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MICROBIOLOGY LEITERS FEMS Microbiology Letters 136 (1996) 123- 127 The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms ’ Francisco Diez-Gonzalez a, James B. Russell b,c3 * a Department of Food Science, Cornell l/nice&y, Ithaca, NY 14853, USA b Section of Microbiology, Wing Hall, Cornell Unicersity, Ithaca, NY 14853, USA ’ Agricultural Research Service, USDA, Ithaca, NY 14853, USA Received 20 September 1995; revised 8 November 1995; accepted 10 November 1995 Abstract Clostridium acetobutylicum P262 had phosphotransferase systems for glucose and lactose, and the lactose system was inducible. When C. acetobutylicum P262 was provided with glucose and lactose, the cultures grew in a diauxic fashion, and glucose was used preferentially. Cells grown on lactose took up thiomethylgalactoside, and retained this non-metabolizable lactose analog for long periods of time. Because glucose inhibited thiomethylgalactoside uptake and caused the efflux of thiomethylgalactoside that had already been taken up, it appeared that C. acetobutylicum P262 had inducer exclusion and inducer expulsion mechanisms similar to those found in lactic acid bacteria. Keywords: Clostridium acetoburylicum; Inducer expulsion; Catabolite 1. Introduction It has long been recognized that bacteria can utilize certain energy sources preferentially and grow diauxically [l]. Sequential patterns of substrate utilization minimize the synthesis of unneeded protein and in many cases enhance the growth rate. In enteric bacteria, substrate preferences are mediated * Corresponding author. Tel.: + 1 (607) 255 4508; Fax: + 1 (607) 255 3904. ’ Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable. 0378-1097/96/$12.00 0 1996 Federation SSDI 0378-1097(95)00486-6 of European Microbiological regulation by two different types of regulation. CAMP-dependent regulation of transcription prevents protein synthesis [2], and phosphotransferase system @‘IS)mediated inducer exclusion inhibits the uptake of non-preferred substrates [3]. Reizer and Panos [4] reported that Streptococcus pyogenes was able to regulate lactose catabolism via a different mechanism that they called ‘inducer expulsion’. Inducer expulsion is an energy-dependent process that is mediated by an increase in fructose 1,6-phosphate @BP) and a cascade of phosphorylated proteins [5]. When glucose is added to the culture, PBP accumulates, and an ATP-dependent protein kinase is activated. The protein kinase phosphorylates the histidine-containing protein, HPr, a non-sugar specific phospho-carrier, at serine 46. HPr Societies, All rights reserved C. acetohu~vlicwn strain P262 was provided by Prof. D.R. Woods. University of Cape Town, South Africa. Spores of this organism were maintained at 4°C in anaerobic 20 mM phosphate buffer and were used to inoculate a basal medium [9] supplemented with glucose and lactose (up to 10 mM). The cultures were incubated at 37°C. formation was monitored spectrophotometrically at 340 nm. Assays were performed in 300-p.1 cuvettes at 38°C. The assay mixture contained per ml: 50 prnol Tris. HCI (pH 7.5). 10 prnol phosphoenolpyruvate (PEP), 5 prnol MgCl,. 5 kmol dithiothreitol (DTT). 2 pmol NADP, 20 prnol glucose. 5 units glucose-6-phosphate dehydrogenase and I-3 mg cell protein. /?-Galactosidase was measured by assaying the release of o-nitrophenol from onitrophenyl-P-D-galactoside (ONPG) in 0.5 ml reaction mixture containing (per ml): 50 prnol Tris HCI (pH 7.5), 5 pmol MgCl?. 5 prnol DTT, 5 pmol ONPG and l-2 mg cell protein. At measured times the reaction was stopped with 250 ~1 1 M Na,CO, and absorbance was measured at 420 nm. One unit of absorbance was equivalent to 0.52 pmol of onitrophenol [ 1I]. Lactose PTS activity was estimated by measuring n-nitrophenol formation in the presence of PEP after subtracting the P-galactosidase activity. The reaction mixture was the same as for ,&galactosidase but 10 mM PEP were included. Phospho-P-galactosidase activity was determined as described for @-galactosidase except that 5 pmol ONPG-6-P was substituted for ONPG. 2.2. Chemical umz1y.w.