L-arabinose binding protein from Escherichia coli B-r

A protein which is capable of binding l-arabinose-1-(14)C has been isolated from l-arabinose-induced cultures of Escherichia coli B/r. Analysis for this l-arabinose-binding protein (ABP) in a number of l-arabinose-negative mutants suggests that the ABP is not coded for by any of the known genetic units of the l-arabinose complex yet is under the control of the regulator gene araC. The ABP has been purified and found to bind l-arabinose, d-fucose, d-xylose, and l-ribulose with decreasing affinities. The K(m) for l-arabinose is 5.7 x 10(-6)m. The molecular weight, as determined by equilibrium centrifugation, was found to be 32,000. The protein was observed to have many features that liken it to other recently isolated binding proteins that have been implicated in the active transport of small molecules.     

The L-arabinose-resistance test with salmonella typhimurium strain SV3 selects forward mutations at several ara genes

A new assay has been described for mutagenicity testing using an l-arabinose-sensitive strain of Salmonella typhimurium. The test strain SV3 and several l-arabinose-resistant mutants selected therefrom are characterized in the present study by 3 different criteria: inhibition of growth by l-arabinose, accumulation of keto-sugars, and activities of the enzymes involved in l-arabinose catabolism. Strain SV3 (ara-531) shows high levels of inducible l-arabinose isomerase (EC 5.3.1.4) and l-ribulokinase (EC 2.7.1.16) activities, but is deficient in l-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4), the enzyme encoded in Escherichia coli by gene D in the araBAD operon. Addition of l-arabinose to SV3 growing in glycerol or casamino acids stops growth. d-Glucose only partially reverses this inhibition. Reversion of the ara-531 mutation restores different levels of epimerase activity and resistance to l-arabinose. However, the great majority of the l-arabinose-resistant mutants do not utilize l-arabinose. The physiological and enzymatic properties of these l-arabinose non-utilizing mutants suggest that l-arabinose resistance is due to forward mutations in at least 3 other genes, araA, araB and araC, blocking steps prior to l-ribulose 5-phosphate accumulation.     

D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC

d-Fucose, a nonmetabolizable analogue of l-arabinose, prevents growth of Escherichia coli B/r on a mineral salts medium plus l-arabinose by inhibiting induction of the l-arabinose operon. Mutations giving rise to d-fucose resistance map in gene araC and result in constitutive expression of the l-arabinose operon. Most of these mutations also permit d-fucose to serve as a gratuitous inducer. It is concluded that d-fucose-resistant mutants produce an araC gene product with an altered inducer specificity. Addition of l-arabinose to cells induced with the gratuitous inducer, d-fucose, resulted in severe transient repression of operon expression followed by permanent catabolite repression. Transient repression but no permanent catabolite repression was obtained when cells unable to metabolize l-arabinose were employed. It is concluded that transport of l-arabinose alone is sufficient to achieve transient repression of its own operon, but that metabolism of l-arabinose must occur to achieve permanent catabolite repression of the l-arabinose operon. This general effect has been termed "self-catabolite repression."     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s.

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency responses was of the fluence-square or "two-hit" type at both 365 and 254 nm when revertants were assayed on minimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on minimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.     

Hyperinducibility as a result of mutation in structural genes and self-catabolite repression in the ara operon

Mutations in gene araB producing an l-arabinose-negative phenotype cause either an increase (hyperinducible), decrease (polar), or have no effect at all on the inducible rate of expression of the l-arabinose operon. Fourteen araB gene mutants exhibiting such effects were shown to be the result of: nonsense, frameshift, or missense mutations. All missense mutants were hyperinducible, exhibiting approximately a twofold increase in rate of l-arabinose isomerase production. All frameshift and most nonsense mutants exhibited polar effect. One nonsense mutant was hyperinducible. The cis-dominant polar effect of nonsense and frameshift mutants (as compared to induced wild type) were more pronounced in arabinose-utilizing merodiploids and in araBaraC(c) double mutants where inducible and constitutive enzyme levels are respectively determined. On the other hand, in arabinose-utilizing merodiploids, missense mutations no longer exhibited hyperinducibility but displayed a wild-type level of operon expression. Increases in the wild type-inducible rate of expression of the operon were found when growth rate was dependent on the concentration of l-arabinose. Cyclic 3',5'-adenosine monophosphate also stimulated expression of the operon with the wild type in a mineral l-arabinose medium. These observations are explained on the basis that the steady-state expression of the l-arabinose operon OIBAD is dependent on the concentration of (i) l-arabinose, the effector of this system, which stimulates the expression of the operon, and (ii) catabolite repressors, produced from l-arabinose, which dampen the expression of the operon. We have termed the latter phenomenon "self-catabolite" repression.     

