140 results on '"Mongkolsuk, S."'
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2. Characterization of transcription organization and analysis of unique expression patterns of an alkyl hydroperoxide reductase C gene (ahpC) and the peroxide regulator operon ahpF-oxyR-orfX from Xanthomonas campestris pv. phaseoli
- Author
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Mongkolsuk, S, primary, Loprasert, S, additional, Whangsuk, W, additional, Fuangthong, M, additional, and Atichartpongkun, S, additional
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- 1997
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3. Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX
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Loprasert, S, primary, Atichartpongkun, S, additional, Whangsuk, W, additional, and Mongkolsuk, S, additional
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- 1997
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4. Heterologous growth phase- and temperature-dependent expression and H2O2 toxicity protection of a superoxide-inducible monofunctional catalase gene from Xanthomonas oryzae pv. oryzae
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Mongkolsuk, S, primary, Loprasert, S, additional, Vattanaviboon, P, additional, Chanvanichayachai, C, additional, Chamnongpol, S, additional, and Supsamran, N, additional
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- 1996
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5. Unusual Growth Phase and Oxygen Tension Regulation of Oxidative Stress Protection Enzymes, Catalase and Superoxide Dismutase, in the Phytopathogen Xanthomonas oryzae pv. oryzae
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Chamnongpol, S, primary, Mongkolsuk, S, additional, Vattanaviboon, P, additional, and Fuangthong, M, additional
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- 1995
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6. Expression analysis and characterization of the mutant of a growth-phase- and starvation-regulated monofunctional catalase gene from Xanthomonas campestris pv. phaseoli
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Vattanaviboon, P. and Mongkolsuk, S.
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- 2000
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7. Characterization and mutagenesis of fur gene from Burkholderia pseudomallei
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Loprasert, S., Sallabhan, R., Whangsuk, W., and Mongkolsuk, S.
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- 2000
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8. Characterization of a ferric uptake regulator (fur) gene from Xanthomonas campestris pv. phaseoli with unusual primary structure, genome organization, and expression patterns
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Loprasert, S., Sallabhan, R., Atichartpongkul, S., and Mongkolsuk, S.
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- 1999
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9. Regulation of the oxidative stress protective enzymes, catalase and superoxide dismutase in Xanthomonas - a review
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Loprasert, S., Vattanaviboon, P., Praituan, W., Chamnongpol, S., and Mongkolsuk, S.
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- 1996
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10. Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions
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Ambulos, N P, Mongkolsuk, S, Kaufman, J D, and Lovett, P S
- Abstract
Sequences essential to the chloramphenicol-inducible expression of cat-86, a chloramphenicol acetyltransferase gene, reside in a 144-base pair (bp) regulatory region that intervenes between the cat-86 coding sequence and its promoter. A key regulatory element within the 144-bp segment consists of a pair of inverted-repeat sequences that immediately precede the cat-86 coding region and span the ribosome-binding site for the gene. Because of the location of the inverted repeats, cat-86 transcripts are predicted to sequester the ribosome-binding site in a stable RNA stem-loop structure which should block translation of cat-86 mRNA. Chloramphenicol induction of gene expression is believed to result from ribosome-mediated destabilization of the RNA stem-loop structure, which frees the cat-86 ribosome-binding site, thereby allowing translation. In this study we demonstrated that deletion of 85 bp from the 5' end of the 144-bp regulatory region abolishes inducible expression of cat-86, although the gene is transcribed. This deletion leaves intact both the inverted repeats and the cat-86 coding sequence, and the deletion mutation is not complementable. Therefore, inducible regulation of cat-86 requires the inverted repeats plus an upstream, cis-acting regulatory region. The cis-acting region is believed to control translation of cat-86 mRNA by its essential participation in chloramphenicol-induced opening of the RNA stem-loop. cat-86 deleted for the 85-bp regulatory region and therefore virtually unexpressed was used to select for mutations that restore expression to the gene. An analysis of one mutant plasmid showed that the cat-86 gene is constitutively expressed and that this results from a duplication of the DNA sequence that spans the ribosome-binding site. The duplication provides cat-86 with two ribosome-binding sites. One of these sites is predicted to be sequestered in an RNA stem-loop, and the other is not involved in RNA secondary structure.
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- 1985
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11. Chloramphenicol-inducible gene expression in Bacillus subtilis is independent of the chloramphenicol acetyltransferase structural gene and its promoter
- Author
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Mongkolsuk, S, Ambulos, N P, and Lovett, P S
- Abstract
cat-86 specifies chloramphenicol acetyltransferase and is the indicator gene on the Bacillus subtilis promoter cloning plasmid pPL703. Insertion of promoters from various sources into pPL703 at a site ca. 144 base pairs upstream from cat-86 activates expression of cat-86, and the expression is characteristically inducible by chloramphenicol. Thus, chloramphenicol inducibility of cat-86 is independent of the promoter that is used to activate the gene. To determine whether cat-86 or its products were involved in chloramphenicol inducibility, gene replacement studies were performed. cat-86 consists of 220 codons. The lacZ gene from Escherichia coli was inserted into a promoter-containing derivative of pPL703, plasmid pPL603E, at two locations within cat-86. pPL3lac2 contains lacZ inserted in frame after codon 2 of cat-86. pPL3lac30 contains lacZ inserted in frame after codon 30 of cat-86. In both constructions, all cat coding sequences 3' to the site of the lacZ insertion were deleted. Both plasmids exhibited chloramphenicol inducibility of beta-galactosidase in B. subtilis. These studies provide the first direct demonstration that the transcription and translation products of a chloramphenicol-inducible cat gene are uninvolved in chloramphenicol inducibility of gene expression. The results localize the region essential to inducibility to the 144-base pair segment that intervenes between the site of promoter insertion and the cat-86 gene.
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- 1984
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12. Induction of the chloramphenicol acetyltransferase gene cat-86 through the action of the ribosomal antibiotic amicetin: involvement of a Bacillus subtilis ribosomal component in cat induction
- Author
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Duvall, E J, Mongkolsuk, S, Kim, U J, Lovett, P S, Henkin, T M, and Chambliss, G H
- Abstract
The plasmid gene cat-86 and the cat gene resident on pC194 each encode chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. Chloramphenicol induction has been proposed to result from chloramphenicol binding to ribosomes, which then permits the drug-modified ribosomes to perform events essential to induction. If this proposal were correct, B. subtilis mutants containing chloramphenicol-insensitive ribosomes should not permit chloramphenicol induction of either cat-86 or pC194 cat. However, we and others have been unable to isolate chloramphenicol-resistant ribosomal mutants of B. subtilis 168. We therefore developed a simple procedure for screening other antibiotics for the potential to induce cat-86 expression. One antibiotic, amicetin, was found to be an effective inducer of cat-86 but not of the cat gene on pC194. Amicetin and chloramphenicol each interact with the 50S ribosomal subunit, and the mechanism of cat-86 induction by both drugs may be similar. Amicetin-resistant mutants of B. subtilis were readily isolated, and in none of six mutants tested was cat-86 detectably inducible by amicetin, although the chloramphenicol-inducible phenotype was retained. The ami-1 mutation which is present in one of these amicetin-resistant mutants was mapped by PBS1 transduction to the "ribosomal gene cluster" adjacent to cysA. Additionally, ribosomes from cells harboring the ami-1 mutation contained an altered BL12a protein, as detected in two-dimensional polyacrylamide gel electrophoresis. Lastly, an in vitro protein-synthesizing system that uses ribosomes from an ami-1-containing cell line was more resistant to amicetin than a system that uses ribosomes from an amicetin-sensitive but otherwise isogenic strain. These results indicate that the host mutation, ami-1, which effectively abolished the inducibility of cat-86 by amicetin, altered a ribosomal component.
