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alpha-p-Nitrophenylgalactoside was found to be accumulated by the lactose transport-system of Escherichia coli. This fact may help to resolve the differences in the reported number of sugar binding sites of the lactose transport protein in nonenergized and energized membrane vesicles.
The kinetic parameters Km and V of transported by the lactose permease of Escherichia coli have been explored in the pH range 4.8--9.2. Besides uphill transport of methylthiogalactoside, two other criteria have been used. Downhill transport of o-nitrophenylgalactoside and substrate protection of the carrier against thiol reagents have both been explored in normal aerated cells and in cells inhibited by cyanide plus azide, therefore unable to build up a proton-motive force. V of the transport processes did not exhibit a major pH dependence that would support an essential protonation. Ktransport for methylthiogalactoside and for o-nitrophenylgalactoside in the energized and in the inhibited state did not show a sharp pH dependence between pH 4.8 and 8.0, but increased between pH 8 and 9, as would be expected if there were an essential protonated group with a pK of 8--8.4, depending on the test utilized. Substrate protection allowed the calculation of a Kprotection which was close to the corresponding Ktransport and was also largely independent of pH between 5 and 8 and independent of energy supply. The role of energization in substrate-carrier binding and the role of the essential protonation in the context of the proton symptom model are discussed.
Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog beta-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K(m) for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V(max)) of normal. All of the mutants accumulated methyl-alpha-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.
The process by which an organism changes the composition of its membranal fatty acids in response to growth temperature, so as to maintain optimal membrane functioning, is known as homeoviscous adaptation (HA). One expression of HA is the constancy of the fluorescence polarization (P) of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of cells grown at various temperatures. The P of DPH in the membranes of Escherichia coli was shown by us to be inversely proportional to bacterial growth rate on different carbon sources. This result, implying failure of HA, is now complemented by measurements of DPH lifetimes, which indicate that the dominant variables contributing to the drop in P are (a) the order parameter of the membrane, which goes down, and (b) the fluidity, which may slightly increase. These are then the changes induced by enhanced growth rate. Two additional effects, cell membrane permeability and sensitivity to thermal shock, determined by the diffusion of o-nitrophenylgalactoside (ONPG) and by exposure to 52 degrees C, respectively, are reported to increase with growth rate. We can now conclude that there is a deviation from the principle of HA in E. coli grown at various rates, brought about by controlling the growth media at constant temperatures.
We have studied the potential of several newly cloned alpha-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor. The kinetics of the reactions were followed by in situ NMR spectroscopy. The following thermophilic enzymes have been tested: Aga A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an alpha-galactosidase from Thermus brockianus (strain IT360). Two other non-thermophilic alpha-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enzymes studied, high regioselectivity was observed leading to two (1 --> 6)-disaccharides: 4-nitrophenyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside and methyl alpha-D-galactopyranosyl-(1--> 6)-alpha-D-galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.
During inflow into Escherichia coli substrates must first diffuse through the porin pores in the outer membrane by simple, passive, diffusion and then be translocated across the inner membrane by a specific (active) carrier protein, or permease. A graphical procedure is outlined whereby it is possible to estimate the concentration drop across the outer membrane from simple kinetic measurements of net inflow velocity. Experiments confirm that the concentration drop across the outer membrane is proportional to rate of inflow, as expected from Fick's law. The expected rate of diffusion of 2-nitrophenylgalactoside through the outer membrane was calculated from reported values of pore radius, length and number, and the rate was found to correspond closely with the experimental results. It is pointed out that at low substrate concentrations the outer membrane is rate-limiting and that a large increase in the amount of permease in the inner membrane will cause very little change in net inflow velocity.
Using cells that overproduce DNA photolyase, we found that UV irradiation (3 J/m2) efficiently inactivates accumulation of methylthiogalactoside (TMG) when RexAB proteins of phage lambda are present. The effect requires both formation of photolyase-dimer-DNA (PDD) complexes and expression of the RexAB proteins. It is reversed completely by a flash of visible light if given immediately after UV and becomes irreversible after post-UV incubation for about 15 min. Inactivation is significant after only 5 min of post-UV incubation, is accompanied by a loss of previously accumulated TMG, and does not require de novo protein synthesis. Passive transport of O-nitrophenylgalactoside by inactivated cells is typical of energy-depleted membranes. We suggest that PDD complexes mimic a developmental intermediate of phage superinfection and stimulate formation of the RexB membrane channel recently proposed by others to explain classical "exclusion". This suggestion is supported by additional data showing an inactivation of colony-forming ability by exclusion stimulation and an inability of PDD complexes to inactivate accumulation of TMG if RexB is present in larger relative amounts than RexA (a detail characteristic of exclusion stimulated by phage superinfection).
