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Lactose is not itself an inducer of the lac operon, nor is it converted to an inducer by ebg+ beta-galactosidase of Escherichia coli. We report here the isolation of a mutant Ebg beta-galactosidase which is capable of converting lactose into an inducer of the lac operon.
The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.
The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF [V. Monnet, M. Nardi, A. Chopin, M.-C. Chopin, and J.-C. Gripon, J. Biol. Chem. 269:32070-32076, 1994]) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp. cremoris NCDO763. Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp. lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763. From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters. The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases. Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403. Traces of several recombination events are visible on the lactose-proteinase plasmid. This suggests that the duplication of pepF occurred by recombination from the chromosome of an L. lactis subsp. lactis strain followed by gene transfer. We discuss the possible functions of PepF and the role of its amplification.
Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK2), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK2 complex and that MBP plays a crucial role in triggering the transport process.
THE METABOLISM OF LACTOSE WAS FOUND TO BE CONTROLLED BY THREE GENES: a gene for the synthesis of a beta-galactosidase attacking only phosphorylated galactosides; a gene for a protein permitting concentration of phosphorylated galactosides which probably acts by transferring phosphates to them; and a gene regulating the first two structural genes. The three genes are closely linked and may have the same order as in Escherichia coli. Galactose-6-phosphate was found to be a better inducer of lactose utilization than is galactose or any other inducer. The inhibition of induction by isopropylthiogalactoside was found to occur at the level of the protein permitting the concentration of galactoside phosphates.
For six strains of Bifidobacterium bifidum (Lactobacillus bifidus), fermentation balances of glucose, lactose, galactose, mannitol, and xylose were determined. Products formed were acetate, l(+)-lactate, ethyl alcohol, and formate. l(+)-Lactate dehydrogenase of all strains studied was found to have an absolute requirement for fructose-1,6-diphosphate. The phosphoroclastic enzyme could not be demonstrated in cell-free extracts. Cell suspensions fermented pyruvate to equimolar amounts of acetate and formate. Alcohol dehydrogenase was shown in cell-free extracts. Possible explanations have been suggested for the differences in fermentation balances found for different strains and carbon sources. By enzyme determinations, it was shown that bifidobacteria convert mannitol to fructose-6-phosphate by an inducible polyol dehydrogenase and fructokinase. For one strain of B. bifidum, molar growth yields of glucose, lactose, galactose, and mannitol were determined. The mean value of Y (ATP), calculated from molar growth yields and fermentation balances, was 11.3.
Since 1971 a lactose-positive (lac+) Salmonella typhimurium variety Copenhagen has been endemic in the city of Sao Paulo. The strain is a strong lactose fermenter and resembles Escherichia coli on primary plating media and in triple sugar iron agar. Although most isolates of the strain have uniform properties, some have slightly different antigens, antibiograms, phage types, or fermentation patterns. Most isolates have come from stools of infants under 1 year of age and are probably hospital acquired; however, other isolates are probably community acquired. Eighteen other lac+ Salmonella isolated in the United States were also studied. Most of these strains resembled E. coli on primary plates and triple sugar iron agar; thus their identification would pose a problem for most clinical laboratories. A simple procedure for detecting lac+ Salmonella mixed with lac+ E. coli consists of touching 12 colonies in succession with a straight wire and then inoculating a peptone iron agar tube. H2S production is apparent from lac+ Salmonella even if 11 E. coli and one Salmonella colony are picked. If a positive peptone iron agar tube is observed, then individual colonies are tested to rule out other strong H2S producers. The true incidence of lac+ Salmonella is unknown because they are not isolated and identified in most laboratories.
Four hundred strains of lactose-fermenting Enterobacteriaceae were tested for hydrolysis of p-nitrophenyl-beta-D-glucopyranosiduronic acid, the chromogenic enzyme substrate of beta-glucuronidase. Escherichia coli was found to be homogeneous with respect to beta-glucuronidase: more than 94% of the examined E. coli strains were positive, whereas none of the other lactose-fermenting strains possessed beta-glucuronidase activity. The qualitative beta-glucuronidase test, as rapid and simple as the o-nitrophenyl-beta-D-galactopyranosidase test, proved to be of diagnostic value, especially in the identification of E. coli in primary urine cultures. No significant differences were observed in the results of experiments in which either substrate-impregnated disks prepared in the laboratory or commercially available tablets were used.
Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in LAC9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding. We discuss possible functions of the two other regions of homology. The functional implications of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.
The absorption of fat and the absorption and retention of nitrogen were studied in 20 children with kwashiorkor. 13 of the 20 had lactose malabsorption and severe diarrhoea on milk feeds. Fat absorption was not affected by lactose-induced diarrhoea, but nitrogen absorption was impaired. Despite this, nitrogen retention was unaffected and it is concluded that milk and milk products would continue to be used in programmes to eliminate malnutrition.
With the use of a staining method by which cells in the urine can be differentiated, the effect of oral frusemide, lactose, and urea on the rate of excretion of these cells was investigated in five healthy persons.It is shown that frusemide greatly increases the urinary excretion of red cells, white cells, and renal tubular cells. Similar though not so marked changes were produced by both lactose and urea. Possible reasons for the increased excretion of cells are discussed. One is that it may be the result of an increase in the rate of urinary flow.
A lactose fermenting strain of Salmonella typhimurium was isolated from two calves which died during an outbreak of acute enteritis. The organism was biochemically typical in all other respects. In one calf, uncomplicated by treatment before death, the autopsy findings were those of a severe fibrinous enteritis which was reproduced in another calf dosed orally with culture. Attention is drawn to scattered reports of lactose fermenting salmonelle causing morbidity and mortality in calves and man.
To determine whether prolactin (PRL) could be conclusively demonstrated in human milk, samples from nine puerperal women were examined by radioimmunoassay techniques. Recovery of 78 +/- 5% (mean +/- SE) PRL added to eight milk samples and mean recoveries of 83, 92, and 92% of PRL after 1, 2, and 3 h incubation at 21 degrees C indicated that quantitative recovery was possible. PRL immunoreactivity was not lost by dialysis for 96 h. Serial dilution of milk samples before and after dialysis gave inhibition curves parallel to PRL standards. Milk PRL concentrations were high for the first 3 days after birth, with a peak mean value of 157 +/- 18 ng/ml (SE) on the third day. Milk PRL levels then fell sharply to a mean of 24 ng/ml 13 days after delivery. Milk PRL concentrations showed a significant negative correlation (P less than 0.001) with milk lactose (r = -0.59) and positive correlations (P less than 0.01) with total milk protein (r = 0.49) and alpha lactalbumin (r = 0.33) estimations. We conclude that i) PRL is a normal constituent of human milk, ii) high concentrations of PRL are present in human milk for the first 3 days postpartum but subsequently fall rapidly, and iii) milk PRL levels correlate significantly with milk lactose, total protein, and alpha-lactalbumin values.
Enzymatic activity, biosynthesis, and maturation of lactasephlorizin hydrolase (LPH) were investigated in adult volunteers with suspected lactose intolerance. Mean LPH activity in jejunal biopsy homogenates of these individuals was 31% compared to LPH-persistent individuals, and was accompanied by a reduced level of LPH-protein. Mean sucrase activity in individuals with low LPH was increased to 162% and was accompanied by an increase in sucrase-isomaltase (SI)-protein. Biosynthesis of LPH, SI, and aminopeptidase N (APN) was studied in organ culture of small intestinal biopsy specimens. In individuals with LPH restriction, the rate of synthesis of LPH was drastically decreased, reaching just 6% of the LPH-persistent group after 20 h of culture, while the rate of synthesis of SI appeared to be increased. In addition, maturation of pro-LPH to mature LPH occurred at a slower rate in LPH-restricted tissue. Immunoelectron microscopy revealed an accumulation of immunoreactive LPH in the Golgi region of enterocytes from LPH-restricted individuals and reduced labeling of microvillus membranes. Therefore, lactose intolerance in adults is mainly due to a decreased biosynthesis of LPH, either at the transcriptional or translational level. In addition, intracellular transport and maturation is retarded in some of the LPH-restricted individuals, and this leads to an accumulation of newly synthesized LPH in the Golgi and a failure of LPH to reach the microvillus membrane.