~ 2.4. Transport of TMG (ser-P) then activates a sugar-phosphate phosphatase. and the dephosphorylated sugar exits the cell. Inducer expulsion has been demonstrated in a variety of lactic acid bacteria [6], but to our knowledge it has never been demonstrated in other groups of bacteria. Preliminary experiments indicated that Clostridium ~~crtohu~licwn P262. a strain selected for its ability to utilize lactose [7,8], had a lactose-PTS and was able to expel the non-metabolizable lactose analog, methyl-P-D-thiogalactopyranoside (TMG), when glucose was added. 2. Materials and methods Fluid samples were centrifuged (13000 >( s. 2 min, 22°C) and sugars in the supematant were determined by HPLC (BioRad HPX-87H column, 0.17 N H,SO,, 0.5 ml mm ‘. 50°C; refractive index detector). Protein was determined by the method of Lowry [IO]. Cell concentration was estimated by optical density at 600 nm. Cultures were harvested by centrifugation ( 10 000 5°C 10 min) and washed twice in 50 mM Tris HCI buffer (5 mM MgCl?, 5 mM DTT, pH 7.5). Cells were disrupted by passage through a French press (room temperature, 1000 psi). Cell debris was eliminated by centrifugation (IO 000 X g, 5°C. 10 min) and cell-free extracts were stored at 4°C before assays. PEP-dependent phosphorylation of glucose of cell-free extracts was determined by a coupled assay with glucose-6-phosphate dehydrogenase. NADPH x g, Exponentially growing cells were harvested anaerobically by centrifugation (3500 X g, IO min, 4°C) and resuspended in anaerobic 50 mM Tris HCI buffer (pH 7.0) containing 5 mM MgCl?. Aliquots of 200 ~1 of cell suspension (approx. 200 mg protein I ‘) were incubated under N, tlushing with [I-( C]TMG (8 PM: 50 mCi mmol- ’ ). For expulsion experiments. 5 mM glucose or 2-deoxy-glucose were added. Transport was stopped by the addition of 2 ml ice-cold 0. I M LiCl to the reaction mixture and cells were collected by filtration through 0.45 ,um pore size cellulose nitrate membrane filters. Filters were washed with 2 ml of 0.1 M LiCl. dried for 15 min at 12O”C, and radioactivity was determined by liquid scintillation counting. The transport had first-order kinetics (protein versus activity was linear). 3. Results When C. acetobu~licum P262 was provided with glucose and lactose. the cultures grew diauxically F. Die:-Gonzalez, J.B. Russell/ FEMS Microbiology Letters 136 (1996) 123-127 125 Table 2 P-Galactosidase and phospho-fi-galactosidase activities of cell-free extracts of Clostridium acetobutylicum P262 grown on glucose and lactose Growth substrate P-Galactosidase activity (nmol (mg protein)-’ min- ’ ) Phospho-/3-galactosidase activity fnmol fmg protein)- ’ mitt- ‘) Glucose Lactose < 0.5 30.0 600.0 100.0 All values are the means of duplicate measurements. C. acetobutylicum P262 cells that had been grown on lactose transported the non-metabolizable lactose analog, TMG, at a rapid rate, but no TMG uptake 0 1 2 3 4 5 Time (h) Fig. I. Growth of CIosfridiunt trcetobufy[icum P262 on mixed glucose and lactose. (A) Diauxic curve. (B) Utilization of substrates. (Fig. IA) and glucose was utilized preferentially to lactose (Fig. 1B). Toluene-treated cells had virtually no detectable PTS activity (less than 0.5 nmol (mg protein)- I min- ’ 1, but PEP-dependent glucose and lactose phosphorylations could be measured with cell-free extracts (Table I>. The lactose PTS activity was inducible. Cells grown on lactose were able to hydrolyze the chromogenic lactose analog, ONPG, but much higher activity was observed when ONPG 6-phosphate was provided (Table 2). -10 ii Table 1 PEP-dependent sugar phosporylation activities of cell-free extracts of CIostridium acefobutylicum P262 grown on glucose and lactose Growth substrate Glucose-PTS activity (nmol (mg protein)min-’ ) Glucose Lactose 23.5 28.3 ’ All values are the means of duplicate Lactose-PTS activity (nmol (mg protein)min-‘) < 0.5 88.0 measurements ’ 1 I 0-j 7.5 10 12.5 15 17.5 20 Time(min) Fig. 2. Accumulation of thiomethylgalactoside (TMG) by cells of Clostridium acetobutylicum P262 grown on lactose and glucose. (A) TMG uptake in the presence of glucose and 2-deoxyglucose (2.DG). Glucose or 2-DG (5 n&l) were added before TMG addition. (B) Retention of TMG after addition of glucose or 2-DG by cells grown on lactose and previously incubated with TMG (10 min). Addition of sugar is indicated by the arrow. Values are the means of at least two separate experiments. was observed when the glucose was added to the transport assay (Fig. 2A). Cells retained TMG for at least 10 min, but rapid TMG expulsion was observed when glucose was added (Fig. 2B). The nonmetabolizable glucose analog, 2-deoxyglucose, did not cause TMG expulsion. 4. Discussion PTS activity has traditionally been measured in toluene-treated cells [ 121. Booth and Morris [I 31 demonstrated PTS in toluene-treated C. pasteuriunum cells, but Mitchell et al. [14] only detected PTS activity with cell extracts of C. crcetobu~licum NCIMB 8052. Cell-free extracts of C. untobutylicum P262 had PTS activity, but no activity was detected in toluene-treated cells. Because the toluene-treated cells had butyryl-CoA dehydrogenase and NAD-independent lactic acid dehydrogenase activities (data not shown), the lack of PEP-dependent glucose phosphorylation could not be explained by permeabilization per se. Because even the cell extracts had relatively low PTS activity, it appeared that the PTS of P262 was sensitive to standard methods of cell disruption. Many species of clostridia use lactose [ 151, but to our knowledge a PTS for lactose had never been reported in clostridia. C. ucetobutylicum P262 had more PTS activity for lactose than glucose, and this activity was induced by lactose. The idea that strain P262 was using a lactose-PTS to take up lactose was supported by the observation that the cells had high phospho-P-galactosidase activity and little P-galactosidase activity. When lactose is transported by the PTS, lactose is converted to lactose 6-phosphate, a phosphorylated derivative that is hydrolyzed by phospho-/3-galactosidase but not P-galactosidase [161. C. acetobutylicum P262 accumulated the nonmetabolizable lactose analog, TMG, and was able to retain it for long periods of time. Based on the observation that cells provided with glucose could no longer transport TMG and expelled TMG that had already accumulated, it appeared that the cells had mechanisms of inducer exclusion and expulsion. The independence of these mechanisms was supported by the observation that 2-deoxyglucose, a non-metabo- lizable glucose analog, could promote inducer exclusion but not inducer expulsion. Clostridia were traditionally classified as ‘sporeforming fermentative rods’ [ 151, but recent work has indicated that spores are not a definitive taxonomic trait for clostridia [17]. Further work is needed to define the phylogeny of the clostridia more precisely, but it should be noted that the clostridia and lactic acid bacteria share many common characteristics: (1) cell wall structure; (2) low mol.% G + C DNA; (3) PTS systems; (4) FBP-activated lactic acid dehydrogenases [9,18]; and (5), as shown here, glucose-dependent mechanisms of inducer expulsion. Acknowledgements This research was supported by the US Dairy Forage Research Center, Madison, WI. F.D.-G. is grateful to Consejo National de Ciencia y Tecnologia. Mexico, for their support. References 111Monod. .I. (1949) The growth of bacterial cultures. Annu. Rev. Microbial. 3. 371-394. A. (1976) Cyclic nucleotidea in bacteria. In: Advances in Cyclic Nucleotide Research (Robison, P.G. and G. A., Eds.), pp. l-48, Raven Press, New York. NY. I31 McGinnis, J.F. and Paigen, K. (1969) Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization. J. Bacterial. 100, 902-913. idI Reizer, J. and Panos, C. (1980) Regulation of P-galactoside phosphate accumulation in Streptococcus pyogcnes by an expulsion mechanism. Proc. Natl. Acad. Sci. USA 77, S4975501. El Ye, J.J., Reizer, J.. Cui, X. and Saier, M.H. 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