysK) was observed. After growth with sulfate, the amount of proteins involved in the metabolism of C1 units (SerA, GlyA, FolD), in the biosynthesis of purines (PurQ, Xpt) and pyrimidines (Upp, PyrAA, PyrF) and in the biosynthesis of chorismate (AroF) was increased (Table 2
). However, the alteration in synthesis of these proteins could be due either to the regulation in response to sulfur limitation or to a more general effect related to the reduction of growth rate.
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Cysteine synthase, CysK, catalyses the synthesis of L- cysteine from sulfide and O-acetylserine (Kredich, 1996 ). The amount of this protein was increased fivefold in the presence of glutathione as revealed by 2D gel analysis (Table 2
). To test the regulation of expression of the cysK gene, strain BSIP1207, carrying a cysKlacZ transcriptional fusion, was constructed. This strain was grown in minimal medium in the presence of sulfate or glutathione as sulfur source. ß-Galactosidase activity was 265 U (mg protein)-1 after growth in the presence of glutathione compared to 50 U (mg protein)-1 in the presence of sulfate. A comparable repression was observed after growth in the presence of cysteine [the ß- galactosidase activity was 28 U (mg protein)-1].
The SsuA and SsuD proteins are involved in the utilization of sulfonates in B. subtilis (van der Ploeg et al., 1998 ). SsuA is the binding protein of an ABC permease system for aliphatic sulfonates and SsuD is likely to be a monooxygenase (Eichhorn et al., 1999
). These two proteins were not detected after growth with sulfate (Fig. 1
, Table 2
). The expression of an ssuAlacZ fusion appeared to be upregulated 600-fold in the presence of glutathione and 1000-fold in the presence of methionine when compared with its expression level in the presence of sulfate. The ß-galactosidase activities measured in the presence of these sulfur sources were 1109, 1646 and 1·6 U (mg protein)-1, respectively. The regulation of this operon is therefore likely to occur at the transcriptional level, as previously observed (van der Ploeg et al., 1998
).
DppE, the dipeptide-binding protein of the high-affinity ABC permease of dipeptides (Mathiopoulos et al., 1991 ), was also more abundant in cells grown on glutathione than in those grown on sulfate. Slot-blot analysis performed with a probe covering dppE revealed a slight induction of this gene: its expression was increased only 1·5-fold with glutathione as compared to its expression with sulfate (Fig. 2
).
Two proteins of unknown function, YtmI and YurL, were detected on 2D gels only in extracts obtained after growth with glutathione (Fig. 1). YurL shares sequence similarities with the PfkB family of carbohydrate kinases (Wu et al., 1991
). Using a strain carrying a yurLlacZ fusion the expression of this gene was shown to be induced 40-fold on glutathione [430 U (mg protein)-1] as compared to its expression on sulfate [10 U (mg protein)-1]. Expression of yurL is thus strongly derepressed at the transcriptional level during sulfur limitation.
Coordinated regulation of the ytmI locus
The synthesis of the YtmI protein is strongly increased in the presence of glutathione. The regulation of expression of the corresponding gene and the genetic organization of the ytmI region were therefore studied in more detail. In order to investigate the regulation of the ytmI gene in response to sulfur availability, a fusion between the ytmI and lacZ genes was constructed. Strain BFS71 contains a ytmIlacZ transcriptional fusion and a ytmI gene disruption (Table 1). The effect of several sulfur sources on the expression of this fusion was tested (Table 3
). The ß-galactosidase activity of strain BFS71 after growth on glutathione was 350-fold higher than that after growth on sulfate. This result confirms the data obtained by 2D gel analysis and indicates that expression of the ytmI gene is regulated at the transcriptional level. The expression of this gene was also strongly increased in the presence of taurine, isethionate, methanesulfonate and methionine (Table 3
and data not shown). In contrast, the transcription of ytmI appeared to be very weak in the presence of sulfate, thiosulfate and cysteine (Table 3
).
