Note: Sucrose transport and metabolism in ... - Wiley Online Library

Journal of Applied Microbiology 1998, 84, 914–919

Note: Sucrose transport and metabolism in Clostridium beijerinckii NCIMB 8052 M. Tangney, C. Rousse, M. Yazdanian and W.J. Mitchell Department of Biological Sciences, Heriot-Watt University, Edinburgh, UK 6221/05/97: received 2 May 1997, revised 8 October 1997 and accepted 15 October 1997

Sucrose is the major carbon source in molasses, the traditional substrate employed in the industrial acetonebutanol-ethanol (ABE) fermentation by solventogenic clostridia. The utilization of sucrose by Clostridium beijerinckii NCIMB 8052 was investigated. Extracts prepared from cultures grown on sucrose (but not xylose or fructose) as the sole carbon source possessed sucrose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity. Extract fractionation and reconstitution experiments revealed that the entire sucrose Enzyme II complex resides within the membrane in this organism. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose grown cultures. The fructokinase activity, which is required specifically during growth on sucrose, was shown to be inducible under these conditions. A pathway for sucrose metabolism in this organism is proposed. M . T AN G NE Y, C . R OU S SE , M . YA ZD A NI AN A ND W. J . M IT C HE LL . 1998.

INTRODUCTION

The uptake and metabolism of carbon sources constitutes a fundamental requirement for growth and development of bacteria. Numerous mechanisms have evolved to cope with this demand. One particularly widespread mechanism amongst bacteria is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (Postma et al. 1993). By this method the energy stored in the high energy phosphate bond in PEP is harnessed to drive the translocation of the substrate, with its concomitant phosphorylation trapping the product within the cell. The phosphate comes from PEP and is sequentially transferred via a number of PTS proteins to the substrate. The PTS is composed of two general cytosolic proteins, called Enzyme I and HPr, as well as a substrate specific enzyme complex called Enzyme II. The architecture of the EII complex can vary considerably, but all contain three distinct functional domains, namely IIA, IIB and IIC. The IIA and IIB domains are involved in sequential phosphate transfer, while IIC is involved in substrate translocation. The domains may all be contained within a single membrane-bound polypeptide chain, but it is not uncommon to find IIA as an independent cytoplasmic protein (Saier and Reizer 1992). The PTS transport mechanism has been Correspondence to: W.J. Mitchell, Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK (e-mail: [email protected]).

demonstrated in saccharolytic clostridia (Mitchell et al. 1991 ; Mitchell 1996) where, as in other bacteria, it may not only be involved in transport but also in metabolic regulation. Recent advances in both genetic and fermentation technologies have stimulated fresh interest in the clostridia which were once commercially exploited in the acetone-butanolethanol (ABE) fermentation industry (Woods 1993). In particular, Clostridium acetobutylicum NCIMB 8052, which has recently been reclassified as a strain of Cl. beijerinckii (Keis et al. 1995), has received much attention, as it can be genetically manipulated with comparative ease (Minton et al. 1993). Industrial fermentations commonly used molasses as a growth substrate, the main carbon source in which is sucrose (Jones and Keis 1995). Observations reported from this laboratory have indicated that sucrose utilization may be regulated by glucose in Cl. beijerinckii NCIMB 8052 (Mitchell et al. 1995). Such regulation has industrial significance as it represents a potentially important control point in the fermentation. Significantly, uptake of the disaccharides lactose and cellobiose is also regulated by glucose in this organism (Mitchell et al. 1995) and both inducer exclusion and inducer expulsion mechanisms have recently been demonstrated in clostridia (Diez-Gonzalez and Russell 1996). An understanding of transport systems and catabolite repression mechanisms may therefore play an important role in any future revival of the ABE fermentation industry. Despite the dependence of a productive ABE fermentation © 1998 The Society for Applied Microbiology

S UC RO S E M ET A BO LI S M B Y C L OS TR I DI UM B EI JE R IN CK I I 915

on the efficient utilization of sucrose in molasses, nothing is known about sucrose transport or metabolism in the industrial organisms. The aim of this work was therefore to characterize sucrose transport and metabolism in Cl. beijerinckii NCIMB 8052. MATERIALS AND METHODS Organism and culture conditions

