Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms

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.     

Inducer exclusion by glucose 6-phosphate in Escherichia coli

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA^Glc as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA^Glc leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-β- D -thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA^Glc or were insensitive to inducer exclusion because enzyme IIA^Glc could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA^Glc. In a mutant insensitive to enzyme IIA^Glc-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA^Glc-mediated catabolite repression which embodies repression by non- PTS carbon sources.     

Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Regulation of Lactose Transport by the Phosphoenolpyruvate-Sugar Phosphotransferase System in Membrane Vesicles of Escherichia coli

Regulation of lactose uptake by the phosphoenolpyruvate-sugar phosphotransferase system (PTS) has been demonstrated in membrane vesicles of Escherichia coli strain ML308-225. Substrates of the phosphotransferase system inhibited D-lactate energized uptake of lactose but did not inhibit uptake of either L-alanine or L-proline. This inhibition was reversed by intravesicular (but not extravesicular) phosphoenolpyruvate. Lactose uptake was also inhibited by enzyme III^glc preparations that were shocked into the vesicles, and this inhibition was reversed by phosphoenolpyruvate. Intravesicular HPr and enzyme I stimulated methyl α-glucoside uptake but did not inhibit or stimulate lactose accumulation. Vesicles maintained at 0°C for several days partially lost 1) the ability to take up lactose, 2) the ability to accumulate PTS substrates, and 3) PTS-mediated regulation. Phosphoenolpyruvate addition restored all of these activities. These results support a mechanism in which the relative proportions of phosphorylated and nonphosphorylated forms of a phosphotransferase constituent regulate the activity of the lactose permcase.     

Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

The Ability of 2-Deoxyglucose to Promote the Lysis of Streptococcus bovis JB1 via a Mechanism Involving Cell Wall Stability

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (<5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (>85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture superntant. Received: 11 April 1997 / Accepted: 10 June 1997     

Synthesis of recombinant human interleukin-2 via controlled feed of lactose-complex media in fed-batch cultures of Escherichia coli BL21(DE3)[pT7-G3IL2]

Using lactose as an inducer, recombinant human interleukin-2 (rhIL-2) was synthesized with an N-terminus fusion partner, G3 (three tandem-arranged glucagon peptides) in fed-batch cultures at high cell concentration (60–90 g l^–1) of Escherichia coli BL21(DE3) [pT7-G3IL2]. With batch additions of lactose (4 × 13.5 g), the fusion rhIL-2 was synthesized up to 9.3 g l^–1. However, if all the lactose (54 g) was added at once to the culture, synthesized fusion rhIL-2 decreased to 5.4 g l^–1 with a decreased cell growth rate. A statistical optimization of the production medium containing glucose, yeast extract, and lactose led to fusion rhIL-2 being produced at > 9 g l^–1.     

Enterotoxin A synthesis in Staphylococcus aureus: inhibition by glycerol and maltose

Studies indicated that prior growth of Staphylococcus aureus 196E on glycerol or maltose led to cells with repressed ability to produce staphylococcal enterotoxin A (SEA). A PTS- mutant (196E-MA) lacking the phosphoenolpyruvate phosphotransferase system (PTS), derived from strain 196E, showed considerably less repression of SEA synthesis when cells were grown in glycerol or maltose. Since SEA synthesis is not repressed in the PTS- mutant, repression of toxin synthesis by glycerol, maltose or glucose in S. aureus 196E appears to be related to the presence of a functional PTS irrespective of whether the carbohydrate requires the PTS for cell entry. With lactose as an inducer, glucose, glycerol, maltose or 2-deoxyglucose repressed the synthesis of beta-galactosidase in S. aureus 196E. It is postulated that these compounds repress enzyme synthesis by an inducer exclusion mechanism involving phosphorylated sugar intermediates. However, inducer exclusion probably does not explain the mechanism of repression of SEA synthesis by carbohydrates.     

