t 49 390 b fragilis 638r 2348 234923452 344 23472346 trna 0024 b fragilis ych46 2243 224422392 238 22422240  (ATCC)

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Cloning, Staining, Plasmid Preparation, Derivative Assay, Clone Assay, Expressing, Selection, Construct, Mutagenesis

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Agar dilution determination of minimal inhibitory concentration (MIC μg/mL) of metronidazole (MTZ) and amixicile (AMIX) for Bacteroides species and B. fragilis 638R mutant strains.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Concentration Assay, Mutagenesis, Isolation

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Schematic representation of B. fragilis 638R chromosomal regions for PFOR, PoxB, Kor1AB, and Kor2AEBG as shown in the panels. Each locus tag is depicted below the respective deduced ORF symbolized by an arrow. The designation of the predicted peptide product is depicted above each open arrow gene region respectively. The Arrow direction depicts the transcription orientation. Arrows filled with color represent the functional annotation group assigned to PFOR (dark blue), PoxB (purple), KorA (orang), KorB (dark gray), KorG (light green), KorD (gold) or, KorC (light gray) orthologs, respectively. The deletion construct representation of each chromosomal region mutant is shown below the native chromosome region, respectively. The DNA fragment containing the promoterless kor2AEBG genes cloned into the expression vector pFD340 (pER‐377) was used for genetic complementation studies. ATPase, predicted ATPase AAA+ superfamily; DUF4870, putative membrane protein of unknown function; Kor1A, 2‐ketoglutarate ferredoxin oxidoreductase subunit α (CBW24739); Kor1B, 2‐ketoglutarate ferredoxin oxidoreductase subunit β (CBW24740); Kor2A, 2‐ketoglutarate ferredoxin oxidoreductase subunit α (CBW22186); Kor2B, 2‐ketoglutarate ferredoxin oxidoreductase subunit β (CBW22184); Kor2C, conserved hypothetical protein containing tetratricopeptide repeat (CBW22188); Kor2D, ferredoxin, 2‐ketoglutarate‐acceptor oxidoreductase subunit δ (CBW22187); Kor2E, hypothetical protein (CBW22185); Kor2G, 2‐ketoglutarate ferredoxin oxidoreductase subunit γ (CBW22183); PFOR, pyruvate:ferredoxin oxidoreductase (GenBank accession number CBW23670); PoxB, putative pyruvate dehydrogenase (CBW23720); ThPP, thiamine diphosphate cofactor.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Functional Assay, Construct, Mutagenesis, Clone Assay, Expressing, Plasmid Preparation, Membrane

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Disc diffusion assay sensitivity of B. fragilis strains to metronidazole (MTZ), panels A,B,C or for Amixicile (AMIX), panels D,E,F. A and D: Parent ( B. fragilis 638R). B and E: ΔfrdB (derived from ADB77, isogenic BF638R thy ‐ strain). C and F: B. fragilis BF8 strain ( nimB + ). Strain designations are depicted in each panel. For these experiments, 20 mM succinate and 50 μg/ml thymine were added to the BHIS media. Each bar represents the average zone of inhibition (mm) of at least three independent biological replicates. Vertical error bars denote the standard deviation of the means from two independent experiments in triplicate. The significance of the P value was calculated by one‐way ANOVA followed by multiple comparisons using Dunnett and Bonferroni tests. Only groups with statistical significance in both tests are reported. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). NS, not significant.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Diffusion-based Assay, Derivative Assay, Inhibition, Standard Deviation

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Disc diffusion assays. Susceptibility of B. fragilis 638R strain (Parent) and its isogenic ΔfrdB deletion mutant to metronidazole (MTZ). Panels (a) and (b): in the presence of 2‐hydroxy‐1,4‐napththoquinone (HNQ). Panel (c): in the presence of 1,‐4‐naphthoquinone (NQ). Panel (d): in the presence of 1,4‐benzoquinone (BQ). Panel (e): in the presence of plumbagin (PLG). Panel (f): in the presence of menadione (MD). Panel (g): in the presence of paraquat (PQ). Panel (h): in the presence of benzyl‐viologen (BV). Panels i and j: Susceptibility of B. fragilis 638 R strain to nitrofurantoin (NT), tetracycline (Tet), or chloramphenicol (Cm) exposed to oxygen with no addition (i) or addition of 50 μM HNQ (j). Each bar represents the average zone inhibition (mm) of at least three independent biological replicates. Vertical error bars denote the standard deviation of the means from two independent experiments in triplicate. Panels a–h: The significance of the p value was calculated by one‐way ANOVA followed by multiple comparisons using Dunnett and Bonferroni tests. Only groups with statistical significance in both tests are reported. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). NS, not significant. For panels i and j, no statistical analyzes were performed.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Diffusion-based Assay, Mutagenesis, Inhibition, Standard Deviation

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Metronidazole minimal Inhibitory concentration (MIC μg/mL) for B. fragilis 638R in BHI media containing 5 μg/mL hemin ( l ‐cysteine was omitted) supplemented with 2‐hydroxy‐1,4‐naphthoquinone, or 1,4‐naphthoquinone.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Concentration Assay, Control

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Survival of B. fragilis 638R in rat tissue cage infection following AMIX treatment at 20 mg/Kg/day via intraperitoneal (a) or 0.5 mg/intra‐cage/day (b). AMIX administration started on Day 1 through Day 7 postinfection. Bacteria were grown overnight in BHIS medium and diluted in PBS to approximately 1 × 10 5 CFU/mL. Four milliliters of the suspension were inoculated into the intraperitoneal tissue cage. Fluid samples were aspirated at time points for CFU counts as described in Materials and Methods. Tissue cage fluid was aspirated at Days 1, 2, 4 and 8 postinfection. Data are expressed as the mean CFU per milliliter of intra‐abdominal tissue cage fluid from three rats. The standard errors of the means (SEM) are denoted by vertical error bars. The detection limit of 1 × 10 1 CFU/mL.