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- 1985
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13. Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis
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Duvall, E J, Williams, D M, Mongkolsuk, S, and Lovett, P S
- Abstract
Plasmid pPL603 is a promoter cloning vector for Bacillus subtilis and consists of a 1.1-kilobase fragment of Bacillus pumilus DNA inserted between the EcoRI and BamHI sites of pUB110. The gene cat-86, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, is located on the 1.1-kilobase cloned DNA. When pPL603 is present in B. subtilis, cat-86 is unexpressed during vegetative growth but expressed during sporulation. The regulation of cat-86 in pPL603 is due to sequences within two restriction fragments, designated P1 and R1, that precede the main coding portion of the gene. The P1 fragment promotes transcription of cat-86 only during sporulation, whereas the adjacent R1 fragment lacks promoter function but contains sequences essential to chloramphenicol inducibility. A second B. pumilus gene, cat-66, was cloned in B. subtilis and is expressed throughout the vegetative growth and sporulation cycle. The cat-66 coding region is preceded by two adjacent restriction fragments designated as P2 and R2. P1 and P2 are identical in size and share 95% conservation of base sequence. R1 and R2 are also identical in size and share 91% conservation of base sequence. Fragment substitution experiments demonstrate that R2 can functionally replace R1. The substitution of P2 for P1 promotes cat-86 expression throughout vegetative growth and sporulation. Analysis of a derivative of pPL603 in which P2 has replaced P1 demonstrates that P2 promotes transcription of cat-86 during vegetative growth and that P2 contains the start site for transcription of cat-86. Thus, P1 and P2 differ strikingly in vegetative promoter function, yet they differ by single-base substitutions at only 11 positions of 203.
- Published
- 1984
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14. Selective expression of a plasmid cat gene at a late stage of Bacillus subtilis sporulation.
- Author
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Mongkolsuk, S and Lovett, P S
- Abstract
The cat-86 gene in plasmid pPL603 specifies chloramphenicol acetyltransferase (CAT) and is selectively expressed in Bacillus subtilis at a stage in sporulation in which internal spores are first observed (approximately T8). The gene is unexpressed in vegetatively growing cells. cat-86 expression and spore formation are both blocked when cells are grown in excess glucose. cat-86 expression at T8 is due to selective transcription of the gene, since cat-86 mRNA is undetectable in vegetatively growing cells but is readily demonstrated in sporulating cells. The transcription start site for cat-86 mRNA from sporulating cells is within a 203-base-pair restriction fragment designated P1, which is located upstream from the cat coding region on pPL603 . Deletion of P1 from pPL603 eliminates the sporulation -associated expression of cat-86. Host sporulation genes, whose function is absolutely required for cat-86 expression at T8, include six early sporulation, spo0 , genes and spoIIE . Therefore, pPL603 provides a novel system in which the in vivo expression of a known, plasmid-linked gene is dependent on sporulation-specific changes in B. subtilis.
- Published
- 1984
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15. Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis
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Mongkolsuk, S, Chiang, Y W, Reynolds, R B, and Lovett, P S
- Abstract
Two restriction fragments of Bacillus subtilis DNA were identified which caused the cat-86 gene present on the promoter cloning plasmid pPL703 to be activated predominantly during postexponential growth of host cells. The postexponential increase was observed in both sporulation-positive strains and in a spoOA mutant of B. subtilis. However, the postexponential increase in the cat-86 gene product, chloramphenicol acetyltransferase, was diminished or not observed when the plasmid-containing cells were grown in the presence of excess glucose. The promoter-containing fragment, designated as 33, was mapped to a site on the B. subtilis chromosome adjacent to hisA. The other fragment, 14, mapped to a site adjacent to ctrA. When present on a high-copy vector, both fragments caused a reduction in the sporulation frequency of host cells. Fragment 33 in high copy number conferred on B. subtilis cells three additional phenotypic changes: brown colony color, intracellular inclusions, and, in a protease-deficient mutant, the production of extracellular protease activity. These activities were observed only in postexponential-phase cultures.
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- 1983
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16. Constitutive variants of the pC194 cat gene exhibit DNA alterations in the vicinity of the ribosome binding site sequence
- Author
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Ambulos, N.P., primary, Chow, J.H., additional, Mongkolsuk, S., additional, Preis, L.H., additional, Vollmar, W.R., additional, and Lovett, P.S., additional
- Published
- 1984
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17. Versatile gene cassette plasmids to facilitate the construction of generalized and specialized cloning vectors
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Mongkolsuk, S., Vattanaviboon, P., Rabibhadana, S., and Kiatpapan, P.
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- 1993
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18. Generalized and mobilizable positive-selection cloning vectors
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Mongkolsuk, S., Rabibhadana, S., Vattanaviboon, P., and Loprasert, S.
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- 1994
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19. Antibiotic Resistance Genes and crAssphage in Hospital Wastewater and a canal receiving the treatment effluent.
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Siri Y, Sresung M, Paisantham P, Mongkolsuk S, Sirikanchana K, Honda R, Precha N, and Makkaew P
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Hospital wastewater is a major hotspot for the spread of antimicrobial resistance (AMR) in aquatic ecosystems. This study aimed to investigate the prevalence of antibiotic resistance genes (ARGs) and their correlation with crAssphage in a hospital wastewater treatment plant (HWWTP) and a receiving canal. Water samples were analyzed for 94 ARGs and crAssphage relative to the 16S rRNA using high-throughput quantitative polymerase chain reaction (HT-qPCR). Subsequently, 7 ARGs and crAssphage were selected and quantified using qPCR. The results showed that the detected genes ranged from 85 to 93 out of 94 genes. The raw wastewater (WW) samples had the highest gene diversity compared to the upstream canal, which had less diversity than downstream samples, as determined by HT-qPCR. The bla
GES was the most abundant in WW samples, while qacEΔ1, merA, IS6100, tnpA, and IS26 showed high prevalence throughout the treatment processes. The concentrations of intI1, sul1, blaTEM, blaNDM, blaVIM, tetQ, mcr-1, crAssphage, and 16S rRNA, measured using qPCR, were the highest in WW and significantly reduced in treated water samples. Although some water quality parameters, such as total suspended solids and dissolved oxygen, did not significantly differ before and after treatment, removal efficiency ranged from 0.60 to 3.23 log reduction values (LRV). The highest LRV was observed for the tetQ, whereas the mcr-1 had the lowest of LRV. Strong positive correlations among the absolute concentrations of ARGs and crAssphage were observed (Spearman's rho = 0.6 - 1.0), and biochemical oxygen demand correlated with blaTEM and blaVIM (Spearman's rho = 0.6 and 0.6, respectively). These results indicate that crAssphage and water quality could reflect the distribution of other ARGs throughout the HWWTP. Further studies are needed to underscore the importance of monitoring ARGs and biomarkers such as crAssphage in HWWTPs and their receiving waters to enhance our understanding of ARG distribution., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier Ltd.)- Published
- 2024
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20. Regulation of curcumin reductase curA (PA2197) through sodium hypochlorite and N-ethylmaleimide sensing by TetR family repressor CurR (PA2196) in Pseudomonas aeruginosa.
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Duang-Nkern J, Nontaleerak B, Thongphet A, Asano K, Chujan S, Satayavivad J, Sukchawalit R, and Mongkolsuk S
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- Promoter Regions, Genetic, Curcumin pharmacology, Binding Sites, Oxidoreductases genetics, Oxidoreductases metabolism, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism, Pseudomonas aeruginosa drug effects, Sodium Hypochlorite pharmacology, Bacterial Proteins genetics, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial drug effects, Repressor Proteins genetics, Repressor Proteins metabolism
- Abstract
Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)
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- 2024
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21. Monothiol Glutaredoxin Is Essential for Oxidative Stress Protection and Virulence in Pseudomonas aeruginosa.