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YtlI positively regulates the expression of the ytmI gene in response to sulfur availability
The ytlI gene is located upstream of ytmI and is transcribed divergently. The product of the ytlI gene displays significant similarities with the LysR family of transcriptional regulators. The YtlI protein might therefore encode the transcriptional activator of the ytmI gene. To test this possibility, a kanamycin-resistance cassette was inserted into the ytlI gene. Chromosomal DNA from strain BSIP1214 (ytlI::aphA3) was used to transform strain BFS71 containing a ytmIlacZ fusion, resulting in strain BSIP1223. The ß-galactosidase activity was measured after growth of the wild-type strain and the ytlI mutant in the presence of various sulfur sources (Table 3). Disruption of the ytlI gene led to the complete loss of ytmI expression in the presence of methionine, taurine or glutathione as sulfur source. This indicates that ytlI positively regulates the expression of ytmI in response to the availability of sulfur sources. The ytlI gene probably controls the expression of the entire ytmI locus. Indeed, when ytlI is disrupted, we also observed lack of expression of the ytnJ and ytmO genes in the presence of either taurine, glutathione or methionine (data not shown). The involvement of YtlI in the regulation of the expression of the serA, glyA, ssuA, cysK and yurL genes was also investigated. The inactivation of ytlI did not modify the expression pattern of serAlacZ, ssuAlacZ, yurLlacZ and cysKlacZ fusions (data not shown). Slot-blot analyses revealed that YtlI does not regulate the expression of glyA. Indeed, this expression remained 3·5-fold higher on sulfate than on glutathione if ytlI was inactivated (data not shown). One may therefore conclude that YtlI is probably not involved in the global regulation of transcription of these genes in response to sulfur availability.
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DISCUSSION |
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Using MALDI TOF spectrometry, 15 proteins whose synthesis is up- or down-regulated by sulfate or glutathione have been identified. Nine other sulfur-limitation-regulated (Slr) proteins remained unidentified. For seven of them, this could be attributed to their low molecular mass (25 kDa). In the presence of sulfate, the amount of proteins involved in the metabolism of C1 units (SerA, GlyA, FolD), in the biosynthesis of nucleotides (PurQ, Upp, PyrAA, PyrF, Xpt) and in the synthesis of chorismate (AroF) is increased. However, the increase of expression of the corresponding genes is weak (Fig. 2). At this time, it is difficult to discriminate between a modification of expression due to differences in the growth rate, a general derepression during nutrient starvation or a specific regulation during sulfur limitation.
Analysis of the proteome of the wild-type strain grown in the presence of glutathione revealed that the synthesis of at least six proteins is significantly enhanced. Five of them are related to ABC transport and/or desulfonation systems: SsuA, SsuD, DppE, YurL and YtmI. ABC permeases which actively transport substrates (e.g. sugars, amino acids, peptides) across biological membranes (Quentin et al., 1999 ) probably play an essential role in the adaptation of B. subtilis to its environment.
The yurL gene is located in the yurONML region. These genes encode proteins sharing similarities with sugar ABC permeases and sugar kinases. Surprisingly, YurL contains six cysteine residues while all the other proteins induced by glutathione contain no or one cysteine residue, like the majority of the B. subtilis proteins (Danchin et al., 2000 ). A similar specific induction of proteins containing very few cysteine residues under sulfur limitation conditions had been previously described for E. coli (van der Ploeg et al., 1996
) and Calothrix (Mazel & Marlière, 1989
). The up-regulation of a cysteine-rich protein such as YurL under sulfur- limiting conditions might not be specific for sulfur deprivation but reflect a more general induction due to nutrient limitation.
The dppE gene belongs to an operon encoding an ABC permease for dipeptides (Mathiopoulos et al., 1991 ). This operon, which is upregulated rapidly after induction of sporulation, may facilitate adaptation to nutrient limitation instead of favouring sporulation. The regulation of dppE expression might be a way for the cell to scavenge sulfur-containing peptides in response to sulfur limitation.
Proteins directly related to sulfur metabolism are also more abundant in the presence of glutathione: CysK, SsuA and SsuD. CysK, which catalyses the biosynthesis of cysteine from acetylserine and sulfide, is involved in the sulfate-starvation response of E. coli. In this bacterium, reduced-sulfur limitation is known to derepress the genes of the cysteine biosynthetic pathway (Kredich, 1996 ). Based on sequence similarity with the SsuD protein from E. coli (63% identity), SsuD of B. subtilis is probably a monooxygenase which catalyses the oxygenolytic conversion of sulfonates to sulfite and the corresponding aldehydes (Eichhorn et al., 1999
). SsuA displays 32% identity with the aliphatic sulfonate binding protein of E. coli (van der Ploeg et al., 1999
). In B. subtilis, the expression of the ssu operon is repressed by sulfate and cysteine, while it is strongly induced by glutathione, taurine and methionine, and to a lesser extent by sulfonates (this work; van der Ploeg et al., 1998
). In E. coli, the ssuEADCB operon (utilization of a broad range of sulfonates) and the tauABCD operon (utilization of taurine) are also upregulated in the absence of sulfate and cysteine (van der Ploeg et al., 1996
, 1999
). When sulfate and sulfonates are present, E. coli preferentially uses the inorganic sulfur source and represses the enzymes involved in the degradation of organosulfur compounds. Full expression of the genes involved in sulfonate utilization requires two LysR-type activators, CysB and Cbl (Kredich, 1996
; Iwanicka- Nowicka & Hryniewicz, 1995).
