O0 and strong-polar mutations in the gal operon are insertions

Summary Three dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal ⁺ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.     

Negative control of the galactose operon in E. coli

Summary Non-inducible mutants have been isolated which synthesize the three galactose enzymes with the basal rate both in the absence and in the presence of inducers. These mutations are closely linked to the lysA gene, as are the constitutive mutations in the regulator gene first described by Buttin (1963).The non-inducible mutants are Gal ^– on EMB gal plates. Revertants to the Gal ⁺ phenotpye are constitutive. Heterozygotes have been prepared at the locus of the regulator gene (galR), abd dominance studies involving the different alleles at this locus have been carried out. The non-inducible mutations are dominant over the wildtype, and this in turn is dominant over constitutive mutations in the galR gene.Starting from the non-inducible mutations, deletions have been isolated, which extend from the galR gene into the lysA gene. These are constitutive.The behavior of the non-inducible mutations and of the deletions are strong arguments for negative control of the galactose operon.     

Insertion mutations in the control region of the Gal operon of E. coli. I. Biological characterization of the mutations

Summary Galactose negative mutations are described which reduce the maximum expression of all three gal genes about 100-fold. The residual enzyme synthesis is not or only slightly inducible.These pleiotropic mutations map in the control region of the gal operon. No recombination is observed between these mutations. All mutants revert spontaneously to a Gal⁺ phenotype. In some mutations wildtype-like as well as constitutive revertants are obtained. The frequency of reversion can be increased by nitrosoguanidine (NG) in all mutants. The revertants, induced by this mutagen, are of a constitutive type.     

Analysis of relative reversion frequencies for IS2 insertion mutations in the regulatory region of the galOPETK operon of Escherichia coli

Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP-3 and galOPE-490, contain IS2 insertions only 1 bp apart in the gal regulatory region; yet only the former yields Gal⁺ phenotypic revertants at a detectable frequency. We have shown that the galOPE-490 allele comprises two mutations—an IS2(I) insertion at bp+(2–6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp+59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490^*) which retains the IS2 of galOPE-490 but is galE⁺ reveals a reversion frequency approximately 30-fold higher than that of galOP-3. Reversion of galOPE-490 is at least 10,000-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems.     

Oo mutations in the galactose operon in E. coli

Summary Eleven mutants lacking the three enzymes of galactose fermentation were investigated.Eight of the mutants revert spontaneously to the Gal ⁺ phenotype. These cannot be deletions. Six of these spontaneously reverting mutants do not respond to the mutagens 2-aminopurine, ethyl-methanesulfonate and N-Methyl-N-Nitro-N-nitrosoguanidine. It is concluded that these o ^o mutations cannot be reverted by base substitution.The eleven o ^o mutants are not of the amber or ochre type as shown by their behaviour towards suppressor genes.The possible nature of the mutations is discussed.     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

Strong-polar mutations in the transferase gene of the galactose operon in E.coli

Summary A class of mutations in the transferase gene of the galactose operon in E. coli is described, which is strongly polar for the synthesis of kinase. The latter enzyme is made only to the extent of about 0.1% of the amount made in the induced wildtype. This amount is not dependent on the map position of the mutations and the residual synthesis is non-inducible. The mutants thus resemble 0° mutants in the same operon.Epimerase, which is coded for by the gene proximal to the transferase gene with respect to the operator, is made in normal amounts and its synthesis is normally inducible.The mutants do not seem to belong either to the nonsense or to the frameshift class on the basis of reversion pattern, suppressibility, and degree of polarity. The possible nature of the mutations is discussed.     

Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments

We have worked out a system to obtain mutations that map in the promoter region of the Escherichia coli galactose operon. In order to easily detect small changes in gal promoter activity, we constructed a plasmid containing an operon fusion in which the lactose operon structural genes were controlled by the galactose operon promoter region. In cells harbouring this plasmid, even modest variations in the expression of the lac genes could be detected on MacConkey lactose indicator plates.Enrichment for mutations that map in the promoter segment of the galactose operon was achieved by mutagenesis in vitro of a small fragment of DNA covering the promoter region. After insertion of the mutagenized gal promoter fragment into the gal-lac fusion plasmid, lac^?1 cells were transformed and screened for an altered Lac⁺ phenotype on indicator plates. Several mutants were isolated due to lesions mapping in the small fragment covering the galactose promoter. In these mutants, the level of β-galactosidase was between 15 and 50% of the wild-type level.The mutant promoters were subsequently reinserted into a plasmid containing the intact galactose operon. Cells harbouring such plasmids, reconstituted with mutant galactose promoters, contained decreased levels of galactokinase that paralleled the decreases in β-galactosidase. The biochemical properties of these mutants are reported in the accompanying paper (Busby et al., 1982).     