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Saninjuk K, Romsang A, Duang-Nkern J, Wongsaroj L, Leesukon P, Dubbs JM, Vattanaviboon P, and Mongkolsuk S
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- Animals, Humans, Virulence, Drosophila melanogaster metabolism, Oxidative Stress, Iron metabolism, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism, Glutaredoxins genetics, Glutaredoxins metabolism
- Abstract
Glutaredoxins (Grxs), ubiquitous redox enzymes belonging to the thioredoxin family, catalyze the reduction of thiol-disulfide exchange reactions in a glutathione-dependent manner. A Pseudomonas aeruginosa Δ grxD mutant exhibited hypersensitivity to oxidative stress-generating agents, such as paraquat (PQ) and cumene hydroperoxide (CHP). In vitro studies showed that P. aeruginosa GrxD acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during CHP degradation. The ectopic expression of iron-sulfur cluster ([Fe-S]) carrier proteins, including ErpA, IscA, and NfuA, complements the function of GrxD in the Δ grxD mutant under PQ toxicity. Constitutively high expression of iscR , nfuA , tpx , and fprB was observed in the Δ grxD mutant. These results suggest that GrxD functions as a [Fe-S] cluster carrier protein involved in [Fe-S] cluster maturation. Moreover, the Δ grxD mutant demonstrates attenuated virulence in a Drosophila melanogaster host model. Altogether, the data shed light on the physiological role of GrxD in oxidative stress protection and virulence of the human pathogen, P. aeruginosa. IMPORTANCE Glutaredoxins (Grxs) are ubiquitous disulfide reductase enzymes. Monothiol Grxs, containing a CXXS motif, play an essential role in iron homeostasis and maturation of [Fe-S] cluster proteins in various organisms. We now establish that the human pathogen Pseudomonas aeruginosa GrxD is crucial for bacterial virulence, maturation of [Fe-S] clusters and facilitation of Ohr enzyme activity. GrxD contains a conserved signature monothiol motif (C
29 GFS), in which C29 is essential for its function in an oxidative stress protection. Our findings reveal the physiological roles of GrxD in oxidative stress protection and virulence of P. aeruginosa.- Published
- 2023
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22. Pseudomonas aeruginosa GidA modulates the expression of catalases at the posttranscriptional level and plays a role in virulence.
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Srimahaeak T, Thongdee N, Chittrakanwong J, Atichartpongkul S, Jaroensuk J, Phatinuwat K, Phaonakrop N, Jaresitthikunchai J, Roytrakul S, Mongkolsuk S, and Fuangthong M
- Abstract
Pseudomonas aeruginosa gidA , which encodes a putative tRNA-modifying enzyme, is associated with a variety of virulence phenotypes. Here, we demonstrated that P. aeruginosa gidA is responsible for the modifications of uridine in tRNAs in vivo . Loss of gidA was found to have no impact on the mRNA levels of katA and katB , but it decreased KatA and KatB protein levels, resulting in decreased total catalase activity and a hydrogen peroxide-sensitive phenotype. Furthermore, gidA was found to affect flagella-mediated motility and biofilm formation; and it was required for the full virulence of P. aeruginosa in both Caenorhabditis elegans and macrophage models. Together, these observations reveal the posttranscriptional impact of gidA on the oxidative stress response, highlight the complexity of catalase gene expression regulation, and further support the involvement of gidA in the virulence of P. aeruginosa ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Srimahaeak, Thongdee, Chittrakanwong, Atichartpongkul, Jaroensuk, Phatinuwat, Phaonakrop, Jaresitthikunchai, Roytrakul, Mongkolsuk and Fuangthong.)
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- 2023
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23. Characterisation of the triclosan efflux pump TriABC and its regulator TriR in Agrobacterium tumefaciens C58.
- Author
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Nontaleerak B, Tasnawijitwong N, Eurtivong C, Sirikanchana K, Satayavivad J, Sukchawalit R, and Mongkolsuk S
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- Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA, Intergenic, Gene Expression Regulation, Bacterial, Operon, Promoter Regions, Genetic, Agrobacterium tumefaciens genetics, Agrobacterium tumefaciens metabolism, Triclosan metabolism, Triclosan pharmacology
- Abstract
TriR serves as a repressor for a resistance-nodulation-cell division (RND) efflux pump TriABC involved in triclosan (TCS) resistance in Agrobacterium tumefaciens. The triR gene is transcribed divergently from the triABC operon. TriR specifically bound to the triR-triA intergenic region, at an imperfect 10 bp inverted repeat, 5'-TTGACTAttC-GgtTAGTCAA-3' (TriR box), that was revealed by DNase I footprinting and electrophoretic mobility shift assay. TCS treatment appeared to up-regulate triR and triABC expression, via preventing TriR binding to the triR-triA intergenic region. Promoter-lacZ fusions and β-galactosidase activity assay further demonstrated TriR-mediated repression of triABC and triR autoregulation. Site-directed mutagenesis confirmed the identified TriR box is essential for TriR repression. A. tumefaciens mutant strains disrupting either triR or triA were constructed to determine their biological functions. The triA mutant showed hypersensitivity to TCS and sodium dodecyl sulfate (SDS), whereas the triR mutant was hyper-resistant, compared to wild-type. In addition to TCS and SDS, overproduction of TriABC from a multi-copy plasmid conferred enhanced resistance to a quaternary ammonium compound, benzalkonium chloride. Molecular modelling was able to predict the model of TriR and docking simulations were able to anticipate plausible binding interactions between TriR and TCS ligand., (Copyright © 2022 Elsevier GmbH. All rights reserved.)
- Published
- 2022
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24. Contribution of Stenotrophomonas maltophilia MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitR.
- Author
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Boonyakanog A, Charoenlap N, Chattrakarn S, Vattanaviboon P, and Mongkolsuk S
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- Bacterial Proteins genetics, Bacterial Proteins metabolism, Cysteine metabolism, Diamide metabolism, Diamide pharmacology, Gene Expression Regulation, Bacterial, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Promoter Regions, Genetic, Stenotrophomonas maltophilia genetics, Stenotrophomonas maltophilia metabolism
- Abstract
Stenotrophomonas maltophilia contains an operon comprising mfsB and mfsC, which encode membrane transporters in the major facilitator superfamily (MFS). The results of the topological analysis predicted that both MfsB and MfsC possess 12 transmembrane helices with the N- and C-termini located inside the cells. The deletion of mfsC increased the susceptibility to diamide, a chemical oxidizing agent, but not to antibiotics and oxidative stress-generating substances relative to wild-type K279a. Moreover, no altered phenotype was observed against all tested substances for the ΔmfsB mutant. The results of the expression analysis revealed that the mfsBC expression was significantly induced by exposure to diamide. The diamide-induced gene expression was mediated by DitR, a TetR-type transcriptional regulator encoded by smlt0547. A constitutively high expression of mfsC in the ditR mutant indicated that DitR acts as a transcriptional repressor of mfsBC under physiological conditions. Purified DitR was bound to three sites spanning from position + 21 to -57, corresponding to the putative mfsBC promoter sequence, thereby interfering with the binding of RNA polymerase. The results of electrophoretic mobility shift assays illustrated that the treatment of purified DitR with diamide caused the release of DitR from the mfsBC promoter region, and the diamide sensing mechanism of DitR required two conserved cysteine residues, Cys92 and Cys127. This suggests that exposure to diamide can oxidize DitR through the oxidation of cysteine residues, leading to its release from the promoter, thus allowing mfsBC transcription. Overall, MfsC and DitR play a role in adaptive resistance against the diamide of S. maltophilia., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
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25. Effects of face masks and ventilation on the risk of SARS-CoV-2 respiratory transmission in public toilets: a quantitative microbial risk assessment.