The ytmI gene is located in a locus containing 12 genes which seem to form an operon. The ribR gene encodes a riboflavin kinase (Solovieva et al., 1999 ). Based on sequence similarities a potential function has been assigned to seven of these genes (Fig. 3
). Five of them (ytmJ, ytmK, ytmL, ytmM, hisP) are part of ABC transport systems. Interestingly, two different binding proteins, YtmJ and YtmK, are present, suggesting that this transport system is involved in the uptake of two different substrates. The ytnJ gene product shares similarities with monooxygenases (3040% identity) and that of hipO with amino acid amidohydrolases (3540% identity). The functions of YtmI, YtmO and YtnM are unknown, while YtnI shares weak similarities with a putative glutaredoxin from Thermotoga maritima. The significant alteration in growth on taurine of a ytmI mutant suggests that at least one of these 12 genes plays a role in taurine assimilation. These genes are probably involved in the uptake and oxidation of alternative sulfur sources. The elucidation of their role in sulfur assimilation in B. subtilis deserves further investigation.
The ytmI gene is weakly expressed during growth with sulfate, cysteine and thiosulfate while its expression is strongly increased in the presence of glutathione, taurine and methionine. The ytmJ, ytmK, ytmM, ytnJ and ytnM genes are regulated in the same way. We therefore propose that either some intermediary of the cysteine biosynthetic pathway mediates repression, or the expression of this operon is induced during sulfur limitation (glutathione and taurine) and in the presence of methionine.
Interestingly, YtlI, a LysR-type regulator, is the transcriptional activator of the ytmI operon. YtlI is the first regulator involved in the control of expression in response to sulfur availability to be identified in B. subtilis. In E. coli, three LysR-type proteins play a role in the regulation of sulfur metabolism: Cbl, CysB and MetR (Iwanicka-Nowicka & Hryniewicz, 1995 ; Kredich, 1996
; Greene, 1996
). However, YtlI seems not to be involved in the global regulation of the sulfur limitation response in B. subtilis as it is not responsible for the regulation of serA, glyA, ssuA, cysK and yurL.
Several mechanisms for regulation of sulfur metabolism exist in B. subtilis. The S-box sequence involved in premature termination of transcription is found upstream of several genes involved in the methionine biosynthetic pathway (Grundy & Henkin, 1998 ). In the present work, we have identified genes regulated in response to sulfur availability and the YtlI protein, a positive regulator of expression of the ytmI operon. The cysK, yurL, glyA and serA genes and the ssu operon are regulated by other mechanisms. The identification of other regulators is a challenging question for the future.
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ACKNOWLEDGEMENTS |
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This research was supported by LION BIOSCIENCE AG in the framework of a scientific collaboration, by grants from the Ministère de lEducation Nationale de la Recherche et de la Technologie, the Centre National de la Recherche Scientifique (URA 2171), the Institut Pasteur, the Université Paris 7 and the European Union Biotech Programme (contract ERBB104 CT960655).
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernhardt, J., Büttner, K., Scharf, C. & Hecker, M. (1999). Dual channel imaging of two-dimensional electropherograms in Bacillus subtilis. Electrophoresis 20, 2225-2240.[Medline]
Danchin, A., Guerdoux-Janet, P., Moszer, I. & Nitschke, P. (2000). Mapping the bacterial cell architecture into the chromosome. Philos Trans R Soc Lond B Biol Sci 355, 179-190.[Medline]
Eichhorn, E., van der Ploeg, J. R. & Leisinger, T. (1999). Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli. J Biol Chem 274, 26639-26646.
Ferrari, F. A., Nguyen, A., Lang, D. & Hoch, J. A. (1983). Construction and properties of an integrable plasmid for Bacillus subtilis. J Bacteriol 154, 1513-1515.[Medline]
Greene, R. C. (1996). Biosynthesis of methionine. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 542560. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Grundy, F. J. & Henkin, T. M. (1998). The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in Gram-positive bacteria. Mol Microbiol 30, 737-749.[Medline]
Hummerjohann, J., Kuttel, E. Quadroni, M., Ragaller, J., Leisinger, T. & Kertesz, M. A. (1998). Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates. Microbiology 144, 13751386.[Abstract]
Iwanicka-Nowicka, R. & Hryniewicz, M. M. (1995). A new gene, cbl, encoding a member of the LysR family of transcriptional regulators belongs to Escherichia coli cys regulon. Gene 166, 11-17.[Medline]
Kertesz, M. A., Leisinger, T. & Cook, A. M. (1993). Proteins induced by sulfate limitation in Escherichia coli, Pseudomonas putida or Staphylococcus aureus. J Bacteriol 175, 1187-1190.[Abstract]
Kertesz, M. A., Schmidt-Larbig, K. & Wuest, T. (1999). A novel reduced flavin mononucleotide-dependent methanesulfonate sulfonatase encoded by the sulfur-regulated msu operon of Pseudomonas aeruginosa. J Bacteriol 181, 1464-1473.
