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Applied and Environmental Microbiology, March 2007, p. 1842-1850, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02082-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of the Mechanism and Regulation of Lactose Transport and Metabolism in Clostridium acetobutylicum ATCC 824

Yang Yu,^1, Martin Tangney,² Hans C. Aass,¹ and Wilfrid J. Mitchell^1*

School of Life Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, United Kingdom,¹ School of Life Sciences, Merchiston Campus, Napier University, Edinburgh EH10 5DT, United Kingdom²

Received 4 September 2006/ Accepted 21 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolize a wide range of carbohydrates offers the potential for revival based on the use of cheap, low-grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolyzed by a phospho-ß-galactosidase. These activities are induced during growth on lactose but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho-ß-galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The acetone-butanol-ethanol (ABE) fermentation of Clostridium acetobutylicum was a classical method to produce the commercially important solvents acetone and butanol, which operated successfully at an industrial scale in many countries during the first half of the 20th century. After the Second World War, the fermentation process declined because of the emergence of the competitive petrochemical-based synthesis of the solvents (18). Nevertheless, oil is a finite commodity and global oil prices are on the rise, and this, coupled with a general worldwide interest in exploiting renewable resources, has stimulated research on the biochemistry and physiology of solventogenic clostridia in recent years, with the aim of exploring the potential for revival of the ABE fermentation (17, 22). A major consideration in any bioconversion process is the availability of a cost-effective and high-product-yielding growth medium, whereby there is maximal conversion of the available carbon into the commercial end product. The substrate of choice in the traditional industrial ABE fermentation, molasses, accounted for more than 60% of the overall cost of the process (19). However, the clostridia are metabolically versatile with respect to carbohydrate utilization and the potential therefore exists to exploit alternative, cheaper, low-grade substrates.

Several clostridial strains are able to produce solvents by fermentation of whey (21), which has been shown to be economically superior to the traditional process (19). Despite low reactor productivity, an attractive feature of these fermentations is that the butanol-acetone ratio is considerably greater than the generally observed value of 2:1 (4, 20), which is particularly significant when considering the current impetus toward the development of butanol in preference to ethanol as a potential biofuel source. Relatively little is currently known about the bioconversion of the whey sugar lactose by solventogenic clostridia. A detailed study of the genetics and physiology of lactose uptake and utilization could therefore contribute to improved productivity based on manipulation of both the organisms and the fermentation conditions for a more effective process.

In bacteria, two enzymes involved in lactose metabolism have been recognized; ß-galactosidase hydrolyzes the disaccharide lactose to glucose and galactose, while phospho-ß-galactosidase cleaves lactose 6-phosphate to glucose and galactose 6-phosphate. The latter enzyme, which is commonly found in gram-positive bacteria, is associated with uptake of lactose via the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), which catalyzes the concomitant uptake and phosphorylation and deposits lactose 6-phosphate in the cytoplasm (16, 29, 35). Several strains of C. acetobutylicum were shown to contain both ß-galactosidase and phospho-ß-galactosidase activities when grown on lactose (40), and a gene encoding ß-galactosidase has been cloned from strain NCIB 2951 (15). However, strain ATCC 824 was found to be unique in synthesizing only a phospho-ß-galactosidase enzyme (40). The implication was that this strain relies exclusively on a PTS for lactose uptake, but the presence of a PTS was not demonstrated.

The phosphate and energy for PTS-mediated solute translocation is derived from PEP and is transferred in a phosphorelay via two proteins (enzyme I and HPr) common to all of the phosphotransferases in the cell and a substrate-specific enzyme II complex. The architecture of the enzyme II may vary, but it typically contains a membrane-bound IIC domain and two hydrophilic, cytoplasmically orientated domains IIB and IIA. These domains may be part of a single polypeptide or may exist as a combination of separate interactive proteins; for example, enzyme II of the lactose PTS in Lactococcus lactis is synthesized as a membrane associated, two-domain IICB protein and a free cytosolic IIA protein (9).