Clostridium beijerinckii NCIMB 8052 was maintained as a spore suspension at 4 °C. Spores were heat shocked at 80 °C for 10 min and inoculated into 20 ml of Reinforced Clostridial Medium (RCM, Oxoid, Basingstoke, UK) in an anaerobic cabinet (Forma Scientific, Marietta, OH, USA) to provide starter cultures. Starter cultures were subcultured into RCM or a defined medium supplemented with the appropriate carbon source and incubated anaerobically at 37 °C to provide working cultures. The defined medium contained the following (g l−1) : ammonium acetate, 2·2 ; KH2PO4, 0·5 ; K2HPO4, 0·45 ; MgSO4.7H2O, 0·2 ; MnSO4.4H2O, 0·01 ; NaCl, 0·01 ; FeSO4.7H2O, 0·01 ; p-aminobenzoic acid, 0·01 ; d-biotin, 0·001. Carbon sources were sterilized separately and added to the medium after cooling. Preparation of cell-free extracts

The preparation and fractionation of cell-free extracts was by the method of Mitchell and Booth (1984). Protein concentration in cell extracts was determined by the microbiuret assay, as described by Zamenhof (1957), using bovine serum albumin as the standard. Enzyme assays in cell-free extracts

Sugar phosphorylation assays in cell-free extracts were carried out as described by Mitchell et al. (1991), with radiolabelled substrate at a concentration of 0·2 mmol l−1. Sucrose PTS activity was determined as the PEP-dependent phosphorylation of sucrose. Fructokinase activity was determined as ATP-dependent phosphorylation of fructose. Sucrose-6phosphate hydrolase activity was determined using the coupled enzyme assay described by Martin and Russell (1987). Chemicals

D-[U-14C]-sugars were obtained from Amersham. D-[U14 C]sucrose was analysed by chromatography and found to be free of any detectable amounts of contaminating sugars. Phosphoenolpyruvate [tri(cyclohexylammonium) salt] was purchased from Sigma and ATP from Boehringer (Lewes, UK). All other chemicals were of the highest available purity.

RESULTS

In a previous publication from this laboratory (Mitchell et al. 1995) an experiment was described where cultures of Cl. beijerinckii NCIMB 8052 were initially grown on sucrose and subsequently diluted into a medium containing both glucose and sucrose. It was observed that both sugars were initially metabolized at the same rate, but sucrose utilization subsequently slowed down until such time as the glucose had been exhausted from the medium. At this point sucrose was once again utilized rapidly. It was concluded that glucose could not directly inhibit sucrose uptake. Nevertheless, the reduction in the rate of sucrose utilization in the culture indicated that glucose could regulate sucrose utilization at some level. As a first step in understanding the regulatory mechanism, we wished to establish the route by which sucrose is transported and metabolized in this organism. The presence of PTS activity can be readily revealed as PEP-dependent sugar phosphorylation in cell-free extracts. We assayed cell-free extracts, which were prepared from cultures grown on sucrose as the sole carbon source, for PEP-dependent phosphorylation of [14C]sucrose, as described in Materials and Methods. In the absence of a high energy phosphate donor, there was no detectable phosphorylation of the sugar. However, PEP, but not ATP, stimulated phosphorylation, demonstrating the presence of sucrose PTS activity. In contrast, there was no significant sucrose PTS activity observed in extracts prepared from cultures grown on either fructose or xylose as the sole carbon source (data not shown). The absence of sucrose PTS activity in cultures grown on fructose or xylose further afforded us the possibility of employing extract reconstitution assays in an attempt to determine the cellular organization of the Enzyme II complex for sucrose. Accordingly, extracts were prepared from cultures of Cl. beijerinckii NCIMB 8052 which had been grown on either sucrose or xylose as the sole carbon source. The membrane and cytosol fractions of the cell-free extracts were then separated. Each fraction was assayed separately but all were devoid of sucrose PTS activity (Fig. 1). This is consistent with earlier work on the glucose PTS in this organism, where it was demonstrated that (as for other bacteria) the complete PTS is composed of both soluble and membranebound proteins (Mitchell et al. 1991). The membrane and cytosol fractions were then recombined in various combinations in a series of complementation assays. In one set of assays the cytosol fraction from cultures grown on sucrose was combined with the membrane fraction from an extract of either sucrose or xylose cultures, and assayed for sucrose PTS activity. The results shown in Fig. 1a demonstrate that only the homologous combination of sucrose membrane and sucrose cytosol was active. In the alternative combinations, the membrane fraction from sucrose grown cells was com-