Kredich, N. M. (1996). Biosynthesis of cysteine. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 514527. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Kunst, F. & Rapoport, G. (1995). Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol 177, 2403-2407.[Abstract]
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[Medline]
Mansilla, M. C. & de Mendoza, D. (1997). L-Cysteine biosynthesis in Bacillus subtilis: identification, sequencing, and functional characterization of the gene coding for phosphoadenylylsulfate sulfotransferase. J Bacteriol 179, 976-981.[Abstract]
Mansilla, M. C. & de Mendoza, D. (2000). The Bacillus subtilis cysP gene encodes a novel sulphate permease related to the inorganic phosphate transporter (Pit) family. Microbiology 146, 815-821.
Mathiopoulos, C., Mueller, J. P., Slack, F. J., Murphy, C. G., Patankar, S., Bukusoglu, G. & Sonenshein, A. L. (1991). A Bacillus subtilis dipeptide transport system expressed early during sporulation. Mol Microbiol 5, 1903-1913.[Medline]
Mazel, D. & Marlière, P. (1989). Adaptative eradication of methionine and cysteine from cyanobacterial light-harvesting proteins. Nature 341, 245-248.[Medline]
Meister, A, (1988). Glutathione metabolism and its selective modification. J Biol Chem 263, 1720517208.
Miller, J. H. (1972). Assay of ß-galactosidase. Experiments in Molecular Genetics, pp. 352355. Edited by J. H. Miller. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
OFarrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007-4021.[Abstract]
Pasternak, C. A., Ellis, R. J., Jones-Mortimer, M. C. & Crichton, C. E. (1965). The control of sulfate reduction in bacteria. Biochem J 96, 270-275.[Medline]
van der Ploeg, J. R., Weiss, M. A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A. & Leisinger, T. (1996). Identification of sulfate starvation-regulated genes in Escherichia coli: a gene cluster involved in the utilization of taurine as a sulfur source. J Bacteriol 178, 5438-5446.[Abstract]
van der Ploeg, J. R., Iwanicka-Nowicka, R., Kertesz, M. A., Leisinger, T. & Hryniewicz, M. M. (1997). Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli. J Bacteriol 179, 7671-7678.[Abstract]
van der Ploeg, J. R., Cummings, N. J., Leisinger, T. & Connerton, I. F. (1998). Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates. Microbiology 144, 2555-2561.[Abstract]
van der Ploeg, J. R., Iwanicka-Nowicka, R., Bykowski, T., Hryniewicz, M. M. & Leisinger, T. (1999). The Escherichia coli ssuEADCB gene cluster is required for the utilization of sulfur from aliphatic sulfonates and is regulated by the transcriptional activator Cbl. J Biol Chem 274, 29358-29365.
Quadroni, M., Staudenmann, W., Kertesz, M. & James, P. (1996). Analysis of global responses by protein and peptide fingerprinting of proteins isolated by two-dimensional gel electrophoresis. Application to the sulfate-starvation response of Escherichia coli. Eur J Biochem 239, 773-781.[Abstract]
Quadroni, M., James, P., Dainese-Hatt, P. & Kertesz, M. A. (1999). Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation- induced response in Pseudomonas aeruginosa PAO1. Eur J Biochem 266, 986-996.
Quentin, Y., Fichant, G. & Denizot, F. (1999). Inventory, assembly and analysis of Bacillus subtilis ABC transport systems. J Mol Biol 287, 467-484.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sekowska, A., Kung, H. F. & Danchin, A. (2000). Sulfur metabolism in Escherichia coli and related bacteria, facts and fiction. J Mol Microbiol Biotechnol 2, 145-177.[Medline]
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850-858.[Medline]
Solovieva, I. M., Kreneva, R. A., Leak, D. J. & Perumov, D. A. (1999). The ribR gene encodes a monofunctional riboflavin kinase which is involved in regulation of the Bacillus subtilis riboflavin operon. Microbiology 145, 67-73.[Abstract]
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, 65-78.[Medline]
Uria-Nickelsen, M. R., Leadbetter, E. R. & Godchaux, W. D.III (1993). Sulfonate-sulfur assimilation by yeasts resembles that of bacteria. FEMS Microbiol Lett 114, 73-77.[Medline]
Vagner, V., Dervyn, E. & Ehrlich, S. D. (1998). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 3097-3104.[Abstract]
Wu, L. F., Reizer, A., Reizer, J., Cai, B., Tomich, J. M. & Saier, M. H.Jr (1991). Nucleotide sequence of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of Escherichia coli. J Bacteriol 173, 3117-3127.[Medline]
Received 17 November 2000;
revised 16 February 2001;
accepted 22 February 2001.