A model is proposed for the D-galactoside-H+(-OH) transporter of Escherichia coli that accounts for essentially all the experimental observations established for this system to date. In this model, the functional unit is postulated to be a dimer (consisting of two copies of lac Y-specified polypeptide) which spans the membrane with a 2-fold symmetry axis in the membrane plane (Lancaster, J.R. (1978) J. Theor. Biol. 75, 35-50). The functional dimer is assumed to possess a single pore flanked by an inner gate (gi) and an outer gate (go) and encompassing two oppositely oriented galactoside binding sites, designated m and mu. When go is open and gi is closed under non-energized conditions, binding site m adopts a configuration defined as State A (i.e., moA) exhibiting high affinity toward Class Ga galactosides (thiodigalactoside, melibiose, alpha-p-nitrophenygalactoside) but low affinity for Class Gb galactosides (lactose, beta-o-nitrophenylgalactoside, beta-isopropylthiogalactoside), whereas binding site mu adopts State B (i.e., muoB) displaying relatively high affinity toward Class Gb galactosides but comparatively low affinity for Class Ga galactosides; further, each moA : muoB dimer contains one thiol group whose reaction with N-ethylmaleimide inactivates the transporter unless blocked by galactoside binding at site moA, while the second homologous thiol of the dimer is unreactive toward thiol reagents. Translocation of the moA : muoB dimer involves closing of go followed by opening of gi, and causes the two thiols (as well as sites m and mu) to interchange roles in a symmetrical fashion: moA : muoB in equilibrium miB : muiA. In the presence of a substantial (negative) transmembrane delta potential of muH+, the m : mu dimer is postulated to undergo an electrogenic protein conformation change to a second form, *(m : mu), in which both sites m and mu possess low affinity toward internal Class Gb substrates; galactoside transport in both m : mu and *(m : mu) is assumed to be coupled to H+-symport (-OH-antiport) with a stoichiometry of approximately 1 : 1. Finally, five characteristic predictions of the half-sites model are outlined for further tests of its validity.
A comparison has been made between the physiology and amino acid sequence of the lactose carriers of Klebsiella pneumoniae M5a1 and Escherichia coli K-12. The membrane transport of lactose was much weaker in Klebsiella than in E. coli. On the other hand o-nitrophenylgalactoside uptake by Klebsiella was distinctly greater than with E. coli. In spite of the differences in sugar transport between the two organisms, the amino acid sequences of the respective lactose carriers were remarkably similar (60% of the amino acids are identical).
Seminal plasmin (SPLN) a 47-residue peptide, isolated from bovine seminal plasma, exhibits antibacterial activity against Gram-positive and Gram-negative bacteria. Although SPLN strongly inhibits the transcription of various natural and synthetic templates by E. coli RNA polymerase in vitro, it also associates with model membranes of phosphatidylcholine and phosphatidic acid. We have undertaken experiments to ascertain whether SPLN permeabilizes the bacterial inner membrane and thereby exerts its antibacterial activity, as in the case of recently isolated antibacterial peptides from mammalian sources. Our results show that SPLN affects the permeability properties of the bacterial inner membrane which is reflected by increased uptake of ortho-nitrophenylgalactoside (ONPG), which can normally be translocated only by protein transporters. SPLN has also been shown to act on the outer membrane, since divalent cations inhibit antibacterial activity.
The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.
The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or alpha-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.
The sugar specificity properties of the lactose permease were investigated. Free galactose was shown to competitively inhibit the lactose permease yielding a Ki value of 7.4 mM. This value was severalfold higher than the observed Km for lactose (1.3 mM). A variety of other monosaccharides also showed significant inhibition of lactose transport. With regard to -OH groups along the galactose ring it appears that the relative importance is OH-3 greater than OH-4 greater than OH-6 greater than OH-2 greater than OH-1. In general, galactosides with alpha-linkages exhibited significantly higher affinities compared with their beta-linked counterparts. An optimal size for the aglycone portion of the galactoside was reached with aglycones containing hexose residues or a benzene ring. The preferred size of the aglycone appears to be hexose, benzene ring greater than methyl group greater than no aglycone much greater than disaccharide greater than trisaccharide. However, neither the specific structure of the aglycone nor its relative hydrophobicity appeared to be important factors in permease recognition. For example, the hydrophobic beta-nitrophenyl-galactosides had lower affinities compared with lactose (a beta-galactoside), whereas the alpha-nitrophenylgalactosides generally had higher affinities compared with melibiose (an alpha-galactoside). In addition, no consistent preference was seen when considering the location of the nitro group on the benzene ring. From this work, a model is presented which depicts the binding of galactosides to the lactose permease.
