In Vivo Complementation Between Wild-Type and Mutant β-Galactosidase in Escherichia coli

A comparison of the specific activity of wild-type beta-galactosidase synthesized in a lacZ(-)/lacZ(+) heterogenote has shown that there is 60% more activity in the heterogenote's enzyme than can be accounted for by wild-type subunits alone. It is concluded that wild type beta-galactosidase subunits can complement mutant subunits.     

High-Level Production of β-Galactosidase by Escherichia coli Merodiploids

Two merodiploids of Escherichia coli that contain genes for the lac operon on both chromosome and episome were tested for production of lac enzymes after growth on various carbon sources. The specific activity of beta-galactosidase (and of thiogalactoside transacetylase) was about twice that from haploid cells when grown on glycerol. With succinate as carbon source, the specific activity increased by an additional factor of 3. Up to 25% of the soluble cell protein is beta-galactosidase in these strains, one of which is inducible and the other constitutive. The enzyme is purified easily in high yield by ammonium sulfate fractionation and electrophoresis.     

β-Galactosidase from Termination and Deletion Mutant Strains

β-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the β-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg⁻) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg⁻ and Deg⁺ strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.     

The lon Gene and Degradation of β-Galactosidase Nonsense Fragments

N-terminal beta-galactosidase fragments are rapidly degraded in growing cells of Escherichia coli. Mutations in the lon gene are sufficient to enhance the stability of these polypeptides.     

Effect of Galactose on β-Galactosidase Synthesis in Escherichia coli K-12

In wild-type strains of Escherichia coli K-12, the rate of thiomethylgalactoside (TMG)-induced beta-galactosidase synthesis is decreased in the presence of galactose or glucose. A spontaneous mutant of a K-12 strain, 58-161, which synthesizes beta-galactosidase at a low rate was isolated. In this mutant, galactose, after a lag of about one generation time, evoked the same final differential rate of enzyme synthesis as did the gratuitous inducer TMG. However, constitutive, TMG-induced and galactose-induced synthesis in the mutant were subject to inhibition by exogenous glucose. It is concluded that repression of beta-galactosidase synthesis derived from glucose is distinct from the inhibition derived from galactose.     

Lysis of Escherichia coli by Ethylenediaminetetraacetate and Phospholipases as Measured by β-Galactosidase Activity

A permeaseless mutant of Escherichia coli, which produces beta-galactosidase constitutively, was treated briefly with ethylenediaminetetraacetate and then with the phospholipases of Bacillus cereus. Cell lysis occurred, as indicated by an increase in beta-galactosidase activity and a decrease in absorbancy of the cell suspension. The susceptibility of the cells to attack by ethylenediaminetetraacetate and the phospholipases was markedly affected by the age of the cells when harvested. The results suggest that permeability changes may be associated with the activity of a phospholipase that specifically degrades phosphatidyl ethanolamine. A sonic-treatment method for determining the total beta-galactosidase content of E. coli cells, which is independent of their age when harvested, is described.     

β-Galactosidase of Propionibacterium shermanii

Ten strains of Propionibacterium shermanii were tested for beta-galactosidase (beta-gal) activity. Of these ten strains, five yielded enhanced enzyme activity when cell suspensions were treated with toluene-acetone; on solvent treatment, the remaining five lost a considerable portion of the activity found in whole-cell suspensions. By using a strain yielding decreased activity upon solvent treatment, explanations for the loss in activity were sought through assays for possible alternative beta-galactoside utilization mechanisms. When this strain was assayed for beta-D-phosphogalactoside galactohydrolase by using orthonitrophenyl-beta-D-galactopyranoside-6-P04 as a substrate, the activity was wither lower or indiffernt as compared with beta-gal activity determined simultaneously. Cell suspensions of P. shermanii 7 and 22 (strains chosen for further work) grown separately on the individual substrates (lactose, glucose, galactose, and sodium lactate) did not show significant differences in beta-gal activity. Optimal temperature for beta-gal activity in untreated and toluene-acetone-treated cell suspensions of strain 7 was 52 C. With strain 22, of the temperatures tested, maximal activity in untreated cell suspensions was noted at 58 C and with solvent-treated cells at 32 C. In the cell-free extract (CFE) system, both strains exhibited maximal activity at 52 C. Optimal pH for untreated and solvent-treated cell suspensions of both strains was around 7.5. In the P. shermanii 22 CFE system, maximal activity occurred at pH 7.0; pH had very little effect on enzyme activity in P. shermanii 7 CFE. Sodium or potassium phosphate buffers in the assay system yielded the best activity. In the CFE system of these two strains, Mn2+ was definitely stimulatory, but in untreated and solvent-treated cell systems of these strains presence or absence of Mn2+ in the assay system had variable effects on enzyme activity. Maximal beta-gal activity was noted in P. shermanii 7 cells harvested after 28 h of growth at 32 C in sodium lactate broth. Sulfhydryl-group blocking agents inhibited enzyme activity in P. shermanii 22 CFE; the inhibition was partly reversed by dithiothreitol.     