We have demonstrated the presence in C. acetobutylicum ATCC 824 of PTS transport systems for a number of substrates, including sucrose and maltose, and have further established that these systems are subject to regulation in response to substrate availability (33, 34). Such regulation of catabolic activity has profound implications in industrial fermentations where the maximization of culture growth and product yield is imperative. The objective of the present study was to determine the mechanism by which lactose is transported and metabolized by C. acetobutylicum ATCC 824 and to investigate the regulation of the activities involved.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organism and growth conditions.
C. acetobutylicum ATCC 824 was maintained as a spore suspension in water at 4°C. Aliquots of spores (0.5 to 1 ml) were heat shocked at 80°C for 10 min, transferred into 20 ml of reinforced clostridial medium (Oxoid), and incubated at 37°C overnight under an atmosphere of N₂-H₂-CO₂ (80:10:10) in an anaerobic cabinet (Forma Scientific, Marietta, OH). Aliquots of these starter cultures (5% [vol/vol]) were then transferred to clostridial basal medium (CBM [33]) supplemented with appropriate sugar(s) as required, and incubation was continued under anaerobic conditions.

Analysis of diauxic growth and sugar utilization.
A 100-ml CBM culture containing the required sugar was grown overnight, and cells were harvested by centrifuging them anaerobically at 20°C and 4,000 x g for 10 min in tubes sealed with a rubber Sub-a-Seal. The cell pellets were resuspended and washed three times in CBM without a carbon source before being resuspended in 10 ml of the same medium. The washed cells were then inoculated into CBM containing glucose and lactose to obtain an optical density at 650 nm of around 0.1, followed by incubation at 37°C. Cell density was measured as the optical density at 650 nm. For sugar analysis, 1 ml of culture was centrifuged at 12,000 x g for 2 min, and glucose was assayed by using a Sigma assay kit (510-A). Lactose concentration was estimated after hydrolysis of lactose to glucose and galactose with ß-galactosidase. Thus, 200 µl of culture supernatant was made up to 1 ml with a solution containing 20 mM potassium phosphate buffer (pH 7.3), 4 mM MgSO₄, and 2.5 U of ß-galactosidase. The reaction mixture was incubated at 37°C for 1 h, which was sufficient to ensure complete lactose hydrolysis. Aliquots of the hydrolyzed sugar were then assayed for glucose. The lactose concentration was determined from the difference in the amount of glucose before and after lactose hydrolysis.

Preparation of cell extracts.
Cell extracts were prepared as described previously (23) and were divided into aliquots, flash-frozen in liquid nitrogen, and stored at –70°C.

Fractionation of crude extract was carried out by ultracentrifugation at 230,000 x g at 4°C for 2 h in a bench-top centrifuge (TL-100; Beckman). After the first centrifugation, the supernatant was removed and recentrifuged, and the final supernatant formed the soluble, cytoplasmic fraction. The surface of the membrane pellet from the first centrifugation was washed three times with 50 mM potassium phosphate buffer (pH 7.0) containing 5 mM MgCl₂ and 1 mM dithiothreitol (DTT). The pellet was then resuspended in the same buffer, centrifuged as described above, washed, and resuspended in 1/10 volume of the original crude extract. Aliquots of soluble fraction and membranes were flash-frozen in liquid nitrogen and stored at –70°C. Protein concentration in cell extracts was determined by a microbiuret assay (41).

Enzyme assays.
Phosphorylation of radiolabeled lactose was monitored by the method of Gachelin (13) based on precipitation of sugar phosphate in an ethanolic solution of BaBr₂. A total of 1 ml of reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 2 mM DTT, 5 mM MgCl₂, 12 mM potassium fluoride, 1 mM PEP or ATP, and cell extract (generally 200 µl of crude extract or cytoplasmic fraction and 20 µl of membranes). The mixture was equilibrated at 37°C for 3 min, and then [D-glucose 1-¹⁴C] lactose (9.67 mM, 1.03 Ci/mol) was added to a concentration of 0.2 mM. Samples (150 µl) of reaction mixture were removed at intervals, and the amount of sugar phosphate formed was estimated as described previously (5).

The activities of phospho-ß-galactosidase and ß-galactosidase were determined by hydrolysis of o-nitrophenyl-ß-D-galactopyranoside 6-phosphate (ONPG 6-P) and o-nitrophenyl-ß-D-galactopyranoside (ONPG), respectively. Reaction mixtures (200 µl) assembled in the wells of a microplate contained 50 mM potassium phosphate buffer (pH 7.0), 5 mM MgCl₂, 1 mM DTT, and 1 mM ONPG 6-P or ONPG. After the addition of cell extract, the production of o-nitrophenol (ONP) was determined by measuring the absorbance at 410 nm in a microplate reader (MR7000; DYNA Tech). The amount of ONP formed was estimated by reference to a calibration curve, and the activities of phospho-ß-galactosidase or ß-galactosidase were calculated based on the rate of ONP production.

