Characterization of relationships between transcriptional units and operon structures in Bacillus subtilis and Escherichia coli

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

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Purification and characterization of the SpoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1987.tb13328.x ◽

1987 ◽

Vol 167 (2) ◽

pp. 233-238 ◽

Cited By ~ 3

Author(s):

Toshihiko IKEUCHI ◽

Susumu TSUNASAWA ◽

Fumio SAKIYAMA

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Purification And Characterization

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Characterization of the Bacillus subtilis ywtD Gene, Whose Product Is Involved in γ-Polyglutamic Acid Degradation

Journal of Bacteriology ◽

10.1128/jb.185.7.2379-2382.2003 ◽

2003 ◽

Vol 185 (7) ◽

pp. 2379-2382 ◽

Cited By ~ 58

Author(s):

Takao Suzuki ◽

Yasutaka Tahara

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Glutamic Acid ◽

Recombinant Plasmid ◽

Signal Sequence ◽

High Molecular Mass ◽

Enzymatic Analysis ◽

Polyglutamic Acid ◽

Partial Similarity

ABSTRACT The ywtD gene, which codes for an enzyme that degrades γ-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for dl-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% l-glutamic acid) and an 11-kDa product (with d-glutamic acid and l-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the γ-glutamyl bond only between d- and l-glutamic acids of PGA, and it may be designated γ-dl-glutamyl hydrolase.

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Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties

Annals of Microbiology ◽

10.1007/s13213-010-0167-4 ◽

2010 ◽

Vol 61 (3) ◽

pp. 517-524 ◽

Cited By ~ 21

Author(s):

Yohei Onishi ◽

Shigekazu Yano ◽

Jaruwan Thongsanit ◽

Kazuyoshi Takagi ◽

Kazuaki Yoshimune ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Type Ii ◽

Gene Encoding ◽

Expression In Escherichia Coli

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Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli-Expressed Cyclopentanone 1,2-Monooxygenase

Applied and Environmental Microbiology ◽

10.1128/aem.68.11.5671-5684.2002 ◽

2002 ◽

Vol 68 (11) ◽

pp. 5671-5684 ◽

Cited By ~ 87

Author(s):

Hiroaki Iwaki ◽

Yoshie Hasegawa ◽

Shaozhao Wang ◽

Margaret M. Kayser ◽

Peter C. K. Lau

Keyword(s):

Escherichia Coli ◽

Regulatory Gene ◽

Regulatory Elements ◽

Open Reading Frames ◽

Chromosomal Locus ◽

New Genes ◽

Transcriptional Units ◽

Acid Catalyzed ◽

Reading Frames

ABSTRACT Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas. Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orf3-orf4 [partial 3′ end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Kmr cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of σ54-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-β-thio-d-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

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Characterization of Polyphenols, Flavonoids and Their Anti-microbial Activity in the Fruits of Vangueria madagascariensis J. F. Gmel

European Journal of Medicinal Plants ◽

10.9734/ejmp/2020/v31i1130296 ◽

2020 ◽

pp. 24-37

Author(s):

Peter K. Njenga ◽

Samuel M. Mugo ◽

Ting Zhou

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Phenolic Compounds ◽

Phenolic Acids ◽

Antimicrobial Activities ◽

Diffusion Method ◽

Botany Department ◽

Fruit Extracts ◽

Caffeoylquinic Acid

Aim: The study aimed to characterize phenolic acids, flavonoids, and determine their antimicrobial activities in fruits of Vangueria madagascariensis (Tamarind of Indies). Study Design: The design of the study included picking of Vangueria madagascariensis fruits from Jomo Kenyatta University of Agriculture and Technology (JKUAT) botanical garden and analysis for their antimicrobial activities at the Botany department research laboratory, JKUAT. Characterization of phenolic acids and flavonoids were conducted at MacEwan University Canada. Place and Duration: JKUAT, Kenya and MacEwan University, Edmonton, Alberta Canada between June 2013 and June 2016. Methodology: Phenolic acids and flavonoids from Tamarind of Indies were determined by high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MSN). The antimicrobial assay was determined using the disk diffusion method. Results: Based on the retention time, the UV spectrum, and the tandem MS behavior, the results revealed a profile composed of 25 phenolic compounds. Some of the identified phenolic compounds included: 3-caffeoylquinic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, 4-feruloyl quinic acid, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin, 3,4-di-caffeoylquinic acid, 4, 5-di-caffeoylquinic acid, kaempferol, diosmetin, caffeic acid, epicatechin, kaempferol 3-O-glucoside. The fruit extracts had a probable presence of quercetin 3-O-6’-malonylglucoside, ikarisoside C, epimedin C, unknown epigallocatechin-3-gallate and quercetin conjugate derivatives. Furthermore, the fruit extracts from Vangueria madagascariensis showed appreciable antimicrobial properties against human pathogen strains. Strong antimicrobial activity was observed for Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The Vangueria madagascariensis was found to be highly potent against Escherichia coli and Bacillus subtilis even at low concentrations of 0.1 mg/mL. Conclusion: The research findings may suggest value of the use of Vangueria madagascariensis fruits as a rich source of antioxidants with therapeutic and nutraceutical value.

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