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Denpetkul T, Pumkaew M, Sittipunsakda O, Leaungwutiwong P, Mongkolsuk S, and Sirikanchana K
- Subjects
- Humans, Bathroom Equipment, Cough, Risk Assessment, SARS-CoV-2, Public Health, Thailand, Communicable Disease Control, COVID-19 prevention & control, Masks
- Abstract
Public toilets may increase the risk of COVID-19 infection via airborne transmission; however, related research is limited. We aimed to estimate SARS-CoV-2 infection risk through respiratory transmission using a quantitative microbial risk assessment framework by retrieving SARS-CoV-2 concentrations from the swab tests of 251 Thai patients. Three virus-generating scenarios were investigated: an infector breathing, breathing with a cough, and breathing with a sneeze. The infection risk (95th percentile) was as high as 10
-1 with breathing and increased to 1 with a cough or a sneeze. No significant gender differences for toilet users (receptors) were noted. The highest risk scenario, namely breathing with a sneeze, was further evaluated for risk mitigation measures. Mitigation to a lower risk under 10-3 succeeded only when the infector and the receptor both wore N95 respirators or surgical masks. Ventilation of up to 20 air changes per hour (ACH) did not decrease the risk. However, an extended waiting time of 10 min between an infector and a receptor resulted in approximately 1.0-log10 further risk reduction when both wore masks with the WHO-recommended 12 ACH. The volume of expelled droplets, virus concentrations, and receptor dwell time were identified as the main contributors to transmission risk.- Published
- 2022
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26. NieR is the repressor of a NaOCl-inducible efflux system in Agrobacterium tumefaciens C58.
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Duang-Nkern J, Nontaleerak B, Udomkanarat T, Saninjuk K, Sukchawalit R, and Mongkolsuk S
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- Biological Transport, Hydrogen Peroxide pharmacology, Operon, Agrobacterium tumefaciens genetics, Agrobacterium tumefaciens metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism
- Abstract
The Agrobacterium tumefaciens atu4217 gene, which encodes a TetR family transcription regulator, is a repressor of the atu4218-atu4219-atu4220 operon. The Atu4218 and Atu4219 proteins belong to the HlyD family (membrane fusion protein) and the AcrB/AcrD/AcrF family (inner membrane transporter), respectively, and may form an efflux pump. The atu4220 gene encodes a short-chain dehydrogenase. Quantitative real-time PCR analysis showed induction of atu4217 and atu4218 by NaOCl but not by N-ethylmaleimide or reactive oxygen species (ROS) including H
2 O2 , menadione and cumene hydroperoxide; therefore, the atu4218 and atu4219 were named NaOCl-inducible efflux genes nieA and nieB, respectively. The atu4217 gene, which was named nieR, serves as a repressor of nieA and nieB. DNase I footprinting assays identified 20-bp imperfect inverted repeat (IR, underlined) motifs 5'-TAGATTTAGGATGCAATCTA-3' (box A) and 5'-TAGATTTCACTTGACATCTA-3' (box R) in the intergenic region of the divergent nieA and nieR genes; these motifs were recognized by the NieR protein. Electrophoretic mobility shift assays demonstrated that NieR specifically binds to the 20-bp IR motifs and that NaOCl prevents this NieR-DNA interaction. Promoter-lacZ fusions and mutagenesis of the NieR boxes (A and R) showed a more dominant role for box A than for box R in the repression of the nieA and nieR promoters. However, full repression of either promoter required both operators. The nieR mutant strain exhibited a small colony phenotype and was more sensitive than the wild-type to NaOCl and antibiotics, including ciprofloxacin, nalidixic acid, novobiocin, and tetracycline. By contrast, the nieAB mutant strain showed no phenotype changes under the tested conditions., (Copyright © 2021 Elsevier GmbH. All rights reserved.)- Published
- 2021
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27. Host Chromatin Regulators Required for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Activity in Saccharomyces cerevisiae Model.
- Author
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Denmongkholchai S, Tsuruda K, Sugai M, Mongkolsuk S, and Matangkasombut O
- Subjects
- Bacterial Toxins metabolism, Chromatin metabolism, Gene Expression, Gene Expression Regulation, Host-Pathogen Interactions immunology, Humans, Mutation, Saccharomyces cerevisiae metabolism, Aggregatibacter actinomycetemcomitans physiology, Bacterial Toxins genetics, Chromatin genetics, Host-Pathogen Interactions genetics, Pasteurellaceae Infections genetics, Pasteurellaceae Infections microbiology, Saccharomyces cerevisiae genetics
- Abstract
Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, Aa CdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.
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- 2021
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28. An inexpensive point-of-care immunochromatographic test for Talaromyces marneffei infection based on the yeast phase specific monoclonal antibody 4D1 and Galanthus nivalis agglutinin.
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Pruksaphon K, Intaramat A, Simsiriwong P, Mongkolsuk S, Ratanabanangkoon K, Nosanchuk JD, Kaltsas A, and Youngchim S
- Subjects
- Antigens, Surface urine, Enzyme-Linked Immunosorbent Assay methods, Gold Colloid chemistry, Humans, Limit of Detection, Mannose-Binding Lectin immunology, Mannose-Binding Lectins immunology, Metal Nanoparticles chemistry, Neglected Diseases diagnosis, Neglected Diseases microbiology, Plant Lectins immunology, Talaromyces isolation & purification, Antibodies, Monoclonal immunology, Antigens, Fungal urine, Immunoassay methods, Mycoses diagnosis, Point-of-Care Testing, Talaromyces immunology
- Abstract
Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1-GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA-MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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29. Human Fecal Pollution Monitoring and Microbial Risk Assessment for Water Reuse Potential in a Coastal Industrial-Residential Mixed-Use Watershed.
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Kongprajug A, Denpetkul T, Chyerochana N, Mongkolsuk S, and Sirikanchana K
- Abstract
Rapid economic development has caused industrial expansion into residential communities, leading to higher fecal pollution loads that could be discharged into aquatic environments. However, little is known regarding the potential microbial impact on human health. This study investigated microbial contamination from coastal industrial-residential community areas in nine sampling sites in waterways during three dry events. A general microbial source tracking (MST) marker, GenBac3, was detected in all samples from all three events, indicating continuing fecal pollution in the area, mostly from human sewage contamination. This was shown by the human-specific genetic marker crAssphage (88.9%) and human polyomavirus (HPyVs; 92.6%) detection. Enteric human adenovirus (HAdV40/41) showed three positive results only from residential sites in the first event. No spatial difference was observed for MST markers and traditional fecal indicators (total coliforms and Escherichia coli ) in each event. Still, a significantly lower abundance of GenBac3, HPyVs, and total coliforms in the first sampling event was detected. Spearman's rho analysis indicated a strong correlation among certain pairs of microbial parameters. Multivariate analysis revealed two clusters of samples separated by land use type (industrial vs. residential). According to factor analysis of mixed data, the land use parameter was more associated with physicochemical parameters (i.e., salinity, conductivity, water temperature, and dissolved oxygen). A Quantitative Microbial Risk Assessment (QMRA) was then conducted to estimate the annual infection risks of HAdV40/41 for non-potable water reuse purposes using predicted concentrations from crAssphage and HPyVs. The highest risks (95th percentiles) were ranked by food crop irrigation, aquaculture, and toilet flushing, at 10
-1 , 10-2 , and 10-3 per person per year (pppy). Required treatment levels to achieve a 10-4 pppy annual infection risk were estimated. QMRA-based water treatment scenarios were suggested, including chlorination for toilet flushing reuse and depth filtration prior to chlorination for aquaculture and food crop irrigation. Microbial monitoring combined with a QMRA could provide better insights into fecal pollution patterns and the associated risks, facilitating effective water quality management and appropriate prior treatments for water reuse., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kongprajug, Denpetkul, Chyerochana, Mongkolsuk and Sirikanchana.)- Published
- 2021
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30. One Health drivers of antibacterial resistance: Quantifying the relative impacts of human, animal and environmental use and transmission.