Preparation of hybridization probes.
DNA was isolated from C. acetobutylicum ATCC 824 by using a genomic DNA isolation kit (Gentra) according to the manufacturer's instructions, but with the modification that cells were lysed with lysosyme (80 µg/ml) rather than the lytic enzyme solution provided. Internal primers (Table 1) were used in PCR amplifications to generate labeled hybridization probes specific for each lac gene. Amplification reactions in Taq buffer (Bioline) contained 1.5 mM MgCl₂; 0.2 mM concentrations each of dATP, dCTP, and dGTP; 0.15 mM dTTP; 0.25 mM digoxigenin-11-dUTP (Boehringer); 100 pmol of each primer; and 1 µl of C. acetobutylicum DNA in a 100-µl reaction volume. After the mixture was heated to 95°C for 5 min, 2.5 U of Taq polymerase (Bioline) was added, and the reaction proceeded with 30 cycles of 95°C for 1 min, annealing at 5°C below the melting temperature of the primers for 1 min, and 72°C for 3 min, followed by a final 10 min at 72°C.

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TABLE 1. Oligonucleotide primers used in this study

Analysis of lac gene expression.
Cells of C. acetobutylicum ATCC 824, grown in CBM containing the required sugars, were harvested from 1 to 1.5 ml of culture by centrifugation at 12,000 x g for 2 min in a microfuge. The supernatant was removed, and the pellet was immediately flash-frozen in liquid nitrogen and stored at –70°C. Total RNA was extracted from cell pellets by using a RNeasy Minikit (QIAGEN) according to the manufacturer's instructions, except that the concentration of lysozyme in cell lysis solution was increased from 1 to 5 mg/ml. The RNA concentration was measured by reading the absorbance at 260 nm using a GeneQuant (Pharmacia Biotech). Total RNA samples (1 µg) for slot blotting were applied to a Hybond-N⁺ nucleic acid transfer membrane (Amersham) under vacuum by using a Minifold II system (Schleicher & Schuell) and immobilized on the membrane by irradiation for 1.5 min in a UV cross-linker (UVC-508; Ultra Lum). Membranes were prehybridized at 50°C for 1 h in high sodium dodecyl sulfate (SDS) solution (7% SDS, 50% formamide, 5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2% blocking reagent, 50 mM sodium phosphate [pH 7.0], 0.1% N-lauroylsarcosine). DNA probe (5 to 10 µl per 15 ml), which had been denatured at 100°C for 10 min, was then added, and hybridization proceeded overnight at 50°C. The membrane was then washed twice under low stringency (2x SSC-0.1% [wt/vol] SDS at room temperature) and twice under high stringency (0.5x SSC-0.1% [wt/vol] SDS at 68°C) conditions. The RNA-DNA hybrids were detected by chemiluminescence using CDP-Star (Ambion) according to the manufacturer's instructions.

Primer extension analysis.
RNA prepared as described above was used as a template for cDNA synthesis using reverse transcriptase (Fermentas) according to the manufacturer's instructions. The reaction mixture contained 2 µg of total RNA and 18 pmol of 5'-PET-labeled primer 309 (Table 1) in a volume of 20 µl. After the reaction, 10 µl of formamide and 0.3 µl of GS500 LIZ standard (Applied Biosystems) were added, and the mixture was heated at 95°C for 5 min and chilled on ice. The size of the cDNA was determined with a genetic analyzer (ABI Prism 3100; Applied Biosystems).