beta-Galactosidase has been isolated from Lactobacillus helveticus of a strain isolated from natural starters for the manufacture of Argentine hard cheeses and its properties have been studied. The enzyme was purified 14-fold (by chromatography on DEAE-cellulose and Sepharose 6B-DEAE-cellulose columns and by affinity chromatography in agarose-p-aminophenyl-beta-D-thiogalactoside). The purified extract exhibited a single band following polyacrylamide gel electrophoresis. Maximum enzymatic activity was observed at 42 degrees C and pH 6.5 in 50 mM phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The Km's for o-nitrophenylgalactoside (ONPG) and ONPG + 10 mM of lactose were 4.46 X 10(-5) and 8.9 X 10(-5) M, respectively. Glucose, galactose, galactose 6-phosphate, and lactate acted as noncompetitive inhibitors; MgCl2 protected the enzyme from thermal denaturation. The activation energy of enzymatic hydrolysis of ONPG was 11,400 cal/mol. The Mr was estimated to be 250,000. It is an oligomeric enzyme made of 4 subunits of Mr 65,000.
Glucose transport and glycolysis are two sequential events which are regulated by both physiological and environmental signals in the yeast Saccharomyces cerevisiae. Transcription of the HXT4 gene was found to be regulated by Gcr1p and Gcr2p, transcription factors that are required for the regulated high level transcriptions of glycolytic genes. Transcription of HXT4 decreased about 35-fold in gcr1 mutant and two-fold in gcr2 mutant yeast cells. However, transcription of other HXT genes was not affected at a significant level by gcr1 or gcr2 mutations. Overproduction of Gcr1p from an inducible promoter resulted in a 15-64% increase in transcription of HXT4, depending on the growth conditions. Gel mobility shift assays performed with the purified DNA binding domain of Gcr1p and the UAS region of the HXT4 gene showed that Gcr1p interacts directly with multiple sites on the HXT4 UAS region. These results indicate that Gcr1p and Gcr2p coordinate the transcription of HXT4 and glycolytic genes.
Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with SsbetaGly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but not that of SsbetaGly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than SsbetaGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki approximately 0.3 M), and at 80 degrees C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsbetaGly, respectively. SsbetaGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsbetaGly, compared with CelB. Overall, CelB and SsbetaGly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at >/=70 degrees C.
The kinetic properties of alpha-galactosidase of Mortierella vinacea were investigated in detail using PNPG (p-nitrophenyl-alpha-D-galactopyranoside) as a substrate. Consequently, the enzyme was markedly inhibited not only by the substrate, but also by the galactose hydrolized. The initial rate of reaction at sufficiently high substrate concentrations, however, did not fall to zero and did approach a finite value. Galactose behaved as a mixed inhibitor and was neither totally competitive nor totally noncompetitive. A rate equation was obtained from a generalized equation derived from a kinetic model which took both the inhibitions into consideration. The constants used in the equation were appropriately estimated. The calculated rate agreed fairly well with the observed initial rate. Moreover, the PNPG hydrolysis progressing in a batch system was found to be approximately representable by simple first order kinetics in which the rate constant was dependent on the initial substrate concentration.
Little is known about techniques for applying untreated microbial cells containing enzymes directly to industrial processes as a biocatalyst. The kinetic behavior of alpha-galactosidase-containing spherical pellets which are formed naturally under given conditions in a submerged culture of Mortierella vinacea was studied on the hydrolysis of PNPG (p-nitrophenyl-alpha-D-galactopyranoside). The effect on intraparticle diffusion on the overall reaction rate was assessed by the use of an effectiveness factor, which was calculated by the approximate solution to the equation derived from the mass balance within a pellet. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including such parameters as pellet size, enzyme concentration in the pellet, and substrate concentration. As the diffusional effect became more significant, the marked substrate inhibition as seen for a free enzyme disappeared gradually. The effect of product inhibition on the pellets was much weaker than that for a free enzyme at a given substrate concentration. In the region of diffusion controlled reaction, it was found that the rate is proportional to the square root of the enzyme concentration in the pellet. In addition, similarly to what was reported previously for a free enzyme,the reaction in a batch system was found to be approximately representable as simple first-order kinetics in which the rate constant was dependent on the initial substrate concentration.