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Booton RD, Meeyai A, Alhusein N, Buller H, Feil E, Lambert H, Mongkolsuk S, Pitchforth E, Reyher KK, Sakcamduang W, Satayavivad J, Singer AC, Sringernyuang L, Thamlikitkul V, Vass L, Avison MB, and Turner KME
- Abstract
Objectives: Antibacterial resistance (ABR) is a major global health security threat, with a disproportionate burden on lower-and middle-income countries (LMICs). It is not understood how 'One Health', where human health is co-dependent on animal health and the environment, might impact the burden of ABR in LMICs. Thailand's 2017 "National Strategic Plan on Antimicrobial Resistance" (NSP-AMR) aims to reduce AMR morbidity by 50% through 20% reductions in human and 30% in animal antibacterial use (ABU). There is a need to understand the implications of such a plan within a One Health perspective., Methods: A model of ABU, gut colonisation with extended-spectrum beta-lactamase (ESBL)-producing bacteria and transmission was calibrated using estimates of the prevalence of ESBL-producing bacteria in Thailand. This model was used to project the reduction in human ABR over 20 years (2020-2040) for each One Health driver, including individual transmission rates between humans, animals and the environment, and to estimate the long-term impact of the NSP-AMR intervention., Results: The model predicts that human ABU was the most important factor in reducing the colonisation of humans with resistant bacteria (maximum 65.7-99.7% reduction). The NSP-AMR is projected to reduce human colonisation by 6.0-18.8%, with more ambitious targets (30% reductions in human ABU) increasing this to 8.5-24.9%., Conclusions: Our model provides a simple framework to explain the mechanisms underpinning ABR, suggesting that future interventions targeting the simultaneous reduction of transmission and ABU would help to control ABR more effectively in Thailand., Competing Interests: Declarations of interest: none, (© 2021 The Authors.)
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- 2021
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31. Over-Expression of Hypochlorite Inducible Major Facilitator Superfamily (MFS) Pumps Reduces Antimicrobial Drug Susceptibility by Increasing the Production of MexXY Mediated by ArmZ in Pseudomonas aeruginosa .
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Dulyayangkul P, Satapoomin N, Avison MB, Charoenlap N, Vattanaviboon P, and Mongkolsuk S
- Abstract
Pseudomonas aeruginosa , a well - known cause of nosocomial infection, is frequently antibiotic resistant and this complicates treatment. Links between oxidative stress responses inducing antibiotic resistance through over - production of RND - type efflux pumps have been reported in P. aeruginosa , but this has not previously been associated with MFS - type efflux pumps. Two MFS efflux pumps encoded by mfs1 and mfs2 were selected for study because they were found to be sodium hypochlorite (NaOCl) inducible. Antibiotic susceptibility testing was used to define the importance of these MFS pumps in antibiotic resistance and proteomics was used to characterize the resistance mechanisms involved. The results revealed that mfs1 is NaOCl inducible whereas mfs2 is NaOCl, N - Ethylmaleimide and t - butyl hydroperoxide inducible. Deletion of mfs1 or mfs2 did not affect antibiotic or paraquat susceptibility. However, over - production of Mfs1 and Mfs2 reduced susceptibility to aminoglycosides, quinolones, and paraquat. Proteomics, gene expression analysis and targeted mutagenesis showed that over - production of the MexXY RND - type efflux pump in a manner dependent upon armZ , but not amgRS , is the cause of reduced antibiotic susceptibility upon over - production of Mfs1 and Mfs2. mexXY operon expression analysis in strains carrying various lengths of mfs1 and mfs2 revealed that at least three transmembrane domains are necessary for mexXY over - expression and decreased antibiotic susceptibility. Over-expression of the MFS-type efflux pump gene tetA (C) did not give the same effect. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs1 and Mfs2. Altogether, this is the first evidence of cascade effects where the over - production of an MFS pump causes over - production of an RND pump, in this case MexXY via increased armZ expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dulyayangkul, Satapoomin, Avison, Charoenlap, Vattanaviboon and Mongkolsuk.)
- Published
- 2021
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32. Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications - A systematic review.
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Leifels M, Cheng D, Sozzi E, Shoults DC, Wuertz S, Mongkolsuk S, and Sirikanchana K
- Abstract
Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009-2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μM; incubation on ice or at room temperature (20 - 25 °C) for 5-120 min; and dye activation using LED or high light (500-800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)
- Published
- 2020
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33. A highly sensitive biosensor with a single-copy evolved sensing cassette for chlorpyrifos pesticide detection.
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Chouichit P, Whangsuk W, Sallabhan R, Mongkolsuk S, and Loprasert S
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- Bacterial Proteins genetics, Bacterial Proteins metabolism, Directed Molecular Evolution, Escherichia coli drug effects, Escherichia coli genetics, Escherichia coli metabolism, Glycine analogs & derivatives, Glycine metabolism, Limit of Detection, Piperazines pharmacology, Promoter Regions, Genetic, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sinorhizobium meliloti genetics, Sulfatases genetics, Sulfatases metabolism, Transcription Factors genetics, Transcription Factors metabolism, Biosensing Techniques methods, Chlorpyrifos isolation & purification, Pesticides isolation & purification
- Abstract
A formylglycine-generating enzyme (FGE)-sulfatase-based whole-cell biosensor was genetically improved into a single-copy system by integrating the Sinorhizobium meliloti transcriptional activator ChpR and the chpA promoter-FGE-sulfatase fusion into the Escherichia coli chromosome. The sensitivity was further enhanced through a random mutagenesis of the chpR . The new integrated biosensor offered both a lower detection limit [5 nM chlorpyrifos (CPF)] and fluorescence background. The ready-to-use kit was developed using silica gel for on-field detection. The biosensor kit was stable for 20 days when stored at 4 °C. Moreover, a 1-(1-naphthylmethyl)-piperazine (NMP) efflux pump inhibitor can improve the sensitivity by 57 %.
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- 2020
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34. Roles of RcsA, an AhpD Family Protein, in Reactive Chlorine Stress Resistance and Virulence in Pseudomonas aeruginosa.
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Nontaleerak B, Duang-Nkern J, Wongsaroj L, Trinachartvanit W, Romsang A, and Mongkolsuk S
- Subjects
- Bacterial Proteins metabolism, Oxidants pharmacology, Pseudomonas aeruginosa drug effects, Virulence drug effects, Anti-Bacterial Agents pharmacology, Bacterial Proteins genetics, Disinfectants pharmacology, Hypochlorous Acid pharmacology, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa pathogenicity
- Abstract
Reactive chlorine species (RCS), particularly hypochlorous acid (HOCl), are powerful antimicrobial oxidants generated by biological pathways and chemical syntheses. Pseudomonas aeruginosa is an important opportunistic pathogen that has adapted mechanisms for protection and survival in harsh environments, including RCS exposure. Based on previous transcriptomic studies of HOCl exposure in P. aeruginosa , we found that the expression of PA0565 , or rcsA , which encodes an alkyl hydroperoxidase D-like protein, exhibited the highest induction among the RCS-induced genes. In this study, rcsA expression was dominant under HOCl stress and greatly increased under HOCl-related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid residues Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After allelic exchange mutagenesis in the P. aeruginosa rcsA , the P. aeruginosa rcsA deletion mutant showed significantly decreased HOCl resistance. Ectopic expression of P. aeruginosa rcsA led to significantly increased NaOCl resistance in Escherichia coli Moreover, the pathogenicity of the rcsA mutant decreased dramatically in both Caenorhabditis elegans and Drosophila melanogaster host model systems compared to the wild type (WT). Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful at complementing phenotypes of the rcsA mutant. Overall, our data indicate the importance of P. aeruginosa RcsA in defense against HOCl stress under disinfections and during infections of hosts, which involves the catalytic Cys60, Cys63, and His67 residues. IMPORTANCE Pseudomonas aeruginosa is a common pathogen that is a major cause of serious infections in many hosts. Hypochlorous acid (HOCl) is a potent antimicrobial agent found in household bleach and is a widely used disinfectant. P. aeruginosa has evolved adaptive mechanisms for protection and survival during HOCl exposure. We identified P. aeruginosa rcsA as a HOCl-responsive gene encoding an antioxidant protein that may be involved in HOCl degradation. RcsA has a distinguishing core motif containing functional Cys60, Cys63, and His67 residues. P. aeruginosa rcsA plays an important role in bleach tolerance, with expression of P. aeruginosa rcsA in Escherichia coli also conferring HOCl resistance. Interestingly, RcsA is required for full virulence in worm and fruit fly infection models, indicating a correlation between mechanisms of bleach toxicity and host immunity during infection. This provides new insights into the mechanisms used by P. aeruginosa to persist in harsh environments such as hospitals., (Copyright © 2020 American Society for Microbiology.)