DNA computational analysis.
The lac operon of C. acetobutylicum ATCC 824 was identified from the genome sequence deposited by the Genome Therapeutics Corp. (26). Protein sequences were obtained by using the BLAST service at the National Center for Biotechnology Information (3). Multiple alignment of protein sequences was performed by using CLUSTAL W of the European Bioinformatics Institute (7), and phylogenetic trees were drawn by using Treeview (28).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactose metabolism by C. acetobutylicum.
Preliminary growth experiments established that C. acetobutylicum ATCC 824 can grow on minimal medium (CBM) supplemented with lactose as the sole carbon source, although it is clear that glucose is a preferred carbon source for this organism. Indeed, when cultures growing on lactose were supplemented with glucose, we observed an inhibition of lactose consumption, whereas the added glucose was used rapidly (Fig. 1), indicating that lactose metabolism is subject to regulation by glucose. In order to investigate the mechanism of lactose transport and metabolism in this organism, cell extracts were prepared from cultures grown on CBM containing either glucose or lactose as the sole carbon source, and these extracts were assayed for phospho-ß-galactosidase and ß-galactosidase activities. In agreement with an earlier report (40), a basal level of ß-galactosidase activity was detected in both glucose and lactose extracts, but while phospho-ß-galactosidase activity was also at basal levels in glucose extracts, there was a marked induction of this activity after growth on lactose (Table 2). Phospho-ß-galactosidase activity would be required in cells transporting lactose via a PEP-dependent PTS, where the product of the transport reaction is lactose-phosphate. Extracts were therefore assayed for lactose PTS activity by monitoring the PEP-dependent phosphorylation of radiolabeled lactose. As can be seen in Table 2, phosphorylation of lactose by extracts prepared from cultures grown on lactose as the sole carbon source was stimulated in the presence of PEP but not by ATP, thus providing convincing evidence for a lactose PTS activity in this organism. Therefore, together with phospho-ß-galactosidase, the lactose PTS provides a mechanism for the uptake and initial metabolism of lactose in C. acetobutylicum. As for the phosphohydrolase, the lactose PTS was not detected in extracts prepared from cells grown on glucose (Table 2), suggesting that both are induced by growth on the substrate.

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FIG. 1. Effect of glucose on lactose utilization by C. acetobutylicum. Time of glucose addition is indicated by the arrow. Growth (•) and concentration of glucose () and lactose () were followed as described in Materials and Methods.

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TABLE 2. Lactose phosphorylation and galactosyl hydrolase activities in C. acetobutylicum cell extractsa

The substrate specificity of a PTS lies in the enzyme II complex, which may be a single membrane-bound protein or composed of both membrane-associated and cytoplasmic components. The lack of lactose PTS activity in glucose extracts facilitated the use of in vitro heterologous sugar phosphorylation assays to investigate the architecture of the lactose PTS in C. acetobutylicum. Extracts were prepared from cultures grown on either lactose or glucose as the sole carbon source, and the membrane and cytosol fractions were then separated. Lactose PTS activity was reconstituted by combining the two lactose cell fractions. However, although the glucose extract was active for glucose phosphorylation (data not shown), neither the combination of purified lactose membranes and glucose cytosol nor purified glucose membranes and lactose cytosol was able to reconstitute lactose PTS activity (Fig. 2). The lactose enzyme II complex must therefore comprise membrane-associated and cytosolic substrate-specific components, neither of which is present in glucose extracts and both of which are induced in lactose-grown cells.

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FIG. 2. Lactose phosphorylation by C. acetobutylicum extracts. (A) PEP-dependent phosphorylation was assayed in the presence of a soluble fraction of lactose extract and a membrane fraction of lactose () or glucose () extract. (B) PEP-dependent phosphorylation was assayed in the presence of a membrane fraction of lactose extract and a soluble fraction of lactose () or glucose () extract. The individual lactose extract fractions alone showed basal activity comparable to that in the presence of glucose extract fractions.

Sequence analysis of the C. acetobutylicum lac operon.
Typically, PTS genes constitute an operon also containing genes encoding catabolic enzymes and a regulator of transcription. Biochemical analysis of the lactose PTS in C. acetobutylicum implies the presence in this organism of at least two genes encoding the enzyme II complex to accommodate the membrane-associated and cytosolic specific components, together with a gene encoding a phospho-ß-galactosidase. Analysis of the C. acetobutylicum genome sequence revealed the presence of a putative operon that could potentially encode these proteins. Three clustered genes in the same orientation are, by homology, suggested to encode a cytosolic PTS enzyme IIA domain, a membrane-associated IICB polypeptide, and a phospho-ß-galactosidase. Accordingly, we have designated these genes lacF, lacE, and lacG, respectively, in convention with equivalent genes in other organisms. The system also appears to be associated with a regulator gene, which we designate lacR, giving the lactose gene system lacRFEG depicted in Fig. 3A.

