Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA1
Department of Biochemistry, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan 7552
Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27710, USA3
Author for correspondence: Milton H. Saier, Jr. Tel: +1 858 534 4084. Fax: +1 858 534 7108. e-mail: msaier{at}ucsd.edu
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ABSTRACT |
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Keywords: regulon, transcription, pleiotropic regulators, adenylate cyclase, CysB
Abbreviations: CRP, cAMP-receptor protein; EMB, eosin-methylene blue
a Present address: The Institute for Genomic Research, 9712 Medical Center Drive, Manassas, VA 20850, USA.
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INTRODUCTION |
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The cysteine regulon includes most of the genes required for synthesis of cysteine and genes for uptake of sulfur sources such as L-cystine, sulfate, thiosulfate and taurine. Transcriptional activation of these genes requires CysB, the inducer N-acetyl-L-serine and conditions of sulfur limitation (Kredich, 1992 , 1996
). CysB is a tetrameric LysR-type regulator with an N-terminal DNA-binding domain, a central inducer-binding domain and a C-terminal oligomerization domain that is essential for stability (Lochowska et al., 2001
). Its activity is regulated by an efflux pump specific for cysteine metabolites (Dassler et al., 2000
). CysB is also an autorepressor, preventing expression of its own structural gene, cysB.
E. coli and S. typhimurium can utilize a number of sulfur-containing compounds as sole sulfur source, including sulfate, sulfite, thiosulfate, sulfide, glutathionine, lanthionine, taurine and L-djenkolate [S,S'-methylene bis(cysteine)] as well as cysteine and cystine (Kredich, 1996 ; van der Ploeg et al., 1997
). Although these organisms cannot utilize methionine as a sole sulfur source, Klebsiella strains can (Seiflein & Lawrence, 2001
). Sulfide and thiosulfate are anti-inducers, probably exerting their effects by competing with N-acetyl-L-serine for binding to the CysB activator (Hryniewicz & Kredich, 1991
; Ostrowski & Kredich, 1990
). Cysteine inhibits inducer synthesis, resulting in maximal repression of the sulfur regulon. Growth with poor sulfur sources such as L-djenkolate or glutathione results in maximal derepression of the sulfur regulon. Rapid growth on rich media results in moderate induction of the regulon (Antón, 2000
).
Hartmann & Boos (1993) demonstrated that mutations in phoB, the gene encoding the pleiotropic activator of the pho regulon, affect the carbohydrate fermentation phenotype in E. coli under certain physiological conditions, particularly under conditions of phosphate starvation. CysB, normally considered to be the central pleiotropic regulator of sulfur metabolism in enteric bacteria, has been shown to be required for normal acid induction of arginine decarboxylase (Shi & Bennett, 1994
) as well as for sensitivity to novobiocin (Rakonjac et al., 1991
) and mecillinam (Oppezzo & Antón, 1995
). Thus, pleiotropic effects of phoB and cysB mutations other than those expected on the basis of their involvement in phosphorus and sulfur metabolic regulation, respectively, have been reported. In this report we present the results of our preliminary studies with representative examples of cysB mutant strains and provide analyses of the basis for specific carbohydrate utilization defects observed for these mutants.
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METHODS |
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Cell growth was always at 37 °C. Fermentation was tested on eosin-methylene blue (EMB) plates lacking lactose, but containing the indicated carbon source at 1%, except for rhamnose which was present at 0·5%. These plates, like the rich media described above, are sulfur-limited. Carbon oxidation was assayed using 96-well Biolog plates (Bochner, 1993 ). Cysteine, when present, was added to a final concentration of 0·2 mM. Although cysteine can be growth-inhibiting at high concentrations (Harris & Lui, 1981
), it is not inhibitory at this concentration. For all enzyme assays, cells were harvested during exponential growth.
Enzyme assays.
