Regulatory Mechanisms for Synthesis of Capsular Polysaccharide in Mucoid Mutants of Escherichia Coli K12

Markovitz, Alvin

doi:10.1073/pnas.51.2.239

Cited by 120 publications

(122 citation statements)

References 27 publications

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“…(1 97 I), Markovitz (1964) and Broda (1974), respectively. In the reciprocal transduction (Table 3), selection was made for hem-361+ transductants on GMA containing proline and 5-ALA. Tests for cotransduction with hem-361f of proC and lac Y+ were made by restreaking each hem-361' transductant on glucose minimal medium (with 5-ALA), and on lactose minimal medium (with 5-ALA and proline).…”

Section: Haemin-requiring Mutantsmentioning

confidence: 99%

Isolation of Haemin-requiring Mutants of Escherichia coli K12

McConville

Charles

1979

Journal of General Microbiology

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155Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen I11 cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC. I N T R O D U C T I O NEscherichia coli K12 is impermeable to haemin under normal cultural conditions (SMrman et al., 1968), and genetic analysis of haem biosynthesis has depended on indirect methods. McConville & Charles (1975, 19798) described mutants of E. coli IS12 which are permeable to haemin. The present paper describes the isolation and study of haeminrequiring mutants derived from haemin-permeable mutants. M E T H O D SUnless stated otherwise, the methods were those used by McConville & Charles (1979~). Media. Glucose minimal medium (GM) was medium E of Vogel & Bonner (1956) supplemented with thiamin (5 mg 1-l) and glucose (5 g 1-l). Glucose minimal agar (GMA) was GM containing Difco Bacto agar (13 g P ) , autoclaved separately. When necessary, the glucose in GM was replaced by other sugars (2 g 1-l). Solutions of amino acids and other growth factors were sterilized by membrane filtration and aseptically added to minimal medium to give a final concentration of 40 mg l-l, except isoleucine and valine were added at 12 and 28 mgl-l, respectively, and 5-aminolaevulinic acid (5-ALA) at 1 mg 1-' . TNM contained (g 1-l): NaCl, 4; Difco Bacto nutrient broth, 8 ; MgS04. 7H20, 2.47. TNAM was TNM autoclaved with agar (13 g I-').Stock solutions of haemin (type 111, Sigma) and protoporphyrin IX (PP9; Calbiochem) were prepared by dissolving 0.2 g in 5 ml Tween 80 and slowly diluting to 50 ml with sterile distilled water; a few drops of alkali were added to give clear solutions. The procedure gave sterile solutions which were stored at -18°C and added aseptically to give concentrations of 25 mg 1-1 in TN media and 10 mg 1-1 in GM media.Genetical analysis. The transducing phage was Plkc; 12 min adsorption were allowed. Gene symbols and references to the E. culi linkage map follow Bachmann e f al. (1976). Details of the strains are given in Table 1.Assay ofporphyrins accumulated in cultures. Bacteria were inoculated into two 2 1 flasks containing glucose minimal medium (400 ml) plus sodium thiosulphate (10 mg 1-l) and 5-ALA (50 mg I-l); one flask contained haemin (10 mg 1-l). Proline was added if required. Incubation was for 48 h at 37 "C in an orbital incubator. Bacteria were then washed and resuspended in 10 ml of glass-distilled water, and a 1 ml sample was removed for drying and weighing. Porphyrins were extracted from the supernatant medium and bacteria by the method of Falk (1964). Coproporphyrin and protoporphyrin were extracted fr...

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Section: Haemin-requiring Mutantsmentioning

confidence: 99%

Isolation of Haemin-requiring Mutants of Escherichia coli K12

McConville

Charles

1979

Journal of General Microbiology

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“…This enzyme is a serine protease with an essential ATPase activity (37) and seems to be a heat shock protein (25). Ion mutations in E. coli result in pleiotropic phenotypes such as increased sensitivity to UV irradiation (10,16) and SOS-inducing agents (35), abnormal cell division (filament formation [16]), mucoid formation (12,22), defective lysogeny of phages A and P1 (32,36), and reduced degradation of various abnormal proteins (13) and certain normal proteins. Various observations indicate that protease La catalyzes rate-limiting steps in the degradation of some regulatory proteins such as SulA and RcsA (23,34).…”

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confidence: 99%

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth

1993

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The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.