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FIG. 3. Organization and function of the lactose (lac) operon of C. acetobutylicum ATCC 824. (A) Gene organization in the chromosome. Each gene is represented by an arrow which indicates the direction of transcription. Putative promoter sequences are indicated in bold and underlined, and a presumed ribosome binding site (rbs) and translational start of the lacR gene are shown in boldface italics. A potential cre sequence encompassing the highlighted transcriptional start is shaded. (B) Proposed pathway encoded by the lac operon. The products glucose and galactose 6-phosphate can be further metabolized by hexokinase and the tagatose 6-phosphate pathway, respectively.

The lacF gene is preceded by a putative ribosome-binding site TGGAGG and encodes a protein of 104 amino acids (aa), with a predicted M_r of 11,704. The deduced amino acid sequence of the LacF protein is clearly related to hydrophilic PTS IIA domains. It is most closely related (60% identity) to the putative LacF protein of Staphylococcus haemolyticus and exhibits more than 50% identity to the corresponding proteins of Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis. Downstream of lacF, at a distance of 75 nucleotides, lies lacE, which is preceded by a putative ribosome-binding site (AGGAGG). Analysis of the lacE product reveals a protein of 560 aa (predicted M_r of 61,799), with a predominantly hydrophobic N-terminal region, typical of the integral membrane IIC domain, followed by a hydrophilic domain that resembles the cytoplasmically orientated IIB. It is homologous over its entire length to the LacE proteins of other low-GC gram-positive bacteria, sharing at least 53% identity with the proteins from Streptococcus pyogenes, S. mutans, S. aureus, and L. lactis. The putative C. acetobutylicum lactose PTS proteins contain the conserved residues his78 (in LacF) and cys464 (in LacE), which have been implicated as the phosphorylation sites of PTS IIA^Lac and IIB^Lac domains (1, 11, 12). Furthermore, phylogenetic analysis clearly established that they are members of the lactose-N,N'-diacetylchitobiose-ß-glucoside (Lac) family. This family includes lactose permeases from gram-positive bacteria, as well as the N',N-diacetylchitobiose permeases of Escherichia coli and Borrelia burgdorferi, together with other permeases believed to transport di- and oligosaccharides. As shown in Fig. 4, the C. acetobutylicum IIA^Lac domain is part of a cluster of lactose IIA domains within this family. Similar results were obtained from analysis of the IIB and IIC domains encoded by the lacE gene (results not shown).

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FIG. 4. Phylogenetic analysis of IIA domains of the PTS Lac family. Abbreviations (with accession numbers in parentheses) are as follows: BbuChb, Borrelia burgdorferi N,N'-diacetylchitobiose (NP_046991); BsuLic, Bacillus subtilis lichenan (P46319); CacCel, Clostridium acetobutylicum cellobiose (NP_347023); CacLac, C. acetobutylicum lactose (NP_349567); EcoChb, Escherichia coli N,N'-diacetylchitobiose (P69793); LcaLac, Lactobacillus casei lactose (CAB02558); LlaCel, Lactococcus lactis cellobiose (NP_266570); LlaLac, Lactococcus lactis lactose (AAA25181); SauLac, Staphylococcus aureus lactose (P0A0D6); ShaLac, Staphylococcus haemolyticus lactose (YP_252761); SmuCel, Streptococcus mutans cellobiose (NP_721934); and SmuLac, S. mutans lactose (NP_721840).

The distal gene in the lac system, lacG, lies 57 nucleotides downstream of lacE. It is preceded by the putative ribosome-binding site AGGGAA and encodes a protein of 474 aa, with an M_r of 54,672, which by homology is a phospho-ß-galactosidase. The predicted C. acetobutylicum LacG protein sequence exhibits between 50 and 60% identity to the corresponding phospho-ß-galactosidases of other lactose metabolizing gram-positive bacteria and belongs in the glycosylhydrolase family I (16). The lacG gene is followed by a potential rho-independent transcription terminator, which begins approximately 90 nucleotides downstream of lacG.