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 914–919

916 M . T AN G NE Y E T AL .

given that xylose grown cells were found not to possess a sucrose PTS. The product of sucrose transport via the PTS will most likely be sucrose-6-phosphate, which must be further modified before it can enter central metabolism. We therefore assayed for the presence of the enzymes believed most likely to effect such conversion, i.e. sucrose-6-phosphate hydrolase and fructokinase. Activity of the former enzyme was detected using the coupled enzyme assay described by Martin and Russell (1987), wherein NADPH, generated during the oxidation of glucose-6-phosphate formed in the assay, is measured. Employing this assay, extracts from cultures grown on sucrose generated significant quantities of NADPH only in the presence of PEP (Fig. 2a). This is consistent with the formation of sucrose-6-phosphate by the sucrose PTS and the presence of hydrolase activity for this product in these extracts. The presence of an ATP-dependent fructokinase enzyme in sucrose extracts was demonstrated as ATP-dependent phosphorylation of fructose (Fig. 2b). There was no significant activity detected in extracts from cells grown on either xylose or fructose, implying that fructokinase activity is induced by sucrose, together with the sucrose PTS. These results confirmed that both sucrose-6-phosphate hydrolase and fructokinase activities were present in sucrose grown cultures. DISCUSSION

Fig. 1 Sucrose phosphorylation in reconstituted cell-free

extracts of Clostridium beijerinckii. Extracts were prepared from cultures grown on sucrose or xylose as the sole carbon source. Extracts were fractionated into membrane and cytosol components and assayed as described by Mitchell and Booth (1984). (a) Sucrose membranes were assayed alone () or together with sucrose cytosol (R) ; xylose membranes were assayed alone (r) or together with sucrose cytosol (Ž). Activity is expressed as, nmol sucrose phosphorylated mg membrane protein−1. (b) Sucrose cytosol was assayed alone (r) ; or together with sucrose membranes (R) ; xylose cytosol was assayed alone () ; or together with sucrose membranes (Ž). Activity is expressed as nmol sucrose phosphorylated mg cytosol protein−1

bined with the cytosol from either sucrose or xylose grown cells. In both instances sucrose PTS activity was observed (Fig. 1b). The fractionated and recombined xylose extracts were devoid of activity (data not shown). This is as expected,

Bacteria have evolved mechanisms which allow them to adapt rapidly and respond to variation in their environment. Certain substrates may be preferred to less energetically favourable ones. The key to such metabolic flexibility is often provided via regulation of synthesis or activity of transport systems. Understanding these processes is therefore critical in establishing a productive fermentation, the objective of which is to generate the maximum conversion of a cheap growth substrate into a commercially valuable product. Sucrose has traditionally been an important substrate in industrial ABE fermentations, but this is the first investigation into sucrose transport and metabolism in a solventogenic clostridium. It was found that sucrose is transported via a PTS mechanism. The sucrose PTS was absent in both xylose and fructose grown cells, indicating that the system is inducible by its substrate. It remains possible that fructose may also repress the sucrose PTS (in fact we have preliminary evidence that this may indeed be the case), but it is very unlikely that the absence of sucrose PTS activity in xylose cells is a result of repression, as this is not a favoured substrate in this organism (Ounine et al. 1985). Work in this laboratory has confirmed that mannitol and glucitol are also transported by inducible PTS transport mechanisms in Cl. beijerinckii NCIMB 8052 and that transcription of the genes encoding the glucitol PTS