ß-Galactosidase was assayed by the method described by Miller (1972) . For the data presented in Table 5, which used minimal media, IPTG (1 mM) was present during growth to eliminate any effect of the lac operon repressor. ß-Galactosidase activities are either expressed in Miller units (µmol min-1) (mg protein)-1 (see Table 5) or in relative activities (see Figs 2 and 3). For reporter gene transcriptional fusion assays, cells were grown overnight in 5 ml NB plus 1% glucose, glucitol or formate at 37 °C with agitation. Fresh NB (100 ml) of the same composition was inoculated from the overnight culture and cells were harvested during exponential growth at 37 °C (OD600 approx. 0·5). The cell density was adjusted and two drops of toluene were added to permeabilize the cells during vortexing for 30 s. The samples were incubated at 30 °C and aliquots were transferred to the appropriate buffers for reporter gene product assay. The ß-galactosidase buffer consisted of 2 mM EDTA, 70 mM Na2HPO4, 35 mM NaH2PO4 (pH 7·0), 1 mM MgSO4, 10 mM KCl and 10 mM ß-mercaptoethanol. The same conditions were used to assay native ß-galactosidase as reported in Table 5. The ß-glucuronidase buffer consisted of 50 mM sodium phosphate, pH 7·0, 0·1 % Triton X-100 and 10 mM ß-mercaptoethanol. The assay mixtures were allowed to equilibrate to room temperature for 10 min before addition of 200 µl 0·4% o-nitrophenyl galactopyranoside (gut operon expression) or 100 µl 10 mM p-nitrophenyl-ß-D-glucuronide (fdo operon expression) to give a final volume of 1·0 ml. The nitrophenol released was measured after addition of 400 µl 1 M Na2CO3 (gut operon expression) or 400 µl 2·5 M 2-amino-2-methylpropanediol (fdo operon expression) to stop the reaction. The samples were then read at 420 or 415 nm, respectively (Jefferson et al., 1986
; Miller, 1972
). Protein concentrations were measured by the Lowry method.
Cell extracts for the assay of glucitol-6-phosphate dehydrogenase, Enzyme IIGut and D-alanine dehydrogenase activities were prepared as follows. Cells were grown with shaking in LB broth at 37 °C, harvested by centrifugation, washed three times with minimal medium 63 and resuspended in the same medium. PMSF (0·1 mM) and ß-mercaptoethanol (0·5 mg ml-1) were added to the samples and the cells were lysed using a French press at 10000 p.s.i. (69 MPa). After centrifugation to remove cell debris, the supernatant was used for the assays. For the assay of glucitol-6-phosphate dehydrogenase activity, aliquots of the cell extracts were added to a solution containing 0·1 M Tris/HCl (pH 8·5), 1·5 mM NAD+, 1 mM dithiothreitol and 2 mM glucitol 6-phosphate. The optical density at 340 nm was followed over a period of 2 min. For the assay of Enzyme IIGut, 0, 1, 2, 5, 10 and 25 µl samples of extract were added to 100 µl twofold concentrated assay mix containing 100 mM potassium phosphate buffer, pH 6·0, 20 µM [14C]D-glucitol, 20 mM glucitol 6-phosphate, 50 mM KF, 25 mM MgCl2 and 5 mM dithiothreitol. The samples were brought to 200 µl, final volume, vortexed and incubated at 37 °C for 40 min. One millilitre ice-cold water was then added to each assay tube to stop the reaction. The samples were applied to NaCl pre-washed ion-exchange columns (Saier et al., 1977 ), the columns were washed twice with 12 ml water and the samples were eluted with 12 ml 1 M LiCl into scintillation vials containing Biosafe II scintillation cocktail in preparation for scintillation counting. For the D-alanine dehydrogenase assay, aliquots of cell extracts were added to a solution containing 2 mM D-alanine, 10 mM potassium phosphate, pH 7·5, and 0·33 mg dinitrophenyl hydrazine ml-1. At time intervals of 10 min, the reaction was stopped by addition of 1·67 ml 10% NaOH and the absorbance was read at 445 nm as described by Wild et al. (1974
; see also Saier et al., 1980
).
Transport assays.
Cells were grown with shaking at 37 °C in LB medium, harvested during exponential growth by centrifugation, washed three times with minimal medium 63 and resuspended in the same medium. The cell suspensions were pre-warmed at 37 °C for 10 min before 14C-labelled glucitol or D-alanine was added to a final concentration of 0·1 mM. At intervals, samples were transferred to pre-washed 0·2 µm (pore diameter) millipore filters. Cells were washed three times with medium 63 before the filters were dried, transferred to scintillation vials containing Biosafe-NA scintillation cocktail and counted.
cAMP assays.