The lac gene system encodes all of the identified activities and proteins implicated in the PTS transport of lactose and the subsequent hydrolysis of the translocated product by phospho-ß-galactosidase to generate intracellular glucose and galactose 6-phosphate. The former can be metabolized directly via the glycolytic pathway, while the latter can be isomerized to tagatose 6-phosphate and subsequently metabolized via the tagatose 6-phosphate pathway. An examination of the C. acetobutylicum genome sequence reveals, as expected, the presence of putative genes encoding tagatose 6-phosphate kinase and tagatose bisphosphate aldolase in an operon with galactose 6-phosphate isomerase. This allows for the complete metabolism of lactose by the pathway depicted in Fig. 3B.

Regulation of expression of the C. acetobutylicum lac operon.
A general characteristic of PTS transport systems is that they are induced in the presence of the PTS substrate. This is typically achieved at the level of transcription via a specific regulator protein, which may itself be encoded by a gene associated with the target PTS gene system. The presence, upstream of lacF, of the gene that we have designated lacR is therefore significant. The lacR gene, which is separated from lacF by 58 nucleotides, encodes a protein of 254 aa, with a predicted M_r of 29,246. It has low but significant homology to transcriptional regulators of the DeoR family, sharing 40% identity with LacR of S. mutans (31) and 34 and 33% identity, respectively, with the LacR proteins of L. lactis and S. aureus, each of which has been demonstrated to act as a repressor of the associated lac operon in these bacteria (27, 37, 38). In common with these studied LacR proteins, the N-terminal end of the C. acetobutylicum LacR has a predicted helix-turn-helix DNA-binding domain characteristic of the DeoR family of transcription regulators, and an alignment of these proteins clearly identifies the C. acetobutylicum LacR as a member of this family of regulator proteins (Fig. 5). Upstream of lacR we have identified a putative promoter sequence with good agreement to the consensus, having a –35 sequence TTGACA and a –10 sequence TATTAT that are separated by 17 nucleotides (Fig. 3A). Primer extension analysis produced a cDNA of 132 nucleotides in length, positioning the transcriptional start site 14 bp downstream of the proposed –10 region, which is in agreement with the designation of the promoter. Interestingly, this start site is situated within a sequence, TGTAAACGAAAACA, that closely resembles a catabolite responsive element (cre) central to catabolite repression in low-GC gram-positive bacteria (24).

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FIG. 5. Alignment of LacR of C. acetobutylicum with LacR proteins of other gram-positive bacteria. Identical residues are within black boxes, and conserved residues are shaded in gray. Abbreviations (with accession numbers in parentheses) are as follows: S.mut, Streptococcus mutans (P26422); S.aur, Staphylococcus aureus (P0A0Q0); L.lac, Lactococcus lactis (AAA25186); and C.ace, Clostridium acetobutylicum (AAK80908).

In order to investigate the regulation of lactose metabolism, the expression of the lac operon was monitored in a culture containing both glucose and lactose. At intervals, samples were removed for analysis of culture growth, sugar utilization, gene expression, and enzyme activity. Culture growth followed a diauxic pattern, with glucose utilized preferentially during the first phase of growth, followed by a brief lag prior to a second growth phase where lactose was utilized (Fig. 6A). The pattern of growth and sugar utilization was independent of whether cells had been pregrown in medium containing glucose or lactose. In cultures inoculated with glucose-grown cells, there was no detectable lactose PTS activity in extracts prepared from cells harvested during the initial phase of growth, whereas activity was detected in extracts prepared from cells in the second phase, during which lactose was being depleted from the medium (Fig. 6B). A similar pattern of activity was observed for phospho-ß-galactosidase, which was present at the basal level in the glucose utilization phase and increased fivefold thereafter. Furthermore, PTS reconstitution assays using purified soluble and membrane fractions of cell extracts revealed that both soluble and membrane components of the lactose PTS were regulated coordinately during diauxic growth (data not shown). The appearance of lactose PTS activity correlated with the expression of the lac operon, as revealed by slot blot analysis using hybridization of individual lac gene-specific probes against total mRNA isolated at intervals throughout the growth experiment. In the case of each of the C. acetobutylicum lacREFG genes, significant expression was only detected during growth on lactose, as shown for the lacR and lacF genes in Fig. 6C, and Northern analysis confirmed that these genes were coexpressed on a polycistronic mRNA (data not shown). Significantly, we found a similar pattern for the putative tagatose gene system when we probed for lacA (galactose 6-phosphate isomerase) gene expression (results not shown). It would therefore appear that all of the genes necessary for complete metabolism of the galactose moiety of lactose are coordinately expressed under conditions in which lactose is being utilized by C. acetobutylicum.