An assay for the detection of superoxide dismutase in individual Escherichia coli colonies

A method for detecting superoxide dismutase activity in individual colonies of Escherichia coli was developed. The assay involves the lysis of individual cells in colonies on filter papers by a series of lysozyme, chloroform, and freeze-thaw treatments. Filters are placed on agar plates to allow diffusion of cellular enzymes into a solid matrix. A nitroblue tetrazolium overlay is applied to detect superoxide dismutase activity. Colonies possessing activity produce achromatic zones against a dark Formazan background. The assay can detect the presence of superoxide dismutase and the relative amount of enzyme as well. This assay provides a method for screening a population of cells for mutants deficient in or overproducing superoxide dismutase.     

Map locations and functions of Salmonella typhimurium men genes

Menaquinone (men) mutants of Salmonella typhimurium isolated on the basis of their inability to produce trimethylamine were characterized with respect to mutation site, the ability to cross-feed each other and be cross-fed by known Escherichia coli men mutants, and response to intermediates of the menaquinone biosynthetic pathway. Cross-feeding tests were based on the requirement of menaquinone for hydrogen sulfide production. Genotypes corresponding to the menA, B, C, D, and possibly E genes described in E. coli were all identified. Additional studies of deletions in the menBCD area revealed that this cluster lies between ack/pta and glpT, as in E. coli. The ack and pta mutants were also defective in the production of trimethylamine and failed to produce gas in the absence of added formate.     

Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase.

A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes.     

Characterization of strong polar mutations in a region immediately adjacent to the L-arabinose operator in Escherichia coli B-r

Seven l-arabinose-negative mutations are described that map in three genetically distinct regions immediately adjacent to the araO (operator) region of the l-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dominant, trans-recessive polarity effect upon the expression of l-arabinose isomerase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initiator region of the l-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens diethyl sulfate and ICR-191. Three of these mutants are suppressible by two independently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara(+) revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppressors, and their partially polar revertants are discussed in terms of the mechanism of initiation of expression of the l-arabinose operon.     

Arabinose-leucine deletion mutants of Escherichia coli B-r

The control of ara gene expression was studied in mutants of Escherichia coli B/r containing deletions which fused the l-arabinose gene complex with the leucine operon (the normal gene order being araDABIOC...leuDCBAO). Complementation experiments with stable merodiploids showed that expression of ara genes cis to araC-leu deletions was controlled by the trans-acting product of the araC gene. Expression of ara genes cis to araB-leu deletions was under leucine control. These studies confirm the existence of a region between genes araC and araB essential for normal activator controlled expression of the ara structural genes. One deletion was characterized as an araO-leu deletion. Its effect on ara gene expression was unique in that ara genes were susceptible to potential regulation by both l-arabinose and leucine. These experiments suggest that two different species of messenger ribonucleic acid (mRNA) may be produced for the ara-leu region as a result of this deletion. One, under l-arabinose-activator control, is initiated in the l-arabinose region; the other, under leucine control, is initiated in the leucine region. The latter indicates that araI can be transcribed. Whether araI is transcribed in the former instance (mRNA made under activator control) remains to be established.     

Does polymyxin B nonapeptide increase outer membrane permeability in antibiotic supersensitive enterobacterial mutants?