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- 2020
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35. Wss1 homolog from Candida albicans and its role in DNA-protein crosslink tolerance.
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Homchan A, Sukted J, Mongkolsuk S, Jeruzalmi D, Matangkasombut O, and Pakotiprapha D
- Subjects
- Amino Acid Sequence, Candida albicans genetics, DNA Repair, DNA, Fungal genetics, DNA-Binding Proteins genetics, Fungal Proteins genetics, Genomic Instability, Small Ubiquitin-Related Modifier Proteins metabolism, Candida albicans metabolism, DNA Damage, DNA-Binding Proteins metabolism, Fungal Proteins metabolism
- Abstract
Candida albicans is an opportunistic yeast that can cause life-threatening systemic infection in immunocompromised individuals. During infections, C. albicans has to cope with genotoxic stresses generated by the host immune system. DNA-protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential for C. albicans to maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) in C. albicans (CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed that CaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain of CaWss1 is essential for its cellular function. Interactions of CaWss1 with Cdc48 and small ubiquitin-like modifier, although not strictly required, contribute to the function of CaWss1 in the suppression of the growth defects under DPC-inducing conditions. This report is the first investigation of the role of CaWss1 in DPC tolerance in C. albicans., (© 2020 John Wiley & Sons Ltd.)
- Published
- 2020
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36. Detection of superoxide dismutase (Cu-Zn) isoenzymes in leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl orchid by comparative proteomic analysis.
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Huehne PS, Bhinija K, Srisomsap C, Chokchaichamnankit D, Weeraphan C, Svasti J, and Mongkolsuk S
- Abstract
Typically, biological systems are protected from the toxic effect of free radicals by antioxidant defense. Extracts from orchids have been reported to show high levels of exogenous antioxidant activity including Bulbophyllum orchids but so far, there have been no reports on antioxidant enzymes. Therefore, differences in protein expression from leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl and Dendrobium Sonia Earsakul were studied using two-dimensional gel electrophoresis and mass spectrometry (LC/MS/MS). Interestingly, the largest group of these stress response proteins were associated with antioxidant defense and temperature stress, including superoxide dismutase (Cu-Zn) and heat shock protein 70. The high expression of this antioxidant enzyme from Bulbophyllum morphologlorum Kraenzl was confirmed by activity staining on native-PAGE, and the two Cu/Zn-SODs isoenzymes were identified as Cu/Zn-SOD 1 and Cu/Zn-SOD 2 by LC/MS/MS. The results suggested that Bulbophyllum orchid can be a potential plant source for medicines and natural antioxidant supplements., Competing Interests: The authors declared that they have no conflict of interest., (© 2020 The Author(s).)
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- 2020
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37. Cefoperazone induces esterase B expression by EstR and esterase B enhances cefoperazone activity at the periplasm.
- Author
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Toewiwat N, Whangsuk W, Ploypradith P, Mongkolsuk S, and Loprasert S
- Subjects
- Escherichia coli genetics, Gene Expression Regulation, Bacterial, Periplasm drug effects, Protein Sorting Signals, Sphingomonadaceae enzymology, Sphingomonadaceae genetics, Cefoperazone pharmacology, Cephalosporins pharmacology, Periplasm enzymology, Serine Endopeptidases genetics, Sphingomonadaceae drug effects, Transcription Factors genetics
- Abstract
The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected β-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to β-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)
- Published
- 2020
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38. Crystal structure and catalytic mechanism of the essential m 1 G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa .
- Author
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Jaroensuk J, Wong YH, Zhong W, Liew CW, Maenpuen S, Sahili AE, Atichartpongkul S, Chionh YH, Nah Q, Thongdee N, McBee ME, Prestwich EG, DeMott MS, Chaiyen P, Mongkolsuk S, Dedon PC, Lescar J, and Fuangthong M
- Subjects
- Catalysis, Crystallography, X-Ray, Kinetics, Protein Binding, Protein Conformation, RNA, Transfer metabolism, S-Adenosylmethionine metabolism, Substrate Specificity, tRNA Methyltransferases chemistry, tRNA Methyltransferases isolation & purification, Pseudomonas aeruginosa enzymology, tRNA Methyltransferases metabolism
- Abstract
The tRNA (m
1 G37) methyltransferase TrmD catalyzes m1 G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa ( Pa TrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of Pa TrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as Pa TrmD substrates. Analysis of steady-state kinetics with S -adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that Pa TrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to Pa TrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM ( Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA ( Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric Pa TrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into Pa TrmD as a potential target for the development of antibiotics., (© 2019 Jaroensuk et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)- Published
- 2019
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39. Inactivation of the Agrobacterium tumefaciens ActSR system affects resistance to multiple stresses with increased H 2 O 2 sensitivity due to reduced expression of hemH .
- Author
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Kullapanich C, Dubbs JM, and Mongkolsuk S
- Subjects
- Agrobacterium tumefaciens genetics, Agrobacterium tumefaciens metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites, Catalase metabolism, Gene Expression Regulation, Bacterial, Oxidation-Reduction, Promoter Regions, Genetic, Protein Kinases genetics, RNA Helicases genetics, RNA Helicases metabolism, Stress, Physiological, Transcription Factors genetics, Transcription, Genetic, Virulence, Adaptation, Physiological physiology, Agrobacterium tumefaciens physiology, Ferrochelatase genetics, Hydrogen Peroxide metabolism, Protein Kinases metabolism, Transcription Factors metabolism
- Abstract
The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl
3 ), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H2 O2 ) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH , encoding ferrochelatase, restored wild-type catalase activity and H2 O2 resistance levels. Gel mobility shift and hemH promoter -lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H2 O2 resulting from a reduction in catalase activity due to reduced expression of hemH .- Published
- 2019
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40. TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa.
- Author
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Thongdee N, Jaroensuk J, Atichartpongkul S, Chittrakanwong J, Chooyoung K, Srimahaeak T, Chaiyen P, Vattanaviboon P, Mongkolsuk S, and Fuangthong M
- Subjects
- Amino Acid Sequence, Guanosine genetics, Humans, Hydrogen Peroxide chemistry, Oxidation-Reduction, Oxidative Stress drug effects, Pseudomonas Infections genetics, Pseudomonas Infections microbiology, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa pathogenicity, RNA, Transfer drug effects, RNA, Transfer genetics, Bacterial Proteins genetics, Catalase genetics, Oxidative Stress genetics, tRNA Methyltransferases genetics
- Abstract
Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
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41. Transcriptional regulation of the Pseudomonas aeruginosa iron-sulfur cluster assembly pathway by binding of IscR to multiple sites.