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FIG. 6. Diauxic growth of C. acetobutylicum on CBM containing glucose and lactose. Cells were pregrown in CBM containing glucose and inoculated into medium containing 5 mM concentration of each sugar. (A) Diauxic growth curve. Samples were withdrawn periodically for measurement of the growth (•) and concentration of lactose () and glucose (), and at the times shown by the arrows, for analysis of gene expression and lactose metabolic activities. (B) Lactose PTS activity in cell extracts. Extracts were prepared from samples taken at points 2 (), 3 (), and 6 () as indicated in panel A. (C) Slot blot analysis of lac gene expression. RNA was prepared from samples taken at points 1 to 7 as indicated in panel A and probed for the expression of lacR (top) and lacF (bottom).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The redevelopment of the ABE process will depend to a significant degree on the exploitation of inexpensive growth substrates and the manipulation of high-yield production strains of solventogenic clostridia and in particular C. acetobutylicum. In this regard, the uptake of a specific substrate by a cell represents an early control point in the fermentation process, and it has been established that clostridia, like other bacteria, possess the capacity to discriminate between carbon sources. In C. acetobutylicum, for example, sucrose is required to induce the expression of the sucrose PTS; however, this induction is prevented in the presence of glucose which can effect catabolite repression of PTS gene systems (33). To maximize carbon flow in the ABE process, it is therefore essential to develop an understanding of these early events in metabolism.

Lactose represents a potentially important substrate for the industrial ABE fermentation process using C. acetobutylicum. Although a low level of ß-galactosidase activity was detected in extracts of strain ATCC 824, it was not induced by growth on lactose and thus represents a basal hydrolysis of the substrate ONPG. In contrast, synthesis of phospho-ß-galactosidase was stimulated severalfold in lactose-grown cells. Concurrently with phospho-ß-galactosidase, a lactose phosphotransferase system was induced which during cell growth would catalyze accumulation and phosphorylation of the disaccharide, thus providing the intracellular substrate for the phosphohydrolase. These results establish that lactose is transported by this organism via a PEP-dependent phosphotransferase system, and the translocated product is further metabolized by the enzyme phospho-ß-galactosidase. A gene system was identified encoding these proteins, and the appearance of these enzyme activities was correlated with the expression of this lac operon. Lactose PTS activity was not detected in glucose-grown cells, allowing us to establish by in vitro PTS assays that the enzyme II complex for lactose in C. acetobutylicum is composed of unique, lactose-specific soluble and membrane-associated components. This contrasts with the sucrose PTS in this organism, where the substrate specificity is contained entirely in the membrane because the enzyme II complex exists as a complete IIBCA protein encoded by a single gene (33). Analysis of the lac genes reveals that there is a cytoplasmic IIA component and a membrane-bound IICB protein. The same two-gene arrangement for the lactose enzyme II complex has been found in other gram-positive bacteria (6, 10, 14, 31).

The products of lactose uptake and hydrolysis are glucose and galactose 6-P. Glucose may be phosphorylated and incorporated into the glycolytic pathway, whereas galactose 6-P is generally metabolized by the tagatose 6-P pathway. In both S. aureus and the lactic acid bacteria S. mutans and L. lactis the genes encoding the enzymes of this pathway form part of a large operon together with the genes encoding the lactose PTS and phospho-ß-galactosidase (30, 31, 39). A putative tag gene system encoding the tagatose 6-phosphate pathway in C. acetobutylicum was identified from the genome sequence but, in contrast to these other organisms, the lac and tag genes in C. acetobutylicum are within two operons separated by a distance of 8 kbp. Nevertheless, our expression studies demonstrate that these systems may be coordinately regulated since neither was expressed during the glucose utilization phase in a diauxic growth culture, whereas the induction of both occurred during the ensuing lag phase in response to the availability of lactose and the alleviation of catabolite repression by glucose. These results are consistent with the pathway presented in Fig. 3B, whereby the complete catabolism of lactose is proposed to involve the lactose PTS, phospho-ß-galactosidase, and both the tagatose 6-phosphate and glycolytic pathways.