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Infection, Bacteria, Suspension

Journal: MicrobiologyOpen

Article Title: New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

doi: 10.1002/mbo3.1429

Figure Lengend Snippet: Schematic diagram of B. fragilis 638R (a) central metabolism, (b) degradation of branched amino acids pathway, and (c) initial steps of menaquinol biosynthetic pathway. The thiamine diphosphate binding enzymes are depicted with ThPP in red font beside the enzyme designation. The enzyme subunits with predicted stable protein‐protein interaction with Pir1 are highlighted with a blue box and interaction with Pir2 with a green box. The compilation of the pathway reactions was based on public database https://www.genome.jp/pathway/bfg00020,/pathway/bfg00010,/pathway/bfg00620,/pathway/bfg00640,/pathway/bfg00280,/pathway/bfg00130 ), B. fragilis 638R genome (GenBank accession number FQ312004), and Refs.: Allison & Robinson, ; Allison et al., ; Butler et al., ; Harris & Reddy, ; Ito et al., ; Macy et al., ; Rios‐Covian et al., ; Schofield et al., ; Sund et al., ;. Zhang et al., . Orange dash/dot arrows indicate there is no report that 2‐ketoglutrate or l ‐glutamate is formed from isocitrate dehydrogenase (Icd) pathway under anaerobic conditions (Allison & Robinson, ; Allison et al., ; Schofield et al., ). AAs, amino acids; AckA, acetate kinase (BF638R_0490); AcnA, aconitase (BF638R_3569); AcoA/AcoB, 2‐Ketoisovalerate dehydrogenase, a single peptide containing alpha‐ and beta‐subunit domains (BF638R_1637); Acs, acetate,CoA ligase (BF638R_4449); AEP‐AT, 2‐aminoethylphosphonate aminotransferase (BF638R_1869) and (BF638R_3511); BCAAT, branched‐chain amino acid transferase (BF638R_3846); CitS_II/ACL, citrate synthase/2‐methylcitrate synthase/ACL family (BF638R_3567); Fp, flavoprotein; Frd, fumarate reductase, FrdCAB (BF638R_4499‐4501); FumB, fumarate hydratase class I, anaerobic (BF638R_0646); Icd, isocitrate dehydrogenase (BF638R_3568); IM, inner cytoplasmic membrane; Kor1AB, 2‐Ketoglutarate ferredoxin oxidoreductase (BF638R_4321‐4322); Kor2CDAEBG, 2‐Ketoglutarate ferredoxin oxidoreductase (BF638R_1660‐1665); LdhA, d ‐Lactate dehydrogenase (BF638R_1473); Lpd1, dihydrolipoamide dehydrogenase‐E3 (BF638R_0023); Lpd2, dihydrolipoamide dehydrogenase‐E3 (BF638R_1634); MaeB, malate dehydrogenase [oxaloacetate‐decarboxylating NADP + ‐dependent] (BF638R_3435); Mdh, malate dehydrogenase (BF638R_0537); MenD, 2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylic‐acid synthase (BF638R_1316); MenF, isochorismate synthase (BF638R_1315); MK, menaquinone; MMCE, methylmalonyl‐CoA epimerase (BF638R_3151); MmdA1 (PccB), propionyl‐CoA carboxylase beta‐chain (BF638R_1625); MmdA2 (PccB), propionyl‐CoA carboxylase beta‐chain (BF638R_3367); MutAB, methylmalonyl‐CoA mutase, small (A) and large (B) subunits (BF638R_3616‐3615); Nad2, NADH dehydrogenase, FAD‐containing subunit (BF638R_1612); Nqr, Na+‐translocating NADH‐quinone oxidoreductase, NqrABCDE, (BF638R_2136_2140); Nuo, NADH,ubiquinone oxidoreductase, NuoABC/DHIJKLMN (BF638R_0850‐0841); OadA1, pyruvate/oxaloacetate carboxyltransferase (BF638R_2828); OM, outer membrane; PckA, phosphoenolpyruvate carboxykinase (BF638R_4326); PEP mutase, phosphoenolpyruvate mutase (BF638R_1867); PFLA, phospholipid fatty acids; PflA, pyruvate formate‐lyase 1, activating enzyme (BF638R_1338); PflB, pyruvate formate‐lyase 1 [formate acetyltransferase 1] (BF638R_1339); PflC, pyruvate formate‐lyase 2, activating enzyme (BF638R_4262); PflD, pyruvate formate‐lyase 2 [formate acetyltransferase 2] 9BF638R_4263); PFOR, pyruvate ferredoxin oxidoreductase (BF638R_3194); Pnpyr‐DC, phosphonopyruvate decarboxylase (BF638R_1868); PoxB, an inner membrane enzyme that catalyzes oxidative decarboxylation of pyruvate to form acetate + CO 2 (BF638R_3245); PpdK, pyruvate orthophosphate dikinase (BF638R_2565); PtaA, phosphate acetyltransferase (BF638R_0489); PycB, pyruvate carboxylase biotin‐containing subunit (BF638R_1927); Pyk, pyruvate kinase (BF638R_4359); SucDC, succinyl‐CoA synthase alpha‐ and beta‐chains (BF638R_2360‐2361); YgfH, succinate‐CoA transferase subfamily (BF638R_0025).

Article Snippet: IB263 , 638R hydrogen peroxide resistant, hpr , Rif r , Rocha & Smith ( ) .

Techniques: Binding Assay, Membrane