The identity of a sulfate ester in rat milk has been determined to be N-acetylneuramin lactose sulfate. This sulfate ester is present in rat mammary tissue and in human milk. The presence of this compound offers an explanation for the simultaneous delivery of sulfate and calcium via the milk, two essential nutrients in early life, without precipitation of calcium sulfate in the milk. N-acetylneuramin lactose sulfate is hydrolyzed in the gut of the neonate and absorbed as inorganic sulfate. This is the first report suggesting that this ester may be of nutritional importance.
The modified extended Hansen method was tested for the first time to determine partial solubility parameters of non-polymeric pharmaceutical excipients. The method was formerly tested with drug molecules, and is based upon a regression analysis of the logarithm of the mole fraction solubility of the solute against the partial solubility parameters of a series of solvents of different chemical classes. Two monosaccharides and one disaccharide (lactose monohydrate, saccharose and mannitol) were chosen. The solubility of these compounds was determined in a series of solvents ranging from nonpolar to polar and covering a wide range of the solubility parameter scale. Sugars do not absorb at the UV-vis region, and the saturated solutions were assayed with a recent chromatographic technique coupled to an evaporative light scattering detector. This technique was suitable to determine the concentration dissolved in most solvents. The modified extended Hansen method provided better results than the original approach. The best model was the four parameter equation, which includes the dispersion delta d, dipolar delta p, acidic delta a and basic delta b partial solubility parameters. The partial solubility parameters obtained, expressed as MPa1/2, were delta d = 17.6, delta p = 28.7, delta h = 19, delta a = 14.5, delta b = 12.4, delta T = 32.8 for lactose, delta d = 16.2, delta p = 24.5, delta h = 14.6, delta a = 8.7, delta b = 12.2, delta T = 32.8 for mannitol and delta d = 17.1, delta p = 18.5, delta h = 13, delta a = 11.3, delta b = 7.6, delta T = 28.4 for saccharose. The high total solubility parameters delta T obtained agree with the polar nature of the sugars. The dispersion parameters delta d are quite similar for the three sugars indicating that the polar delta p and hydrogen bonding parameters (delta h, delta a, delta b) are responsible for the variation in the total solubility parameters delta T obtained, as also found for drugs. The results suggest that the method could be extended to determine the partial solubility parameters of other non-polymeric pharmaceutical excipients.
Eleven oligosaccharides formed by a transglycosylation reaction during lactose hydrolysis with Bacillus circulans beta-galactosidase were purified by gel permeation chromatography, charcoal chromatography, and HPLC. From the results of methylation analysis, and MS and NMR studies, it was concluded that these oligosaccharides were beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->2)-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-D-Glc, beta-D-Galp-(1-->6)-[beta-D-Galp-(1-->2)]-D-Glc, beta-D-Galp-(1-->6)-[beta-D-Galp-(1-->4)]-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->2)-D-Glc, beta-D-Galp-(1-->4)-[beta-D-Galp-(1-->2)]-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->6)[beta-D-Galp-(1-->3)]-D-Glc. The last five are newly observed oligosaccharides. The results of a use test (in vitro) by human intestinal bacteria showed that the oligosaccharides containing lactose units were predominantly used by human intestinal bifidobacteria.
A thermostable beta-glucosidase from Thermus sp. Z-1 that not only hydrolyzes beta-glucosides but also beta-galactosides was shown to efficiently produce oligosaccharides during hydrolysis of lactose. The yield of oligosaccharides was more than 40% for 0.88 M lactose solution at 70 degrees C at pH 7.0. The major product was a trisaccharide, 3'-galactosyllactose, formed by a galactosyltransfer reaction.