Lactose Uptake Driven by Galactose Efflux in Streptococcus thermophilus: Evidence for a Galactose-Lactose Antiporter

Galactose-nonfermenting (Gal^-) Streptococcus thermophilus TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal^- cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated [¹⁴C]lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force (Δp) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a Δp of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in Gal^-S. thermophilus is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system.     

Specialized transduction of the biotin region of Escherichia coli by phage T1.

Summary Phage T1 transduces Bio⁺ by special mechanism which leads to a higher efficiency of Bio⁺ transduction than other bacterial markers. Efficient Bio⁺ transduction depends on a site located between the galactose operon and the bacterial attachment site for phage . Evidence is presented which supports the hypothesis that the site is essential for efficient Bio⁺ transduction because at the site phage T1 initiates head filling in a polar (unidirectional) fashion leading to increased pickup of the Bio⁺ marker.     

Second-element turn-on of gene expression in an IS1 insertion mutant

Summary To learn more about the ways in which genes silenced by insertion mutations can be reactivated, we have undertaken a systematic investigation of Gal⁺ revertants of the polar mutant galOP-306::IS1 in Escherichia coli K12. The selective conditions used excluded reversion to wild type by precise excision of IS1. In this system (which resisded on a multi-copy plasmid) reversion to the Gal⁺ phenotype occurred with a frequency of about 10^-7 per cell and per generation. Analysis of the revertants revealed that — with the single exception of the previously published chromosomal mutant sis1 — alterations in the structure of IS1 lead to reactivation of gal operon expression. These events fall into four classes: (I) insertion of IS2 at position 327 in IS1, insertion of IS2 at position 687 in IS1, (III) insertion of a hitherto undetected mobile element, IS150, at position 387, (IV) a 16-bp deletion encompassing IS1 coordinates 553–568. Of some 200 independent reversion events studied, all but one were of types I–III i.e. they involved the intervention of a second mobile element.     

Galactose regulation in Saccharomyces cerevisiae. The enzymes encoded by the GAL7, 10, 1 cluster are co-ordinately controlled and separately translated.

The enzymes for galactose metabolism in Saccharomyces cerevisiae are encoded by three tightly linked genes. Data presented in this paper show that, in contrast to enzymes encoded by other gene clusters in yeast, these three enzymes are translated as separate polypeptides. First, two of the enzymes encoded by the cluster, galactokinase and uridylyl transferase. purified to near homogeneity, are separate polypeptides. Second, no precursor polypeptide-containing sequences common to both these enzymes is detectable in extracts from galactose-induced yeast cells. Third, no partial or absolute polarity of expression of the enzymes is observed in strains containing nonsense mutations in any of the genes of the cluster.Expression of the three galactose metabolic enzymes is co-ordinate, both during induction and during steady-state synthesis. This is true both for wild-type yeast strains and for strains carrying the long-term galactose adaptation mutation, gal3. In GAL3⁺ strains mutations within the galactose gene cluster have no effect on this co-ordinate expression. However, in gal3^? strains, mutations in any of the genes of the cluster completely eliminate expression of the other two genes. These results suggest that the GAL3 gene product is responsible for inducer synthesis and that the actual inducer is an intermediate in galactose metabolism.     

mRNA distal to polar nonsense and insertion mutations in the gal operon of E. coli

Summary mRNA of the galactose operon of E. coli was measured in wildtype E. coli and in gal operon amber and insertion mutants. The mRNA coded by the distal half of the operon is reduced in the mutants. This reduction is more pronounced in the insertion mutants than in the amber mutants. It was compared with the polar effects of the mutations on the enzymes of the operon.     

Integration of IS3 into IS2 generates a short sequence duplication.

Summary The Gal⁺ allele IS2-43 is known to segregate Gal^- clones. Among 11 Gal^- segregants, one was shown to be due to the integration of IS3 into IS2-43. Precise excision of the integrated IS3 element occured at a rate of 5x10^-9/cell/generation. DNA sequence analysis revealed that the termini of the IS3 element have the relation of imperfect inverted repeats and it is now flanked by a 3bp or 4bp duplication, a size which has not been seen before with other elements.     

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

The soluble and insoluble fractions obtained after sonication and centrifugation of Bifidobacterium adolescentis M101–4 cells were examined, and both of these fractions exhibited mitogenic activity in art assay of murine splenocytes and Peyer’s patch cells in vitro. The soluble fraction was further treated by a 6-step procedure involving proteinase K-treatment, ultrafiltration with a 50-kDa cut-off molecular-sieving membrane, anion-exchange chromatography, dialysis, ultrafiltration through a 6-kDa cut-off membrane filter, and gel-filtration to yield a soluble high molecular weight fraction (SHF) which was effective for stimulating the proliferation of murine splenocytes. Almost three quarters of this fraction by weight was found to consist of carbohydrates containing glucose and galactose as major constituents, and the average molecular weight was estimated to be between 60,000 and 2,460,000, with the main peak at 1,550,000 Da, by the retention time of gel permeation chromatography. A structural analysis by ¹H- and ¹³C-nuclear magnetic resonance and methylation indicated that SHF contained polysaccharides consisting of -4Galp1-, -4Glcp1-, and -6Glcp1- as the major residues, and Galf1- and -6Galf1- as the minor residues. Immunopotentiating SHF was found to contain galactofuranosyl residues as characteristic constituents which had not been previously detected in other soluble fractions from Gram-positive bacteria.     

