Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi

Servos, Spiros; Chatfield, Steven N.; Hone, David M.; Levine, Myron; Dimitriadis, G J; Pickard, Derek; Dougan, Gordon; Fairweather, Neil; Charles, Ian G.

doi:10.1099/00221287-137-1-147

Cited by 22 publications

(17 citation statements)

References 42 publications

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“…The aroD gene encodes 3-dehydroquinate dehydratase, an enzyme involved in the shikimate pathway used to synthesize chorismic acid, a central precursor for aromatic compounds (31). This is a housekeeping function for the cell and therefore, the gene should be highly conserved.…”

Section: Figmentioning

confidence: 99%

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

et al. 2017

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Staphylococcus hyicus and Staphylococcus agnetis are two coagulasevariable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus, S. agnetis, and Staphylococcus aureus. Isolates (n ϭ 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus, S. agnetis, and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis (n ϭ 43) or S. hyicus (n ϭ 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulasepositive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus. Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections.KEYWORDS Staphylococcus, cattle, milk S taphylococci are the most common bacteria isolated from the cow's mammary gland and are frequently associated with subclinical or mild clinical mastitis (1-3). Staphylococci can be differentiated into groups using the coagulase test. Hemolytic coagulase-positive isolates are often presumptively identified as Staphylococcus aureus, a so-called "major" contagious mastitis pathogen, and coagulase-negative staphylococci are frequently grouped together as "minor" pathogens (4).Currently, there are two coagulase-variable staphylococcal species that have been associated with bovine mastitis, Staphylococcus hyicus and Staphylococcus agnetis. Staphylococcus hyicus was the first staphylococcal species to be described as coagulase variable, with coagulase production being found in approximately 24 to 56% of strains (5). The coagulation reaction of S. hyicus was described as often having weak activity,

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Section: Figmentioning

confidence: 99%

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

et al. 2017

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“…Some of these type I enzymes, for example those from E. coli and S. cyphi [19,20], occur as monofunctional enzymes while others, for example those from N. crassa, A. nidulans and S. cerevisiae, occur as the fourth domain of the pentafunctional arom protein [14][15][16]17,18]. It has been…”

Section: Introductionmentioning

confidence: 99%

“…and Neurospora crassa [12,1416] and studied in other species such as Aspergillus nidulans [ 171, Saccharomyces cerevisiae [18] and Salmonella typhi [19,20] are heat labile, have a mechanism involving an imine intermediate and use a histidine residue as a general base [13]. Some of these type I enzymes, for example those from E. coli and S. cyphi [19,20], occur as monofunctional enzymes while others, for example those from N. crassa, A. nidulans and S. cerevisiae, occur as the fourth domain of the pentafunctional arom protein [14][15][16]17,18].…”

Section: Introductionmentioning

confidence: 99%

The characterisation of the shikimate pathway enzyme dehydroquinase fromPisum sativum

et al. 1994

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Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase.Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of F! sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase cDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.

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“…Escherichia coli DHQase is monofunctional (encoded by the aroD gene) and has been cloned, sequenced and overexpressed [7]. Salmonella typhi [8,9], Enterococcus faecalis [10] and Bacillus subtilis [11] all possess monofunctional DHQases. In plants such as Physcomitrella patens [12], Nicotiana tabacum [13] and Pisum sati um [14], DHQase is part of a bifunctional polypeptide that also carries shikimate dehydrogenase activity, the next enzyme in the pathway.…”

Section: Introductionmentioning

confidence: 99%

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

Bottomley

Clayton²,

Chalk³

et al. 1996

Biochemical Journal

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A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coliaroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

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Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi

Cited by 22 publications

References 42 publications

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

The characterisation of the shikimate pathway enzyme dehydroquinase fromPisum sativum

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

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