Factors Affecting the Antibacterial Activity of the Ruminal Bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h⁻¹) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2–1.2 h⁻¹) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h⁻¹) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h⁻¹) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml⁻¹) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application. Received: 6 February 2002 / Accepted: 27 March 2002     

IPTG can replace lactose in auto‐induction media to enhance protein expression in batch‐cultured Escherichia coli

Auto‐induction media containing glucose, lactose, and glycerol are a simple and efficient approach for high‐throughput protein expression in Escherichia coli with lac‐derived expression systems. Its principle is based on inducer exclusion between glucose and lactose, preventing the induction by lactose before the depletion of glucose. Isopropyl‐β‐d ‐1‐thiogalactopyranoside (IPTG)—at least in typically used millimolar concentrations—is thought to be unsuitable for this purpose since it can enter the cell by diffusion independently of inducer exclusion. In this study, using parallel batch cultivations in stirred‐tank bioreactors on a milliliter scale, we show that the induction by micromolar concentrations of IPTG is prevented in the presence of glucose. With up to 40 μM IPTG, full induction and heterologous protein expression start only after the depletion of glucose. Thus, auto‐induction is possible with either lactose or IPTG, and the expression greatly depends on the type and concentration of the inducer. The best expression of enhanced green fluorescent protein was achieved with 40 μM IPTG in stirred‐tank bioreactors on a milliliter scale. The IPTG‐based auto‐induction was also reproduced in shaking flasks. Therefore, IPTG can be used in auto‐induction media for protein expression in batch‐cultured E. coli. Furthermore, we show that acetate or arabinose can have significant effects on the auto‐induction mechanism.     

Molecular mechanisms controlling fructose‐specific memory and catabolite repression in lactose metabolism by Streptococcus mutans

Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long‐term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose‐phosphate metabolites, F‐1‐P and F‐6‐P, could modulate the DNA‐binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal‐6‐P induced the formation of different LacR‐DNA complexes. Deletion of lacR resulted in strain‐specific growth phenotypes on lactose, but also on a number of mono‐ and di‐saccharides that involve the glucose‐PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose‐PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.     

Assessing Glucose Uptake through the Yeast Hexose Transporter 1 (Hxt1)

The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1^N370A and HXT1^W473A are resistant to such degradation. Hxt1^N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1.     

High-level succinic acid production and yield by lactose-induced expression of phosphoenolpyruvate carboxylase in ptsG mutant Escherichia coli

Escherichia coli strains with foreign genes under the isopropyl-β-d-thiogalactopyranoside-inducible promoters such as lac, tac, and trc were engineered and considered as the promising succinic acid-producing bacteria in many reports. The promoters mentioned above could also be induced by lactose, which had not been attempted for succinic acid production before. Here, the efficient utilization of lactose as inducer was demonstrated in cultures of the ptsG, ldhA, and pflB mutant strain DC1515 with ppc overexpression. A fermentative process for succinic acid production at high level by this strain was developed. In flask anaerobic culture, 14.86 g l⁻¹ succinic acid was produced from 15 g l⁻¹ glucose with a yield of 1.51 mol mol⁻¹ glucose. In two-stage culture carried out in a 3-l bioreactor, the overall yield and concentration of succinic acid reached to 1.67 mol mol⁻¹ glucose and 99.7 g l⁻¹, respectively, with a productivity of 1.7 g l⁻¹ h⁻¹ in the anaerobic stage. The efficient utilization of lactose as inducer made recombinant E. coli a more capable strain for succinic acid production at large scale.     

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria (Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic.     

Production and physicochemical characterization of β-glucan produced by<Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JB115

This study was conducted to develop a bacterial glucan as an animal feed additive. A novel glucan-producing bacterium.Paenibacillus polymyxa JB115, was isolated from Korean soil. The glucan, JB115-BG, produced byP. polymyxa JB115, was confirmed by TLC to be composed of glucose only. By examining FT-IR,¹H NMR, and¹³C NMR spectra, it was proven that JB115-BG has a β-(1→3)- and β-(1→6)-linked glucan structure. The particle size of JB115-BG was distributed in the range of 4–800 μm, with a mean value of 149.1 μm, and its molecular distribution ranged from 6.9∼3,103.7 kDa. It was also observed that 80% of the purified JB115-BG had a molecular distribution above 100 kDa. The obtained results suggest that the glucan JB115-BG can be used as an animal feed additive for the purpose of enhancing immunity.