Effects of various inhibitors on β-galactosidase purified from the thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis> subsp. <Emphasis Type="Italic">Rittmannii</Emphasis> isolated from Antarctica

β-Galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica is a member of the GH42 family. The enzyme was not effected by various concentrations of its reaction product glucose, but was greatly inhibited by the other reaction product galactose using both substrates, ONPG and lactose. Linewever-Burk plot analysis derived from both ONPG and lactose hydrolysis results showed that galactose is a mixed-type inhibitor of the purified β-galactosidase. The enzyme was slightly activated by Mg²⁺ (13% at 20 mM), while inhibited at higher concentrations of Ca⁺² (33% at 10 mM), Zn⁺² (86% at 8 mM) and Cu⁺² (87% at 4 mM). The enzyme activity was not significantly altered by the metal ion chelators EDTA and 1,10-phenanthroline up to 20 mM, indicating that this enzyme is not a metalloenzyme. 2-Mercaptoethanol and DTT were found to enhance β-galactosidase activity, while p-chloromercuribenzoic acid (PCMB) completely inhibited enzymatic activity (97% at 1 mM; 99.7% at 2 mM), indicating at least one essential Cys residue modified by the reagents in the active site of β-galactosidase. Iodoacetamide and Nethylmaleimide had little effect on the β-galactosidase. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme strongly (19.8% at 1 mM; 71.9% at 10 mM), also showing the participation of serine for enzyme activity.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Isolation of Rec? mutants from an F-prime merodiploid strain of Escherichia coli K-12

Summary This paper describes a method of screening mutagenised populations of an E. coli gal _A ^– gal _B ^– F-prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal⁺ papillae. With a suitable choice of gal ^–mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.     

Sodium ion transport in isolated intestinal epithelial cells. The effect of actively transported sugars on sodium ion efflux

A technique to measure Na⁺ efflux from isolated intestinal epithelial cells has permitted us to examine the mechanisms responsible for Na⁺ transport in absorptive cells without contamination by other cell types. We examined the effect of actively transported sugars on Na⁺ efflux from isolated rat jejunal epithelial cells to evaluate the mechanism by which actively transported non-electrolytes stimulate Na⁺ absorption. Glucose, galactose and 3-O-methylglucose, sugars known to be actively transported by the small intestine, stimulate total Na⁺ efflux from isolated epithelial cells. This stimulation results from an increase of active Na⁺ transport, since it is inhibited by ouabain. Glucose stimulation is significantly greater than that produced by galactose or 3-O-methylglucose, 2-Deoxyglucose, a sugar that is not actively transported, has no effect on total Na⁺ efflux from isolated cells. Phloridzin, which has no effect on Na⁺ efflux in a sugar-free medium, completely abolishes the effect of galactose. These findings (a) support the hypothesis that the increase in intestinal absorption of Na⁺ in the presence of actively transported non-electrolytes occurs by a transcellular route; and (b) are consistent with the ion-gradient model. The results are not compatible with the direct energy-coupling model.     

Ion regulation in potassium-sensitive mutants of paramecium tetraurelia

Two recessive mutations of Paramecium tetraurelia confer sensitivity to potassium: While wild-type cells survive when up to 30 mM KCI is added to their growth medium, mutants cease to grow and die when levels of added KCl reach 20–25 mM. Similar sensitivities are seen to Rb⁺ and Cs⁺, but not to Na⁺. Swimming behavior of mutants is indistinguishable from wild type when place in stimulating solutions containing Na⁺, K⁺, or Ba²⁺. Behavioral adaptation to low levels of K⁺ also is indistiguishable from wild type. Flame photometry reveals that one mutant is unable to keep out K⁺ when that ion is at high levels in the medium, while the other mutant readily leaks K⁺ and Na⁺ when those ions are at low levels in the medium. Both mutants have markedly lower internal Na⁺ than does wild type. Problem with K⁺ permeability account for the sensitivity of the one mutant to elevated external K⁺, but the basis of sensitivity in the other mutant is unclear. These mutants expand the range of ion regulation mutants in Paramecium and demonstrate that lesions in cellular ion regulation in this organism need not result in changes in swimming behavior.