Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella.

Klebsiella sp. strain CT-200 lacks both its plasmid-borne lac operon, which specifies beta-galactosidase I, and its chromosomal lac operon, which specifies beta-galactosidase II, but it expresses a gene for a third beta-galactosidase, beta-galactosidase III, constitutively. CT-200 was examined to determine whether there was a beta-galactoside permease associated with the beta-galactosidase III gene. The failure of CT-200 to transport thiomethyl-beta-galactoside, o-nitrophenyl-beta-D-galactopyranoside, phenyl-beta-galactoside, lactulose, or galactosyl-arabinose was taken as evidence that beta-galactoside permease is not part of a beta-galactosidase III operon. Optimal assay conditions for beta-galactosidase II, whose activity was used as a measure of beta-galactoside transport, are reported here, as are an improved purification method and some physical and catalytic properties of the enzyme not previously reported.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Expression of the lactose transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.     

The lactose carrier of Escherichia coli functionally incorporated in Rhodopseudomonas sphaeroides obeys the regulatory conditions of the phototrophic bacterium

Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton-motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.     

Operon Coordination in Different Bacterial Hosts

Coordination of gene expression in the lac operon was compared in Escherichia coli and Salmonella typhimurium as an approach to detecting possible differences in protein synthesis or membrane structure between organisms. Either a wild-type F' lac pro(AB) episome or the same episome with a polar mutation in one of the lac genes was introduced into pro(-) derivatives of the two strains of bacteria. Activity assays showed that the beta-galactosidase levels were only slightly lower in the S. typhimurium cells than in E. coli cells, whereas the transacetylase levels were significantly higher in S. typhimurium for all of the lac markers tested. Galactoside transport activities were always comparable in the two strains of bacteria; this latter result indicates that the cell envelopes of E. coli and S. typhimurium do not differ sufficiently to affect the membrane-associated lac transport system. It was found, however, that the specific transport activity is very sensitive to culture age in both bacteria, and decreases rapidly in cultures past the mid-exponential phase of growth.     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Physiological studies of beta-galactosidase induction in Kluyveromyces lactis

We examined the kinetics of beta-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. Enzyme activity began to increase 10 to 15 min, about 1/10 of a cell generation, after the addition of inducer and continued to increase linearly for from 7 to 9 cell generations before reaching a maximum, some 125- to 150-fold above the basal level of uninduced cells. Thereafter, as long as logarithmic growth was maintained, enzyme levels remained high, but enzyme levels dropped to a value only 5- to 10-fold above the basal level if cells entered stationary phase. Enzyme induction required the constant presence of inducer, since removal of inducer caused a reduction in enzyme level. Three nongratuitous inducers of beta-galactosidase activity, lactose, galactose, and lactobionic acid, were identified. Several inducers of the lac operon of Escherichia coli, including methyl-, isopropyl- and phenyl-1-thio-beta-d-galactoside, and thioallolactose did not induce beta-galactosidase in K. lactis even though they entered the cell. The maximum rate of enzyme induction was only achieved with lactose concentrations of greater than 1 to 2 mM. The initial differential rate of beta-galactosidase appearance after induction was reduced in medium containing glucose, indicating transient carbon catabolite repression. However, glucose did not exclude lactose from K. lactis, it did not cause permanent carbon catabolite repression of beta-galactosidase synthesis, and it did not prevent lactose utilization. These three results are in direct contrast to those observed for lactose utilization in E. coli. Furthermore, these results, along with our observation that K. lactis grew slightly faster on lactose than on glucose, indicate that this organism has evolved an efficient system for utilizing lactose.     

Glucose-lactose diauxie in Escherichia coli

Growth of Escherichia coli in medium containing glucose, at a concentration insufficient to support full growth, and containing lactose, is diauxic. A mutation in the gene, CR, which determines catabolite repression specific to the lac operon, was found to relieve glucose-lactose but not glucose-maltose diauxie. Furthermore, a high concentration of lactose was shown to overcome diauxie in a CR(+) strain. Studies on the induction of beta-galactosidase by lactose suggested that glucose inhibits induction by 10(-2)m lactose. Preinduction of the lac operon was found to overcome this effect. The ability of glucose to prevent expression of the lac operon by reducing the internal concentration of inducer as well as by catabolite repression is discussed.     