Fig. 2 Enzyme activities in sucrose extracts. (a) Sucrose-6-phosphate hydrolase activity. Extracts were assayed using the coupled assay

described by Martin and Russell (1987), whereby sucrose-6-phosphate hydrolase activity can be detected indirectly by the generation of NADPH. Assays were performed in the presence (Ž) and absence (ž) of 1 mmol l−1 PEP. Enzyme activity is expressed as nmol NADPH mg protein−1. (b) Fructokinase activity. Extracts from cultures grown on sucrose (Ž), fructose (ž) or xylose (R) were assayed for ATP-dependent phosphorylation of fructose. Assays were performed in the presence (closed symbols) and absence (open symbols) of 1 mmol l−1 ATP. Enzyme activity is expressed as nmol fructose phosphorylated mg protein−1

is catabolite repressed by glucose in this organism (Mitchell 1996 ; Tangney et al. 1998). Induction and repression of PTS transport mechanisms is a common regulatory feature in other bacteria and it would now appear that Cl. beijerinckii behaves similarly in this respect, although the underlying mechanisms have yet to be elucidated. Sucrose-specific Enzyme II proteins in bacteria have been identified which form one of two arrangements. Either the IIA, IIB and IIC domains are found in a single polypeptide (Sato et al. 1989), or alternatively the protein comprises only IIB and IIC domains (Fouet et al. 1987 ; Wagner et al. 1993). The fact that sucrose membranes were both necessary and sufficient to bestow a xylose cytosol with sucrose PTS activity demonstrates that all of the sucrose specific PTS information in Cl. beijerinckii NCIMB 8052 is contained within the induced sucrose membranes. Were there a separate cytosolic sucrose specific component (as in a IIA sucrose protein) then this would have been absent from the xylose cytosol, and hence this combination would not have possessed sucrose PTS activity. While it is clear that there is no soluble sucrose specific IIA component, it cannot be concluded with certainty that Enzyme II contains all three recognized domains. It is possible that a non-specific IIA component provides this function. This has been observed in Escherichia coli where the plasmid encoded Enzyme II for sucrose requires the soluble IIA

domain of the glucose PTS for sucrose PTS activity (Lengeler et al. 1982) and a similar energization of the Enzyme IIBCScr permease by IIAGlc has been demonstrated in B. subtilis, even though the IIAGlc domain is fused to the IIBC glucose permease in this organism (Sutrina et al. 1990). Therefore the IIAGlc, which has been detected by virtue of complementation of crr mutants of E. coli (Mitchell et al. 1991) could conceivably fulfil a similar role in Cl. beijerinckii. The cloning and sequencing of the sucrose PTS gene(s) would clarify this situation. Irrespective of the source of the IIA function, the product of sucrose transport via the PTS is likely to be sucrose-6phosphate, which must be further modified before it can enter central metabolism. Sucrose-6-phosphate hydrolase activity was detected in extracts of sucrose grown cells, using an indirect assay which is dependent on the presence of an active sucrose PTS. It was, therefore, not feasible to use this method to look for the activity in extracts of uninduced cells grown on xylose or fructose, and so no conclusions can be drawn as to the regulation of the synthesis (induction) of this enzyme. In contrast, it was clearly demonstrated that fructokinase activity was only significant in sucrose induced cells and not in cells grown on either xylose or fructose, as was found for sucrose PTS activity. It is worthwhile noting that fructokinase is specifically required during metabolism of sucrose in this strain, since fructose itself is taken up by a fructose


918 M . T AN G NE Y E T AL .

Fig. 3 Schematic representation of the pathway for sucrose

transport and metabolism in Clostridium beijerinckii NCIMB 8052. Abbreviations : cm = cell membrane ; PTS = phosphotransferase system ; S6PH = sucrose-6-phosphate hydrolase ; FK = fructokinase. The pathway is described in detail in the text

PTS, the product of which is fructose-1-phosphate (Mitchell 1996). In conclusion, we propose the pathway depicted in Fig. 3 for sucrose utilization in Cl. beijerinckii NCIMB 8052. By this scheme, sucrose is translocated into the cell and converted (as shown) to fructose-6-P and glucose-6-P (which can also be generated by the glucose PTS) both of which can be metabolized via the Embden-Meyerhof pathway.

ACKNOWLEDGEMENTS

This work was supported in part by Research Grant no. T04089 from the Biotechnology and Biological Sciences Research Council, UK. REFERENCES Diez-Gonzalez, F. and Russell, J.B. (1996) The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by

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