- Author
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Saninjuk K, Romsang A, Duang-Nkern J, Vattanaviboon P, and Mongkolsuk S
- Subjects
- Base Sequence, Binding Sites, Iron-Sulfur Proteins metabolism, Models, Biological, Mutation genetics, Promoter Regions, Genetic, Protein Binding, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Iron-Sulfur Proteins genetics, Pseudomonas aeruginosa genetics, Transcription, Genetic
- Abstract
Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5'AWWSSYRMNNWWWTNNNWSGGNYWW3'. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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42. The Agrobacterium tumefaciens atu3184 gene, a member of the COG0523 family of GTPases, is regulated by the transcriptional repressor Zur.
- Author
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Khemthong S, Nuonming P, Nookabkaewb S, Sukchawalit R, and Mongkolsuk S
- Subjects
- Agrobacterium tumefaciens growth & development, DNA Footprinting, DNA, Bacterial, Escherichia coli, Gene Expression Regulation, Bacterial, Molecular Chaperones, Operon, Promoter Regions, Genetic, Recombinant Proteins, Virulence genetics, Zinc metabolism, Agrobacterium tumefaciens genetics, Bacterial Proteins genetics, DNA-Binding Proteins genetics, Escherichia coli Proteins genetics, GTP Phosphohydrolases genetics, Repressor Proteins genetics, Transcription Factors genetics
- Abstract
Analysis of the Agrobacterium tumefaciens C58 genome revealed a potential Zur (zinc uptake regulator) binding site (5'-GATATGTTATTACATTAC-3', the underlined letters are the center of symmetry of the inverted palindrome) located in the upstream region of atu3184, whose gene product is a member of the COG0523 subfamily of G3E GTPases. The specific interaction of the Zur protein with the 18-bp inverted repeat operator motif in the presence of zinc was demonstrated in vitro by a DNA band shift assay and a DNase I footprinting assay. A LacZ reporter fusion assay further confirmed that Zur negatively regulates atu3184 promoter activity in vivo. The expression of atu3184 was upregulated in response to zinc limitation in the wild-type strain, but the zur mutant strain exhibited high-level constitutive expression of atu3184 under all conditions, irrespective of the zinc levels. It is likely that A. tumefaciens Zur senses zinc and directly regulates the atu3184 promoter by a molecular mechanism similar to that of Escherichia coli Zur, where the operator DNA is surrounded by four Zur monomers forming two dimers bound on the opposite sides of the DNA duplex. Disruption of atu3184 did not affect cell growth under metal-limited conditions and had no effect on the total cellular zinc content. Furthermore, an A. tumefaciens strain lacking atu3184 caused a tumor disease in a host plant., (Copyright © 2019 Elsevier GmbH. All rights reserved.)
- Published
- 2019
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43. Genome-Wide Identification of Host Genes Required for Toxicity of Bacterial Cytolethal Distending Toxin in a Yeast Model.
- Author
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Denmongkholchai S, Katare P, Choochuay S, Thanyasrisung P, Tsuruda K, Sugai M, Mongkolsuk S, and Matangkasombut O
- Abstract
Background: Aggregatibacter actinomycetemcomitans , a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity., Objective: To investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model., Methods: We transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type., Results: Out of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity., Conclusion: The genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication.
- Published
- 2019
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44. Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation.
- Author
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Wongsaroj L, Saninjuk K, Romsang A, Duang-Nkern J, Trinachartvanit W, Vattanaviboon P, and Mongkolsuk S
- Subjects
- Animals, Bacterial Proteins genetics, Cell Movement, Drosophila melanogaster microbiology, Ethylmaleimide pharmacology, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Mutation, Oligopeptides genetics, Oligopeptides metabolism, Oxidants chemistry, Paraquat pharmacology, Pseudomonas Infections, Pseudomonas aeruginosa genetics, Pyocyanine genetics, Pyocyanine metabolism, Virulence Factors genetics, Virulence Factors metabolism, Bacterial Proteins metabolism, Biofilms, Genes, Bacterial, Glutathione biosynthesis, Pseudomonas aeruginosa metabolism, Virulence
- Abstract
Pseudomonas aeruginosa PAO1 contains gshA and gshB genes, which encode enzymes involved in glutathione (GSH) biosynthesis. Challenging P. aeruginosa with hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increased the expression of gshA and gshB. The physiological roles of these genes in P. aeruginosa oxidative stress, bacterial virulence, and biofilm formation were examined using P. aeruginosa ΔgshA, ΔgshB, and double ΔgshAΔgshB mutant strains. These mutants exhibited significantly increased susceptibility to methyl viologen, thiol-depleting agent, and methylglyoxal compared to PAO1. Expression of functional gshA, gshB or exogenous supplementation with GSH complemented these phenotypes, which indicates that the observed mutant phenotypes arose from their inability to produce GSH. Virulence assays using a Drosophila melanogaster model revealed that the ΔgshA, ΔgshB and double ΔgshAΔgshB mutants exhibited attenuated virulence phenotypes. An analysis of virulence factors, including pyocyanin, pyoverdine, and cell motility (swimming and twitching), showed that these levels were reduced in these gsh mutants compared to PAO1. In contrast, biofilm formation increased in mutants. These data indicate that the GSH product and the genes responsible for GSH synthesis play multiple crucial roles in oxidative stress protection, bacterial virulence and biofilm formation in P. aeruginosa., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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45. Characterization and regulation of AcrABR, a RND-type multidrug efflux system, in Agrobacterium tumefaciens C58.
- Author
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Nuonming P, Khemthong S, Dokpikul T, Sukchawalit R, and Mongkolsuk S
- Subjects
- Anti-Bacterial Agents metabolism, Binding Sites, DNA Footprinting, DNA, Bacterial chemistry, DNA, Bacterial metabolism, Drug Resistance, Multiple, Bacterial, Drug Tolerance, Electrophoretic Mobility Shift Assay, Gene Expression Profiling, Indoles metabolism, Promoter Regions, Genetic, Protein Binding, Real-Time Polymerase Chain Reaction, Agrobacterium tumefaciens genetics, Agrobacterium tumefaciens metabolism, Gene Expression Regulation, Bacterial drug effects, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Repressor Proteins genetics, Repressor Proteins metabolism
- Abstract
Agrobacterium tumefaciens AcrR is the transcriptional repressor of the acrABR operon. The AcrAB efflux pump confers resistance to various toxic compounds, including antibiotics [ciprofloxacin (CIP), nalidixic acid (NAL), novobiocin (NOV) and tetracycline (TET)], a detergent [sodium dodecyl sulfate (SDS)] and a biocide [triclosan (TRI)]. The sequence to which AcrR specifically binds in the acrA promoter region was determined by EMSA and DNase I footprinting. The AcrR-DNA interaction was abolished by adding NAL, SDS and TRI. Quantitative real time-PCR analysis showed that induction of the acrA transcript occurred when wild-type cells were exposed to NAL, SDS and TRI. Indole is a signaling molecule that increases the antibiotic resistance of bacteria, at least in part, through activation of efflux pumps. Expression of the A. tumefaciens acrA transcript was also inducible by indole in a dose-dependent manner. Indole induced protection against CIP, NAL and SDS but enhanced susceptibility to NOV and TRI. Additionally, the TET resistance of A. tumefaciens was not apparently modulated by indole. A. tumefaciens AcrAB played a dominant role and was required for tolerance to high levels of the toxic compounds. Understanding the regulation of multidrug efflux pumps and bacterial adaptive responses to intracellular and extracellular signaling molecules for antibiotic resistance is essential. This information will be useful for the rational design of effective treatments for bacterial infection to overcome possible multidrug-resistant pathogens., (Copyright © 2018 Elsevier GmbH. All rights reserved.)
- Published
- 2018
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46. Pseudomonas aeruginosa nfuA: Gene regulation and its physiological roles in sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence.