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“…2 Independent experiments of Howard-Flanders, Simson, and Theriot,4 and Adler and Hardigree5 demonstrated that a mutation designated lon-caused E. coli K12 to become very sensitive to ultraviolet (UV) and ionizing radiation. These lon-strains are mucoid4 and after X-ray radiation form filaments that die.5 It is now clear that the phenotype of capR and lon mutants are identical, i.e., mucoid and UV sensitive," 2, 4-7 and arise from the same mutational event.…”

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confidence: 99%

“…2 3 8 The sugar components of the capsule are D-glucose, D-galactose, D-glucuronic acid, and L-fucose." 2 Independent experiments of Howard-Flanders, Simson, and Theriot,4 and Adler and Hardigree5 demonstrated that a mutation designated lon-caused E. coli K12 to become very sensitive to ultraviolet (UV) and ionizing radiation. These lon-strains are mucoid4 and after X-ray radiation form filaments that die.…”

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confidence: 99%

Derepression of GDP-α-D-Mannose and UDP-Glucose Pyrophosphorylases by a Regulator Gene Mutation; Episomal Dominance in Partial Diploids

Lieberman

Buchanan

Markovitz

1970

Proc. Natl. Acad. Sci. U.S.A.

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Abstract. 1\Jutants of Escherichia coli K12 at the capR locus are mucoid, overproduce capsular polysaccharide, and are derepressed for synthesis of several enzymes involved in capsular polysaccharide synthesis'-' including GDP-mannose pyrophosphorylase.8 UDP-glucose pyrophosphorylase is also derepressed in a haploid capR9 mucoid mutant. Heterozygous mucoid partial diploids with the capR9 allele on the episome and the wild-type (capR+) allele on the chromosome (F'capR9/capR+) are derepressed for UDP-glucose pyrophosphorylase and GDP-mannose pyrophosphorylase, while the reciprocal nonmucoid heterozygotes (F'capR+/capR9) are repressed for these enzymes. These results provide evidence that the episomal capR allele is dominant with respect to synthesis of these two enzymes.A regulator gene (designated capR; previously designated R,' 2) controls the synthesis of capsular polysaccharide (mucoid) and several enzymes involved in capsular polysaccharide synthesis in Escherichia coli K12.1-3 The wild type is nonmucoid and contains low levels of the enzymes, while mutants at the capR locus are mucoid and contain derepressed levels of several of the enzymes of capsular polysaccharide synthesis.2 3 8 The sugar components of the capsule are D-glucose, D-galactose, D-glucuronic acid, and L-fucose." 2 Independent experiments of Howard-Flanders, Simson, and Theriot,4 and Adler and Hardigree5 demonstrated that a mutation designated lon-caused E. coli K12 to become very sensitive to ultraviolet (UV) and ionizing radiation. These lon-strains are mucoid4 and after X-ray radiation form filaments that die.5 It is now clear that the phenotype of capR and lon mutants are identical, i.e., mucoid and UV sensitive," 2, 4-7 and arise from the same mutational event."1 4, The lon-or capR mutants are not defective in repair of irradiated T14 or T7 bacteriophage (Uretz, unpublished data). The mechanism by which capR controls radiation sensitivity is unknown at the present time.Genetic studies by Markovitz and Rosenbaum have demonstrated that a specific mutation, capR9, is dominant when on an episome but recessive on a chromosome. Thus a partial diploid strain with the genotype F'capR9/capR+ is mucoid, but a partial diploid strain with the genotype F'capR+/capR9 is nonmucoid. On the basis of this observation, a model for the capR gene product, was formulated.' The model, termed the active oligomer model, postulates that 625

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Regulatory Mechanisms for Synthesis of Capsular Polysaccharide in Mucoid Mutants of Escherichia Coli K12

Cited by 120 publications

References 27 publications

Isolation of Haemin-requiring Mutants of Escherichia coli K12

Isolation of Haemin-requiring Mutants of Escherichia coli K12

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth

Derepression of GDP-α-D-Mannose and UDP-Glucose Pyrophosphorylases by a Regulator Gene Mutation; Episomal Dominance in Partial Diploids

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