The complete C. acetobutylicum lac operon, in addition to the three structural genes, also encodes a transcription regulator in the form of LacR, a repressor protein of the DeoR family. Analysis of mRNA in induced cells confirmed that the four genes of the operon are coordinately expressed and on a single polycistronic mRNA. Interestingly, the lac and tag operons both appear to be associated with closely related regulators, suggesting that these proteins may provide the basis of a common induction mechanism. The C. acetobutylicum LacR proteins are homologous to the corresponding repressor proteins in S. aureus, S. mutans, and L. lactis (27, 31, 37), although there are differences in gene organization and regulation in these organisms. In the lac operons of S. aureus and S. mutans, the lacR gene is in the same orientation as the structural genes, as is observed in C. acetobutylicum, but the tag genes are inserted between lacR and lacF (27, 31). The lac and tag genes are also found in an operon in L. lactis, but in this case the lacR gene is transcribed divergently from the structural genes, and its expression is repressed in the presence of lactose (37). This is clearly different from C. acetobutylicum, in which the expression of lacR is induced coordinately with the structural genes. C. acetobutylicum is not unique in having a discrete lac operon; for example, Lactobacillus casei also possesses a distinct lactose PTS operon, although the gene order lacEGF is different, and it is under the control of a PTS-modulated antiterminator, LacT, rather than a repressor protein (2, 14). While all of these operons respond to the availability of external lactose, the intracellular inducers are galactose 6-P, which has been implicated as the inducer of lac operon expression in Staph. aureus (25), or tagatose 6-P, which has been demonstrated to be the inducer of the lac operon in L. lactis (9, 36). The identity of the intracellular inducer in C. acetobutylicum remains to be determined.

Expression of the lac operon is clearly subject to carbon catabolite repression (CCR) by glucose, as is evident from the diauxic growth analysis (Fig. 6A). This is not unprecedented in this organism, since in addition to lactose, glucose is known to inhibit the utilization of other carbon sources such as sucrose (33) and maltose (34). It seems likely that a global mechanism of CCR is at least partly responsible for these observations. In a number of low-GC gram-positive bacteria, CCR is mediated by a metabolite-activated protein kinase that phosphorylates the PTS phosphocarrier protein HPr on a serine residue. The resultant HPr(ser-P) then forms a ternary complex with the regulatory protein CcpA which can bind to a specific regulatory sequence, the catabolite responsive element (cre), to modulate expression of catabolic operons (8). In a recent publication we described HPr kinase activity and associated phosphorylation of HPr in C. acetobutylicum and postulated that a similar mechanism of catabolite repression is operational in this organism (32). The presence of a potential cre sequence upstream of the first gene of the lac operon is therefore significant. Primer extension analysis places the start point of transcription within this putative cre and evokes a conceptually simple strategy whereby in the presence of glucose, CcpA-mediated catabolite repression of transcription could override the derepression of the operon (via LacR) in response to the availability of lactose in the medium. Nevertheless, it is likely that inhibition of uptake of lactose by glucose (inducer exclusion), which is demonstrated by the slowing of lactose utilization in growing cultures on addition of glucose (Fig. 1) and the occurrence of a normal glucose-lactose diauxie after pregrowth on lactose, also contributes substantially to regulation of lactose metabolism. It will be of interest to confirm experimentally the relative importance and precise roles of the CcpA and LacR proteins in modulating the expression of the lac operon, thus providing further insight into the mechanism(s) of catabolite repression in C. acetobutylicum. These molecular studies can make a significant contribution to the revival and sustainability of ABE fermentation as an industrial process.

 
We are grateful to Alex Reid for advice and technical assistance.

Y.Y. acknowledges receipt of a scholarship from Heriot-Watt University.

 
* Corresponding author. Mailing address: School of Life Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, United Kingdom. Phone: 44 131 451 3459. Fax: 44 131 451 3009. E-mail: w.j.mitchell{at}hw.ac.uk.

Published ahead of print on 5 January 2007.

Present address: Mascoma Corporation, 16 Cavendish Court, Suite 2A, Lebanon, NH 03766.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, March 2007, p. 1842-1850, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02082-06
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