- Author
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Romsang A, Duang-Nkern J, Saninjuk K, Vattanaviboon P, and Mongkolsuk S
- Subjects
- Amino Acid Motifs, Bacterial Proteins genetics, Cysteine chemistry, Iron-Sulfur Proteins genetics, Mutagenesis, Site-Directed, Oxidants chemistry, Phenotype, Promoter Regions, Genetic, Pseudomonas aeruginosa genetics, Reactive Oxygen Species metabolism, Stress, Physiological, Transcription Factors metabolism, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Iron-Sulfur Proteins physiology, Pseudomonas aeruginosa physiology
- Abstract
The role of the nfuA gene encoding an iron-sulfur ([Fe-S]) cluster-delivery protein in the pathogenic bacterium Pseudomonas aeruginosa was investigated. The analysis of nfuA expression under various stress conditions showed that superoxide generators, a thiol-depleting agent and CuCl2 highly induced nfuA expression. The expression of nfuA was regulated by a global [2Fe-2S] cluster containing the transcription regulator IscR. Increased expression of nfuA in the ΔiscR mutant under uninduced conditions suggests that IscR acts as a transcriptional repressor. In vitro experiments revealed that IscR directly bound to a sequence homologous to the Escherichia coli Type-I IscR-binding motifs on a putative nfuA promoter that overlapped the -35 element. Binding of IscR prevented RNA polymerase from binding to the nfuA promoter, leading to repression of the nfuA transcription. Physiologically, deletion of nfuA reduced the bacterial ability to cope with oxidative stress, iron deprivation conditions and attenuated virulence in the Caenorhabditis elegans infection model. Site-directed mutagenesis analysis revealed that the conserved CXXC motif of the Nfu-type scaffold protein domain at the N-terminus was required for the NfuA functions in conferring the stress resistance phenotype. Furthermore, anaerobic growth of the ΔnfuA mutant in the presence of nitrate was drastically retarded. This phenotype was associated with a reduction in the [Fe-S] cluster containing nitrate reductase enzyme activity. However, NfuA was not required for the maturation of [Fe-S]-containing proteins such as aconitase, succinate dehydrogenase, SoxR and IscR. Taken together, our results indicate that NfuA functions in [Fe-S] cluster delivery to selected target proteins that link to many physiological processes such as anaerobic growth, bacterial virulence and stress responses in P. aeruginosa., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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47. Pseudomonas aeruginosa ttcA encoding tRNA-thiolating protein requires an iron-sulfur cluster to participate in hydrogen peroxide-mediated stress protection and pathogenicity.
- Author
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Romsang A, Duang-Nkern J, Khemsom K, Wongsaroj L, Saninjuk K, Fuangthong M, Vattanaviboon P, and Mongkolsuk S
- Subjects
- Amino Acid Sequence, Animals, Catalase genetics, Drosophila melanogaster microbiology, Oxidative Stress drug effects, Virulence genetics, Bacterial Proteins genetics, Genes, Bacterial genetics, Hydrogen Peroxide pharmacology, Iron-Sulfur Proteins genetics, Oxidative Stress genetics, Pseudomonas aeruginosa genetics, RNA, Transfer genetics
- Abstract
During the translation process, transfer RNA (tRNA) carries amino acids to ribosomes for protein synthesis. Each codon of mRNA is recognized by a specific tRNA, and enzyme-catalysed modifications to tRNA regulate translation. TtcA is a unique tRNA-thiolating enzyme that requires an iron-sulfur ([Fe-S]) cluster to catalyse thiolation of tRNA. In this study, the physiological functions of a putative ttcA in Pseudomonas aeruginosa, an opportunistic human pathogen that causes serious problems in hospitals, were characterized. A P. aeruginosa ttcA-deleted mutant was constructed, and mutant cells were rendered hypersensitive to oxidative stress, such as hydrogen peroxide (H
2 O2 ) treatment. Catalase activity was lower in the ttcA mutant, suggesting that this gene plays a role in protecting against oxidative stress. Moreover, the ttcA mutant demonstrated attenuated virulence in a Drosophila melanogaster host model. Site-directed mutagenesis analysis revealed that the conserved cysteine motifs involved in [Fe-S] cluster ligation were required for TtcA function. Furthermore, ttcA expression increased upon H2 O2 exposure, implying that enzyme levels are induced under stress conditions. Overall, the data suggest that P. aeruginosa ttcA plays a critical role in protecting against oxidative stress via catalase activity and is required for successful bacterial infection of the host.- Published
- 2018
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48. PCR data and comparative performance of Bacteroidales microbial source tracking genetic markers.
- Author
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Somnark P, Chyerochana N, Kongprajug A, Mongkolsuk S, and Sirikanchana K
- Abstract
We reported modified endpoint PCR results analyzed by universal and human-, swine-, and cattle-specific Bacteroidales gene markers with human sewage and animal fecal samples (i.e., swine, cattle, chicken, goat, sheep, buffalo, and duck) from Tha Chin and Chao Phraya watersheds. Annealing locations of PCR primers were illustrated by maps of 16s rRNA Bacteroidales genes. We also summarized previously published work on the performance of the PCR assays. For further discussion of the data presented here, please refer to Somnark et al., Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds, Environ. Pollut. 236 (2018) 100-110.
- Published
- 2018
- Full Text
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49. Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds.
- Author
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Somnark P, Chyerochana N, Mongkolsuk S, and Sirikanchana K
- Subjects
- Agriculture, Animals, Cattle, DNA, Bacterial genetics, Feces, Humans, Real-Time Polymerase Chain Reaction, Sewage, Swine, Thailand, Water Microbiology, Bacteroidetes genetics, Environmental Monitoring methods, Genetic Markers
- Abstract
Microbial source tracking (MST) DNA-based assays have been used to successfully solve fecal pollution problems in many countries, particularly in developed nations. However, their application in developing countries has been limited but continues to increase. In this study, sixteen endpoint and quantitative PCR (qPCR) assays targeting universal and human-, swine-, and cattle-specific Bacteroidales gene markers were modified for endpoint PCR, evaluated for their performance with sewage and fecal samples from the Tha Chin watershed and subsequently validated with samples from the Chao Phraya watershed, Thailand. Sample sizes of 81 composite samples (from over 1620 individual samples) of farm animals of each type as well as 19 human sewage samples from the Tha Chin watershed were calculated using a stratified random sampling design to achieve a 90% confidence interval and an expected prevalence (i.e., desired assay's sensitivity) of 0.80. The best universal and human-, swine-, and cattle-specific fecal markers were BacUni EP, HF183/BFDrev EP, Pig-2-Bac EP, and Bac3 assays, respectively. The detection limits for these assays ranged from 30 to 3000 plasmid copies per PCR. The positive predictive values were high in universal and swine- and cattle-specific markers (85-100%), while the positive predictive value of the human-specific assay was 52.2%. The negative predictive values in all assays were relatively high (90.8-100%). A suite of PCR assays in Thailand was established for potential MST use in environmental waters, which supports the worldwide applicability of Bacteroidales gene markers. This study also emphasizes the importance of using a proper sample size in assessing the performance of MST markers in a new geographic region., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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50. The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide.
- Author
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Panmanee W, Charoenlap N, Atichartpongkul S, Mahavihakanont A, Whiteside MD, Winsor G, Brinkman FSL, Mongkolsuk S, and Hassett DJ
- Subjects
- DNA Footprinting, Electrophoretic Mobility Shift Assay, Reverse Transcriptase Polymerase Chain Reaction, Bacterial Proteins genetics, Genes, Bacterial, Pseudomonas aeruginosa drug effects, Pseudomonas aeruginosa genetics, tert-Butylhydroperoxide pharmacology
- Abstract
The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.
- Published
- 2017
- Full Text
- View/download PDF
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