Cultures were grown for net cAMP production in minimal salts medium containing 1% lactate or 1% lactate plus 0·5% glucose for 5 h before samples were removed for extraction of cAMP (Castro et al., 1976 ). The optical density of the cultures (600 nm) was approximately 0·6. Samples were placed in a boiling water bath for 5 min, chilled to 0 °C and centrifuged to remove cell debris. Total cAMP was determined using partially purified cAMP-binding protein from beef muscle (Gilman, 1970
), essentially as described previously (Castro et al., 1976
; Feucht & Saier, 1980
). The amount of cAMP produced (Table 6) is expressed in nmol (mg dry wt)-1. No background activity was subtracted from the values presented.
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RESULTS |
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Use of reporter gene transcriptional fusions to study the effects of a cysB mutation on gene expression
A plasmid bearing a gutBlacZ transcriptional fusion was transformed into wild-type and cysB mutant strains of E. coli. Cells bearing this multicopy plasmid were then grown in nutrient medium under non-inducing, repressing and inducing conditions as described in Methods. The lack of induction by glucitol presumably reflects the multiple copies of the gut operon control region since the chromosomally encoded regulatory proteins, GutM and GutR (Yamada & Saier, 1988 ), may not be present in sufficient amounts to regulate all plasmid operators. The results depicted in Fig. 2(a)
clearly show that when cells were grown under either non-inducing conditions (NB alone) or inducing conditions (NB plus 1% glucitol), the presence of the cysB mutation drastically reduced glucitol operon expression (Yamada & Saier, 1987
, 1988
; Yamada et al., 1990
). CRP is present in E. coli cells in large amounts. Consequently, catabolite repression would not be expected to be altered by expression of a target gene on a multicopy plasmid. As can be seen, strong catabolite repression is observed. Unexpectedly, however, there was little effect of the cysB mutation under glucose-repressing conditions.
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Effect of exogenous cAMP on gene expression
The results reported in Fig. 2 suggested that the cysB defect might interfere with the function of the cAMPCRP complex that normally activates catabolite-sensitive genes. To test this possibility, expression of the two reporter gene fusions (gutBlacZ and fdoGuidA) was studied as a function of the concentration of exogenous cAMP. The results are summarized in Fig. 3.
As can be seen, cAMP increased expression of both fusions in the cysB genetic background, but decreased expression of both fusions in the wild-type background. The mild repressive effect in the wild-type background suggests that, in the absence of exogenous cAMP, these cells synthesize sufficient cAMP for maximal transcription. The results suggest that a primary deficiency, giving rise to defective expression of carbon catabolic enzyme-encoding genes, is related to depressed levels of cytoplasmic cAMP in the cysB mutant relative to the wild-type strain.
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DISCUSSION |
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Recently, evidence for a connection between the cAMPCRP-regulated modulon and the stringent response in E. coli has been reported (Johansson et al., 2000 ). Moreover, the homologous nucleoid proteins, H-NS and StpA, were shown to play a positive role in expression of stringently regulated genes as well as in cell growth. The results reported suggest that catabolite repression, the stringent response and control of gene expression by structural nucleoid proteins are all interconnected (Johansson et al., 2000
). The relationship between these observations and those reported here, if any, have yet to be investigated, but the results of Johansson et al. (2000)
corroborate our postulate that the different regulons in the bacterial cell are interrelated.
In this communication, we not only document the effects of cysB mutations in E. coli and S. typhimurium on carbon oxidation and carbohydrate fermentation, but we also pursue the effect of the cysB mutation on carbon utilization to the mechanistic level. The results reported revealed that the effects were at the transcriptional level for both the gut and the fdo operons. Both effects appear to be partially reversed by exogenous cAMP or a sulfur source such as cysteine or djenkolate. Furthermore, we provide evidence that the effect is on regulation of the cAMP biosynthetic enzyme, adenylate cyclase, by the IIAGlc protein of the PTS, and not on the inherent activity of adenylate cyclase itself. Our current postulate is that these effects are indirect, that they affect the regulation of adenylate cyclase as a primary target and that the level and/or activity of this enzyme will prove to be hypersensitive to the metabolic state of the cell. Such sensitivity would allow the bacterium to coordinate carbon/energy metabolism with the availability of other macro- and micronutrients. The mechanistic details of these interactions are currently under study in our laboratory.
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ACKNOWLEDGEMENTS |
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Received 5 March 2001;
revised 12 July 2001;
accepted 23 August 2001.