Lactose metabolism in Erwinia chrysanthemi.

Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.     

Elucidation of enzyme control mechanisms using macroscopic measurements in a mixed substrate fermentation system

The objective of this work was to relate macroscopically measurable on-line fermentation parameters such as dissolved oxygen, off-gas oxygen and carbon dioxide, and cell mass, to the controlled production of key intracellular enzymes under carbon limited conditions. Both batch and perturbed batch aerobic fermentations were performed using two different strains of Escherichia coli, with glucose and lactose as the sole carbon sources. The two strains differed from each other only in the lac operon region of their genome. The parent strain, E. coli 3000, was inducible for the enzyme beta-galactosidase. The other strain, E. coli 3300, was a constitutive mutant in the production of beta-galactosidase. In all experiments, off-line assays of sugars and beta-galactosidase activity were performed. It was observed that there is a clear relationship between the macroscopic on-line measurements, dissolved oxygen tension, carbon dioxide evolution rate and oxygen uptake rate, and the microscopic control phenomena of catabolite repression, catabolite inhibition, and inducer repression.     

Selection procedure for mutants defective in the beta-methylgalactoside transport system of Escherichia coli utilizing the compound 2R-glyceryl-beta-D-galactopyranoside

A procedure has been devised that allows selection of mutants defective in the beta-methylgalactoside transport system (mgl) of Escherichia coli. This procedure utilizes the compound 2R-glyceryl-beta-d-galactopyranoside (glycerylgalactoside), which is known to be transported by only two transport system in E. coli, namely, the lactose and the beta-methylgalactoside transport systems. Mutants lacking glycerol-3-phosphate dehydrogenase (glpD) are sensitive to glycerol. Similarly, mutants lacking uridine diphosphate-galactose-4-epimerase (galE) are sensitive to galactose. Glycerylgalactoside is an inducer of the lactose operon and also a substrate for beta-galactosidase. Thus, a mgl(+)glpD galE lacY strain will not grow in the presence of glycerylgalactoside owing to accumulated glycerol-3-phosphate, galactose-1-phosphate, and uridine diphosphate-galactose. We have constructed such a strain and shown that mgl mutants can be obtained by selecting for those that grow in the presence of glycerylgalactoside.     

A mutant Ebg enzyme that converts lactose into an inducer of the lac operon.

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 lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators.

The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino-terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95-amino-acid protein with a sequence similar to the amino-terminal portion of beta-galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis.     

In silico evolved lac operons exhibit bistability for artificial inducers, but not for lactose

Bistability in the lac operon of Escherichia coli has been widely studied, both experimentally and theoretically. Experimentally, bistability has been observed when E. coli is induced by an artificial, nonmetabolizable, inducer. However, if the lac operon is induced with lactose, the natural inducer, bistability has not been demonstrated. We derive an analytical expression that can predict the occurrence of bistability both for artificial inducers and lactose. We find very different conditions for bistability in the two cases. Indeed, for artificial inducers bistability is predicted, but for lactose the condition for bistability is much more difficult to satisfy. Moreover, we demonstrate that in silico evolution of the lac operon generates an operon that avoids bistability with respect to lactose, but does exhibit bistability with respect to artificial inducers. The activity of this evolved operon strikingly resembles the experimentally observed activity of the operon. Thus our computational experiments suggest that the wild-type lac operon, which regulates lactose metabolism, is not a bistable switch. Nevertheless, for engineering purposes, this operon can be used as a bistable switch with artificial inducers.     

Selective inhibition of carbohydrate transport by the local anesthetic procaine in Escherichia coli.

Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli. Procaine mildly inhibited growth on lactose. The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease. However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities. In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited. Coordinate inhibition of soluble and membrane products was not observed with the lac operon. beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced. Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction.