Journal: Applied and Environmental Microbiology

Article Title: Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

doi: 10.1128/AEM.01381-18

Figure Lengend Snippet: The transposon integration sites were confirmed by amplification of the region of the genome flanking the insertion sites. Panel A shows the genomic PCR amplification scheme for the AFKI1 strain. The primers flanking the insertion site were designed from the published genome sequence for ATCC 23270. In the wild-type genome, the PCR amplicon is of a known size. In the transposon-integrated strains, the same PCR primers were used to amplify the transposon region as well, resulting in a 3.5-kb increase in amplicon size. Panel B shows the results of the method applied to the three transposon locations identified in the mutant strains and visualized on an agarose gel, confirming the successful identification of integration sites.

Article Snippet: Using the NCBI Nucleotide blastx program, sequences were compared against the published ATCC 23270 genome to identify integration loci. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Primer Sequence (5′–3′) Tn5Fwd TAT TAT CTG CGG CCG CCA TCG ACT GCA CGG TGC AC Tn5Rev AGA TAT CTG CGG CCG CTG TCA CTT T Tn5Rev2 AGA TAT CTC GCG GCC GCA AAA AGG CCA TCC GTC AGG ATG HypTnpFwd TAC ACA AGT AGC GTC GCA TGC CAT CGA CTG CAC HypTnpRev TTA GGC GGG CTA CTA TCT AGA TGT CAC TTT GCT TGA TAT ATG AGA ATT ATT TAA C pBAMFwd GAC GCT ACT TGT GTA CTG TCT CTT ATA CAC ATC TGA CGT CTT GTG T pBAMRev TAG TAG CCC GCC TAA TGA GCG pBAM2F AAG CGG GGT AAG CGC AAG AAT pBAM2R ATC GCC CAT GTT ATG CAG AAA tn1RFwd CCA CTA CCG GCA AGT TCT CCG tn2RFwd CAG TTC ACC GAC ACC AAA GGT G tn1RRev TAT GAA GAT GCA TGA GCC GGT C tn1LFwd TCG TCG ACC GAG CTT TTG C tn1LRev GAA AGA GGA TGC GCC GAA AGT G tn2LRev GGG AAA GCT CTT CGC CGA AC tnFSeq TGC ACA GCC ATA CCA CAG CTT C tnRSeq GGC TAC AGC TCG TTT CAC GCT G AFKI1Fwd TCG CCG TTC GTT TTC TCG AFKI1Rev GCC ACC GCA TCC AGT AAT C AFKI2Fwd ATG GTT CAC ACC GAA ATC AAT GC AFKI2Rev CAT CCA TGC TAC AGC CTA AGT TGC C AFKI3Fwd CCT GAT GTA GTC GTT GGC GTC C AFKI3Rev GTT CGT CAA CAG CAA AGT GGA AC Open in a separate window Primers used in this study (iii) Confirmation of integration loci.

Techniques: Amplification, Sequencing, Mutagenesis, Agarose Gel Electrophoresis

Journal: Applied and Environmental Microbiology

Article Title: Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

doi: 10.1128/AEM.01381-18

Figure Lengend Snippet: Location of the chromosomal integration sites. Panel A shows the integration loci for KDC-integrated strains. The insertion locus identifies the 9-bp sequence duplicated by the transposase to insert the transposon. Panel B shows the approximate locations of the transposon insertions for the mutant strains in relation to the whole A. ferrooxidans 23270 genome.

Article Snippet: Using the NCBI Nucleotide blastx program, sequences were compared against the published ATCC 23270 genome to identify integration loci. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Primer Sequence (5′–3′) Tn5Fwd TAT TAT CTG CGG CCG CCA TCG ACT GCA CGG TGC AC Tn5Rev AGA TAT CTG CGG CCG CTG TCA CTT T Tn5Rev2 AGA TAT CTC GCG GCC GCA AAA AGG CCA TCC GTC AGG ATG HypTnpFwd TAC ACA AGT AGC GTC GCA TGC CAT CGA CTG CAC HypTnpRev TTA GGC GGG CTA CTA TCT AGA TGT CAC TTT GCT TGA TAT ATG AGA ATT ATT TAA C pBAMFwd GAC GCT ACT TGT GTA CTG TCT CTT ATA CAC ATC TGA CGT CTT GTG T pBAMRev TAG TAG CCC GCC TAA TGA GCG pBAM2F AAG CGG GGT AAG CGC AAG AAT pBAM2R ATC GCC CAT GTT ATG CAG AAA tn1RFwd CCA CTA CCG GCA AGT TCT CCG tn2RFwd CAG TTC ACC GAC ACC AAA GGT G tn1RRev TAT GAA GAT GCA TGA GCC GGT C tn1LFwd TCG TCG ACC GAG CTT TTG C tn1LRev GAA AGA GGA TGC GCC GAA AGT G tn2LRev GGG AAA GCT CTT CGC CGA AC tnFSeq TGC ACA GCC ATA CCA CAG CTT C tnRSeq GGC TAC AGC TCG TTT CAC GCT G AFKI1Fwd TCG CCG TTC GTT TTC TCG AFKI1Rev GCC ACC GCA TCC AGT AAT C AFKI2Fwd ATG GTT CAC ACC GAA ATC AAT GC AFKI2Rev CAT CCA TGC TAC AGC CTA AGT TGC C AFKI3Fwd CCT GAT GTA GTC GTT GGC GTC C AFKI3Rev GTT CGT CAA CAG CAA AGT GGA AC Open in a separate window Primers used in this study (iii) Confirmation of integration loci.

Techniques: Sequencing, Mutagenesis

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Enzymatic activity of AWAT2 toward geometric isomers of retinol. A: Western blot detection of mouse AWAT2 in microsomal fraction isolated from yeast cells transfected with YepM containing the enzyme’s cDNA. The control sample represents membranes from the cells transfected with empty vector. B: HPLC chromatograms show separation of retinols and products of their AWAT2-dependent esterification. Peaks 1 and 1’ represent 9-cis-retinol and 9-cis-retinyl myristyl, respectively; peaks 2, and 2’ represent 11-cis-retinol and the corresponding ester, respectively; peaks 3 and 3’ represent 13-cis-retinol and its ester, respectively; and peaks 4 and 4’ represent all-trans-retinol and all-trans-retinyl ester, respectively. The inset shows characteristic UV/Vis spectra for each of the isomers. C: Comparison of the time courses of retinyl ester formation from retinol and its geometric isomers (blue line, 9-cis-retinol isomer; red line, 11-cis-retinol isomer; green line, 13-cis-retinol isomer; black line, all-trans-retinol isomer). D: Determination of kinetic parameters for the enzymatic reaction in the presence of retinol and its isomers. The curves represent dependence of the initial velocity of retinyl myristate formation on the substrate concentrations. The kinetic parameters of the reaction were determined by fitting the experimental points to the Michaelis-Menten model.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay, Western Blot, Isolation, Transfection, Control, Plasmid Preparation, Comparison

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Kinetic parameters of AWAT2-dependent esterification of retinol isomers

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques:

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: AWAT2 activity toward long-chain alcohol. A: LC-MS-based detection of myristate ester. The extracted ion chromatogram represents elution of product of the enzymatic reaction (myristyl myristate) detected at m/z 425.3 [MH]+. In addition to these ions, the MS spectrum averaged across this chromatography peak displays ions corresponding to palmityl myristate (m/z 453.3 [MH]+) that served as an internal standard for the purpose of quantification. B: Rate of myristyl myristate formation as a function of concentration of myristyl alcohol. By analogy to production of retinyl esters, the experimental points were used to determine the kinetic parameters for the enzymatic wax formation.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Chromatography, Concentration Assay