Nature of Ribosome-Bound β-Galactosidase

Properties of the ribosome-bound β-galactosidase were examined in Escherichia coli cells after prolonged induction. This fraction of enzyme was not chased from ribosomes by removal of inducer, or by treatments with hydroxylamine, puromycin, chloramphenicol, and azide. However, the metabolic turnover of this fraction could be demonstrated by means of a pulsed exposure to the phenylalanine analogue β-2-thienylalanine, and this fraction was enriched in heavy forms relative to the soluble enzyme. These observations indicated a tight coupling of the release of ribosome-bound enzyme to nascent enzyme synthesis, and it is suggested that the ribosome-bound enzyme is related to an intermediate stage in the assembly of quarternary enzyme structures.     

Modulation of In Vivo Migratory Function of α2β1 Integrin in Mouse Liver

We report herein that expression of α2β1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that α2β1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of α2β1 on KX2C2 and RDX2C2 cells using a α2β1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated α2β1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn²⁺, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that α2β1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that α2β1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.     

Localization of β-Galactosidase in Cells of Escherichia coli by Low Voltage Electron Bombardment

By using low voltage electrons to bombard dried cells of Escherichia coli, the inactivation of the enzyme β-galactosidase as a function of depth of electron penetration has been studied. There is little inactivation for a penetration of 100 A, but considerable for a penetration of 300 A. An analysis of the data for six initial electron energies shows that there exist outer and inner bounds of the enzyme region which are approximately 300 and 700 A below the cell surface, respectively.     

Kinetic Studies of β-Galactosidase Induction

The kinetics of β-galactosidase induction in E. coli ML 3 have been studied. Following addition of inducer, the rate of enzyme synthesis accelerates from the uninduced to a steady-state rate. At saturating concentration of inducer the time constant (T_c) for this process is 2.5 to 3 minutes. With decreasing inducer concentration (I), increasing time constants are observed. I/I + K′ approximates I/T_c. The steady-state rate of β-galactosidase synthesis is approximated by I²/I² + K². K′ and K have been estimated for IPTG and TMG. The kinetics of β-galactosidase production after the removal of inducer by dilution or after the addition of glucose have been investigated. A transition time of 2.5 to 3 minutes is observed before enzyme synthesis slows or stops. These results are consistent with the hypothesis that the enzyme-forming unit is unstable.     

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR₉₂-1-216 (normally used as the source for α- and β-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase K_m for α-ketoisovaleric acid, 6 × 10⁻⁵ M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K_m for α-ketoisovaleric acid, 1.1 × 10⁻³ M; transaminase B K_m for α-ketoisovaleric acid, 2 × 10⁻³ M).     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

β-β′ Subunits of Ribonucleic Acid Polymerase in Episome-Free Minicells of Escherichia coli

Episome-free minicells of Escherichia coli, previously shown to lack ribonucleic acid polymerase activity, do contain the beta-beta' subunits of the polymerase.     

Comparative Study of Isoenzyme Formation of Bacterial β-Galactosidase

The enzyme beta-galactosidase was studied in crude extracts of Escherichia coli 3300, E. coli grown on a selenium and sulfur medium, Salmonella typhimurium F-lac, Serratia marcescens F-lac, S. marcescens P-lac, Proteus mirabilis F-lac, P. mirabilis P-lac, Aeromonas formicans, and Streptococcus lactis. The isoenzymes could be demonstrated by an alternative histochemical technique. Different isoenzyme patterns were found to be determined by the beta-galactosidase structural gene and not by the cytoplasm within which the beta-galactosidase was formed. In addition, the beta-galactosidases from strains which form isoenzymes were more stable to heat and urea treatments than the enzyme formed by those organisms which produce reduced amounts of, or no, isoenzyme.     

Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.