Purification, Characterization, and Biochemical Assays of Biotin Synthase From Escherichia coli

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5ʹ-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.

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Amplification of the put genes and identification of the put gene products in Escherichia coli K12

Canadian Journal of Biochemistry ◽

10.1139/o80-110 ◽

1980 ◽

Vol 58 (10) ◽

pp. 787-796 ◽

Cited By ~ 20

Author(s):

Janet M. Wood ◽

David Zadworny

Keyword(s):

Escherichia Coli ◽

Dehydrogenase Activity ◽

Gene Products ◽

Proline Dehydrogenase ◽

Transport Activity ◽

E Coli ◽

Proline Transport ◽

Escherichia Coli K12 ◽

Biochemical Assays ◽

Transport Defect

The utilization of L-proline as carbon or nitrogen source for the growth of Escherichia coli K12 requires the activities of an L-proline porter (PP-I) and a bifunctional L-proline dehydrogenase – Δ1-pyrroline carboxylate dehydrogenase. PP-I is inactivated by mutations at putP and the bifunctional dehydrogenase is encoded in the adjacent locus, putA, at 22 min on the chromosome map. Two additional loci, proP (at 92 min) and proT (at 82 min), have also been implicated in L-proline transport. We have studied four ColE1/E. coli K12 hybrid plasmids from the plasmid bank prepared by Clarke and Carbon. Each of these plasmids was shown previously to complement an L-proline transport defect in E. coli. Genetic complementation analysis and biochemical assays of L-proline transport and L-proline dehydrogenase activity show that three of these hybrid plasmids bear the putPA region of the E. coli chromosome (plasmids pLC4-45, pLC10-29, and pLC43-41). The fourth plasmid, pLC35-38, specifically enhances the L-proline transport activity of its host bacteria but not their L-proline dehydrogenase activity. It probably encodes putP. We have used these plasmids in an E. coli minicell system to identify the putA and putP gene products.

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Biotin Synthase: Purification, Characterization as a [2Fe-2S]Cluster Protein, and in vitro Activity of the Escherichia coli bioB Gene Product

Biochemistry ◽

10.1021/bi00178a020 ◽

1994 ◽

Vol 33 (12) ◽

pp. 3625-3631 ◽

Cited By ~ 90

Author(s):

Indrajit Sanyal ◽

Gerald Cohen ◽

Dennis H. Flint

Keyword(s):

Escherichia Coli ◽

Gene Product ◽

Vitro Activity ◽

In Vitro Activity ◽

Biotin Synthase

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Genotypic and Phenotypic Characterization of Escherichia coli Isolates from Feces, Hands, and Soils in Rural Bangladesh via the Colilert Quanti-Tray System

Applied and Environmental Microbiology ◽

10.1128/aem.03214-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1735-1743 ◽

Cited By ~ 13

Author(s):

Timothy R. Julian ◽

M. Aminul Islam ◽

Amy J. Pickering ◽

Subarna Roy ◽

Erica R. Fuhrmeister ◽

...

Keyword(s):

Escherichia Coli ◽

False Positive ◽

False Positive Rate ◽

Pathogen Transmission ◽

Environmental Matrices ◽

Content Type ◽

E Coli ◽

Biochemical Assays ◽

Positive Rate ◽

Genotypic Characteristics

ABSTRACTThe increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection ofEscherichia colifrom environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidase-basedE. colidetection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source ofE. coliinfluences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API-20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genessfmH,rfaI, andfucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples.E. coliisolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection ofE. colion hands and in soils with low false-positive rates. Furthermore,E. coliisolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping.

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