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: AWAT2-dependent esterification of 11-cis-retinol in the presence of holo-CRALBP. A: The apo-CRALBP expressed in E. coli cells was purified to homogeneity (inset) and preincubated with 11-cis-retinol to form holo-protein. After repurification, the efficiency of ligand binding was verified by recording UV/Vis absorbance spectra. The spectrum indicates two maxima at 278 nm corresponding to the protein scaffold and at 344 nm that is representative of bound 11-cis-retinol. B: The time course of 11-cis-retinol esterification in the presence of either free (open circle) or CRALBP-bound (filled circle) substrate. C, D: Verification of the isotopic composition of 11-cis-retinol (C) and its deuterated form (D). C: The MS spectrum of unlabeled retinoid reveals the main isotopic peak at m/z 269.3 that corresponds to ionization-driven loss of water from the parent ion. The conforming tandem spectrum (MS2) shows the fragmentation pattern of the retinoid moiety. D: The main peak for heavy atom-labeled 11-cis-retinol is at m/z 274.3 that indicates five deuterium atoms incorporated into the molecule. The ion at m/z 274.3 that corresponds to the molecules labeled with only four heavy atoms represents less than 20% of the mixture. Importantly, the unlabeled retinoid was not detectable. E: HPLC separation of 11-cis-retinol (blue peak) and its esterified form (green peak) after 2 min incubation of AWAT2 with holo-CRALBP and free 5D-11-cis-retinol. The isotopic composition of the substrates (blue) and products (green) are shown in (F), whereas (G) represents temporal changes in the ratio between labeled and unlabeled 11-cis-retinyl esters during the enzymatic reaction (filled circle) and the time course of ligand exchange between holo-CRALBP and free 11-cis-retinol (gray diamonds).

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Purification, Ligand Binding Assay, Labeling, Incubation

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Substrate selectivity of AWAT2 in the presence of a mixture of retinol isomers. A: Theoretical progress of the esterification reaction. Accumulation of 9-, 11-, and 13-cis-retinyl esters shown as dashed line, solid line, and dotted line, respectively, was simulated based on the kinetic parameters summarized in Table 1. The initial concentrations of the substrates resembled thermodynamic equilibrium between retinol isomers and were set below the Km values at 0.1, 1.0, and 2.3 μM for 11-, 9-, and 13-cis isomers, respectively. B: Representative chromatograms indicate changes in the composition of retinol isomers upon incubation with AWAT2. Trace (a) corresponds to separation of 9-cis-, 11-cis-, 13-cis-, and all-trans-retinol (peaks 1, 2, 3, and 4, respectively) from an equimolar mixture. Trace (b) shows the composition of retinoids extracted after 30 min of incubation in the standard condition for AWAT2 enzymatic assay. Disproportional (compared to other isomers) esterification of 11-cis-retinol is evident by diminishing of peak 2 and concomitant appearance of peak 2’ corresponding to 11-cis-retinyl myristate. C: The time course of the enzymatic reaction conducted in the presence of mixture of retinol isomers. Because formation of retinyl esters other than 11-cis was not sufficient for reliable quantification, the data are represented as ratios between substrates 11-cis- or 9-cis-retinol (filled circles) and 13-cis-retinol (open circles). The dashed line corresponds to the expected rate of 9-cis-retinyl myristate formation based on the kinetic parameters determined solely in the presence of 9-cis-retinol (Fig. 1A, C, D; Table 1). There is an apparent discrepancy between the efficiency of 9-cis-retinol esterification in the presence and absence of other retinol isomers.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Incubation, Enzymatic Assay

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: The effect of 11-cis-retinyl esters on AWAT2 activity. A: Inhibition of 9-cis-retinol esterification in the presence of 11-cis-retinyl palmitate. The enzymatic reaction was carried out in the presence of 6 μM 11-cis-retinyl ester and 100 μM 9-cis-retinol. After 20 min of incubation, the retinoid composition was examined by HPLC. The analysis indicated a lower amount of 9-cis-retinyl esters (peak 3’) produced in the presence of 11-cis-retinyl esters (gray trace) as compared with a control reaction (black trace). The chromatography peaks were identified based on the elution time and characteristic UV/Vis spectra (insets). The 11-cis-retinyl ester and the corresponding spectrum with maximum absorbance at 318 nm are marked with asterisks. Peak 3 and confirming UV/Vis spectra with maximum at 324 nm (inset) indicate 9-cis-retinol. B: The 11-cis-retinyl palmitate (11-cis-RP) inhibited esterification of 9-cis-, 13-cis-, or all-trans-retinol, but not 11-cis-retinol. Ten micromoles of selected retinyl esters were preincubated with AWAT2 containing microsomes prior to the addition of the alcohol substrate. C: The 9-cis-, 13-cis-, or all-trans-retinyl palmitates had no effect on esterification of retinol isomers. D: The inhibitory effect of 11-cis-retinyl esters was not dependent on the length of the acyl moiety. Results of the enzymatic assays performed in the presence of 11-cis-retinyl palmitate, laureate (LA), or acetate (AC) revealed a comparable level of inhibition for each of the tested compounds.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay, Inhibition, Incubation, Produced, Control, Chromatography