Abstract The outer-membrane-disorganizing peptide (polymyxin B nonapeptide; PMBN) was able to sensitize even "antibiotic supersensitive" enterobacterial mutants to hydrophobic antibiotics. This resulted in an extreme sensitivity. The mutants included the "deep rough" lipopolysaccharide mutants, as well as the acrA mutant of Escherichia coli and the "class A, B, and C mutants of Salmonella typhimurium . Sensitization factors of approx. 30 or more were found for most antibiotics. Even minimum inhibitory concentrations as low as approx. 0.5 ng/ml (rifampicin), 1.5 ng/ml (erythromycin), 2 ng/ml (fusidic acid), 6 ng/ml (novobiocin), and 30 ng/ml (clindamycin) were achieved in the presence of 30 μg/ml of PMBN. The finding indicates that the mechanisms which mediate the increase in hydrophobic diffusion are different but synergistic in the mutants and in the PMBN-grown cells.     

Genetic analysis of the leucine region in Escherichia coli B-r: gene-enzyme assignments

Genetic mapping by transduction and conjugation using F(-) and F' strains carrying either point mutations in the l-arabinose or leucine regions or ara-leu fusion-deletion mutations has resulted in a detailed genetic map of the arabinose-leucine region of Escherichia coli B/r. These studies have identified four genes in the leucine region having the same order as found in Salmonella typhimurium: ara... leuDCBA.     

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).     

Isolation and preliminary characterization of β-lactamase excretory mutants of Escherichia coli K-12

Abstract Escherichia coli exc mutants able to release the plasmid pBR322-encoded β-lactamase (EC 3.5.2.6) into the extracellular medium have been isolated using a new in situ plate assay.
A preliminary characterization of the exc mutants was carried out: the presence of exc mutations was associated with a specific or pleiotropic pattern of excretion of periplasmic enzymes, an increased sensitivity to different growth inhibitors (EDTA, chloramphenicol, cholic acid) and a poor growth on various carbon sources.
After quantitative analysis, three groups of exc mutants were identified on the basis of their temperature-dependent or -independent pattern of growth and β-lactamase synthesis and excretion.     

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.     

Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.     

Development of a new "GFP hop-on assay" system for insertion sequence transposition in Bacillus subtilis 168 using IS4Bsu1 from B. subtilis (natto)

While most studies involving transposition have focused on analyzing the detailed mechanisms of transposition, the cellular conditions under which transposition occurs remain to be elucidated. In Escherichia coli, papillation assay is a powerful tool for transpositional analysis and the isolation of mutants affecting transposition. On the other hand, while our assay system based on the E. coli papillation assay can detect transpositional events in Bacillus subtilis 168, it is not suitable for quantitating transposition frequency because blue papillae on the transposant colonies of B. subtilis are not countable. We succeeded in developing a new "GFP hop-on assay" system that facilitates quantitative detection of the transposition of the FACS-optimized GFP mutant gene. Our assay system is a step forward in understanding the cellular conditions under which transposition occurs.     

The L-arabinose-resistance test with Salmonella typhimurium strain SV3 selects forward mutations at several ara genes.

A new assay has been described for mutagenicity testing using an L-arabinose-sensitive strain of Salmonella typhimurium. The test strain SV3 and several L-arabinose-resistant mutants selected therefrom are characterized in the present study by 3 different criteria: inhibition of growth by L-arabinose, accumulation of keto-sugars, and activities of the enzymes involved in L-arabinose catabolism. Strain SV3 (ara-531) shows high levels of inducible L-arabinose isomerase (EC 5.3.1.4) and L-ribulokinase (EC 2.7.1.16) activities, but is deficient in L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4), the enzyme encoded in Escherichia coli by gene D in the araBAD operon. Addition of L-arabinose to SV3 growing in glycerol or casamino acids stops growth. D-Glucose only partially reverses this inhibition. Reversion of the ara-531 mutation restores different levels of epimerase activity and resistance to L-arabinose. However, the great majority of the L-arabinose-resistant mutants do not utilize L-arabinose. The physiological and enzymatic properties of these L-arabinose non-utilizing mutants suggest that L-arabinose resistance is due to forward mutations in at least 3 other genes, araA, araB and araC, blocking steps prior to L-ribulose 5-phosphate accumulation.