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: Evaluation of the oligomeric state of AWAT2 by EM. A: A representative cryo-electron micrograph of purified GST-AWAT2. The red squares indicate fusion protein monomers, whereas the green circles indicate fusion protein homodimers. The 2D classes shown were produced using the 1,603 particle dataset. The top row of classes represents fusion protein monomers, whereas the second row corresponds to homodimers. Each row is marked with the percentage distribution of each oligomeric state calculated from the 12,846 particle dataset. B: Three views of the 3D reconstructed density adjusted to a reasonable volume using the volume/mass conversion of 0.81 Da/Å. The crystal structure of GST (purple) is docked into the density using the Chimera fit to map function.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Purification, Produced

Journal: Journal of Lipid Research

Article Title: Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2

doi: 10.1194/jlr.M073692

Figure Lengend Snippet: A schematic representation of the modulation of AWAT2 enzymatic activity. A: In the absence of 11-cis-retinoids, AWAT2 reveals relaxed substrate selectivity allowing for esterification of a variety of geometric isomers of retinol. B: Interaction of the retinoid moiety in the 11-cis configuration with a putative allosteric site (marked with green boundary) leads to changes in the active site (blue and orange boundaries) that result in narrowing substrate specificity of the enzyme and selective production of 11-cis-retinyl esters.

Article Snippet: cDNA of mouse AWAT2 (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_177746","term_id":"594150398","term_text":"NM_177746"}} NM_177746 ) was purchased from OriGene Technologies.

Techniques: Activity Assay

Journal: Nature Communications

Article Title: Pangenome dynamics and population structure of the zoonotic pathogen Salmonella enterica serotype Hadar

doi: 10.1038/s41467-025-68026-3

Figure Lengend Snippet: The networks contain 3384 nodes, connected when Jaccard Index (JI) ≥ 0.988. Eighteen groups (JI-groups A-R) are labeled. Legends are left of each network. a National Center for Biotechnology Information (NCBI) SNP (single-nucleotide polymorphism) cluster across JI-groups (obtained from Pathogen Detection Isolate Browser ). b Condensed allele code across JI-groups. Grey nodes indicate genomes with no assigned allele code. c Plasmid taxonomic units (PTUs) across JI-groups. Grey nodes indicate genomes with no known PTU. d Most common antimicrobial resistance (AMR) genes conferring predicted aminoglycoside and tetracycline resistance. Grey nodes indicate genomes without selected AMR genes. e Genomes determined to be REPTDK01 strains according to Centers for Disease Control and Prevention (CDC)-defined core genome multilocus sequencing typing (cgMLST) allele range. Grey nodes indicate genomes not assigned to REPTDK01. f Genomes isolated from different sources.

Article Snippet: Legends are left of each network. a National Center for Biotechnology Information (NCBI) SNP (single-nucleotide polymorphism) cluster across JI-groups (obtained from Pathogen Detection Isolate Browser ). b Condensed allele code across JI-groups.

Techniques: Labeling, Plasmid Preparation, Control, Sequencing, Isolation

Journal: Nature Communications

Article Title: Pangenome dynamics and population structure of the zoonotic pathogen Salmonella enterica serotype Hadar

doi: 10.1038/s41467-025-68026-3

Figure Lengend Snippet: The network contains 3384 nodes, connected when JI ≥ 0.988. Eighteen groups (JI-groups A-R) are labeled, singleton genomes that do not associate with a JI-group are displayed around the outside of the network. Genomes are colored according to JI-group. Counts and percentages of genomes within each JI-group, along with their dominant National Center for Biotechnology (NCBI) SNP (single nucleotide polymorphism) cluster and plasmid taxonomic unit (PTU) profile are included in the table.

Article Snippet: Legends are left of each network. a National Center for Biotechnology Information (NCBI) SNP (single-nucleotide polymorphism) cluster across JI-groups (obtained from Pathogen Detection Isolate Browser ). b Condensed allele code across JI-groups.

Techniques: Labeling, Plasmid Preparation

Journal: Journal of Clinical Microbiology

Article Title: InhA1, NprA, and HlyII as Candidates for Markers To Differentiate Pathogenic from Nonpathogenic Bacillus cereus Strains ▿

doi: 10.1128/JCM.02123-09

Figure Lengend Snippet: Expression profiles of inhA1, nprA, and hlyII among B. cereus strains. Logarithmic representation of the expression levels of inhA1, nprA, and hlyII among 57 (54 for inhA1) B. cereus nonpathogenic (NP), food-poisoning (FP), and clinical (C) strains normalized to the mean expression value of the two reference genes rpoA and rpoB. For each strain, the mean value of two independent experiments done in duplicate is represented as a cross. For each pathogenic profile, the mean value is represented as a dot and the median value as a bold line. The × symbol represents the nprA expression level of the NVH 0391/98 strain.

Article Snippet: In some strains, if the targeted gene had not been detected with the first primer pair, a second screen was performed with another primer pair (Table ). table ft1 table-wrap mode="anchored" t5 TABLE 2. caption a7 Primer purpose and target gene (locus tag a ) Primer b Primer sequence c (5′-3′) Location within gene a Annealing temp (°C) Product size (bp) Reference or source PCR for inhA1 , nprA , and hlyII detection inhA1 (BC1284) F-inhA1-d1 ACGCITTIAAATTTGCICG 1937-1955 55 257 This study R-inhA1-d1 ACGCGTTGGAGATACAACTT 2193-2174 F-inhA1-d2 CCAGCTTGGAAAGTTGTATC 2164-2183 55 166 This study R-inhA1-d2 CAGCTTGTCCTACTACTTCA 2329-2310 nprA (BC0602) F-nprA-d GTATACGGAGATGGTGATGG 1111-1130 55 263 This study R-nprA-d GGATCACTCATAGAGCGAAG 1373-1354 hlyII (BC3523) Fhly-II GATTCTAAAGGAACTGTAG 94-112 48 868 19 Rhly-II GGTTATCAAGAGTAACTTG 961-943 F-hlyII-d CAAGTTACTCTTGATAACC 943-961 48 194 This study R-hlyII-dq TCACCATTTACAAAGATACC 1136-1117 PCR amplification of hlyII probe hlyII (BC3523) F-hlyII-p GCATTTGCAGATTCTAAAGG 85-104 55 1,019 This study R-hlyII-p TCGTAACTGATACCATAACC 1103-1084 RT-qPCR inhA1 (BC1284) F-inhA1-q GATAAAAC(A/G)CCAGCTTGGAAAG 2155-2176 60 189 This study R-inhA1-q TGCAGA(A/T)TTATCATCAGCTTG 2343-2323 nprA (BC0602) F-nprA-q TGCAGCAGCAGTAGATGCTC 951-970 60 188 This study R-nprA-q ATGTTACACCATCACCATC 1138-1120 hlyII (BC3523) F-hlyII-q CTGGAAAAACCATCAAGTTACTC 930-952 60 207 This study R-hlyII-dq TCACCATTTACAAAGATACC 1136-1117 rpoA (BC0158) F-rpoA-q TAACTCCTTACGTCGTATTC 111-130 60 186 This study R-rpoA-q ATTTCCAACGTCTTCTCTTC 296-277 rpoB (BC0122) F-rpoB-q TCAGTGGTTTCTTGATGAGG 111-130 60 176 This study R-rpoB-q CTTTTACACGAAGTGGTGCT 286-267 Open in a separate window a Reference strain Bacillus cereus ATCC 14579 (NCBI accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_004722","term_id":"30018278","term_text":"NC_004722"}} NC_004722 ). b F, forward primer; R, reverse primer. c I, inosine.

Techniques: Expressing