scholarly journals Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1433-1441 ◽  
Author(s):  
Huizhong Chen ◽  
Sherryll L. Hopper ◽  
Carl E. Cerniglia
Keyword(s):  
Staphylococcus Aureus ◽  
Azo Dyes ◽  
Mrna Level ◽  
Hydrophobic Interaction Chromatography ◽  
Consumer Products ◽  
Methyl Red ◽  
Gene Encoding ◽  
Native Molecular Mass ◽  
Ponceau S ◽  
Prosthetic Groups

Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent K m values for NADPH and Methyl Red substrates were 0·074 and 0·057 mM, respectively. The apparent V max was 0·4 μM min−1 (mg protein)−1. Azo1 was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora.

Phylogeny Characterization of Seb Gene Encoding Enterotoxins in Staphylococcus aureus Isolated from Raw Milk and Cheese

2019 ◽  
Vol 9 (o3) ◽  
Author(s):  
Fatima N. Aziz ◽  
Laith Abdul Hassan Mohammed-Jawad
Keyword(s):  
Staphylococcus Aureus ◽  
Food Poisoning ◽  
Raw Milk ◽  
Staphylococcal Enterotoxin B ◽  
Human Pathogen ◽  
Conventional Pcr ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Gene Encoding

Food poisoning due to the bacteria is a big global problem in economically and human's health. This problem refers to an illness which is due to infection or the toxin exists in nature and the food that use. Milk is considered a nutritious food because it contains proteins and vitamins. The aim of this study is to detect and phylogeny characterization of staphylococcal enterotoxin B gene (Seb). A total of 200 milk and cheese samples were screened. One hundred ten isolates of Staphylococcus aureus pre-confirmed using selective and differential media with biochemical tests. Genomic DNA was extracted from the isolates and the SEB gene detects using conventional PCR with specific primers. Three staphylococcus aureus isolates were found to be positive for Seb gene using PCR and confirmed by sequencing. Sequence homology showed variety range of identity starting from (100% to 38%). Phylogenetic tree analyses show that samples (6 and 5) are correlated with S. epidermidis. This study discovered that isolates (A6-RLQ and A5-RLQ) are significantly clustered in a group with non- human pathogen Staphylococcus agnetis.

Penggunaan Limbah Logam Tembaga yang Didaur Ulang untuk Antibakteri dan Degradasi Metil Merah Secara Fotolisis

2019 ◽  
Vol 4 (1) ◽  
pp. 15
Author(s):  
Ariyetti Ariyetti ◽  
Muhammad Nasir ◽  
Safni Safni ◽  
Syukri Darajat
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Silver Nitrate ◽  
Copper Sulfate ◽  
Bacterial Growth ◽  
Methyl Red ◽  
Copper Metal ◽  
Sulfate Hydrate ◽  
Red Dye ◽  
And Staphylococcus Aureus

<p><em>Metil merah merupakan salah satu zat warna golongan azo yang sering digunakan dalam industri dan laboratorium. Penggunaan metil merah dapat menimbulkan efek terhadap kesehatan dan lingkungan. Oleh sebab itu dilakukan metode fotodegradasi dengan menggunakan semikonduktor dan radiasi sinar tampak. Semikonduktor yang digunakan yaitu berbahan dasar tembaga sulfat hidrat dan perak nitrat. Prekusor tembaga sulfat hidrat dibuat dari pengolahan limbah logam tembaga hasil pemotongan tembaga yang ada di bengkel Lembaga Ilmu Pengetahuan Indonesia (LIPI) Bandung. Bahan semikonduktor juga memiliki kemampuan dalam menghambat pertumbuhan bakteri. Hasil optimum yang didapatkan dalam proses fotodegradasi dan antibakteri merupakan gabungan antara kedua prekusor tembaga sulfat hidrat dan perak nitrat dengan bantuan penyinaran. Kemampuan dalam menghambat pertumbuhan bakteri didapatkan persentase kematian 100 % untuk masing-masing bakteri, yaitu Escherichia coli dan Staphylococcus aureus. Aktifitas fotokatalitiknya dengan konsentrasi semikonduktor 10 ppm untuk mendegradasi zat warna metil merah 5 ppm, selama 23 jam, dimana persentase degradasi yang didapatkan dengan penyinaran lebih tinggi dibandingkan dengan tanpa penyinaran. Pengaruh pH larutan terhadap degradasi metil merah yaitu optimum pada pH 12 (basa).</em></p><p><em><br /></em></p><p><em>Methyl red is one of the azo group dyes that is often used in industry and laboratories. The use of methyl red can have an effect on health and the environment. Therefore photodegradation method is done by using semiconductor and visible light radiation. The semiconductor used is based on copper sulfate hydrate and silver nitrate. The copper sulphate hydrate precursor is made from the processing of copper-cut copper metal waste in the workshop of the Indonesian Institute of Sciences (LIPI) in Bandung. Semiconductor materials also have the ability to inhibit bacterial growth. The optimum results obtained in the photodegradation and antibacterial process are a combination of both copper sulfate hydrate precursor and silver nitrate with the help of irradiation. The ability to inhibit bacterial growth obtained 100% mortality for each bacterium, namely Escherichia coli and Staphylococcus aureus. Photocatalytic activity with 10 ppm semiconductor concentration to degrade methyl red dye 5 ppm, for 23 hours, where the percentage of degradation obtained by irradiation is higher than without irradiation. The effect of pH of the solution on the degradation of methyl red is optimum at pH 12 (base).</em></p>

scholarly journals The Prevalence of Virulence Determinants and Antibiotic Resistance Patterns in Methicillin—Resistant Staphylococcus aureus in a Nursing Home in Poland

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 427
Author(s):  
Martyna Kasela ◽  
Agnieszka Grzegorczyk ◽  
Bożena Nowakowicz-Dębek ◽  
Anna Malm
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Variable Number Tandem Repeat ◽  
Multidrug Resistant ◽  
Variable Number ◽  
Pulse Field ◽  
Virulence Determinants ◽  
Gene Encoding ◽  
Methicillin Resistant ◽  
Pulse Field Gel Electrophoresis

Nursing homes (NH) contribute to the regional spread of methicillin-resistant Staphylococcus aureus (MRSA). Moreover, residents are vulnerable to the colonization and subsequent infection of MRSA etiology. We aimed at investigating the molecular and phenotypic characteristics of 21 MRSA collected from the residents and personnel in an NH (Lublin, Poland) during 2018. All MRSA were screened for 20 genes encoding virulence determinants (sea-see, eta, etb, tst, lukS-F-PV, eno, cna, ebpS, fib, bbp, fnbA, fnbB, icaADBC) and for resistance to 18 antimicrobials. To establish the relatedness and clonal complexes of MRSA in NH we applied multiple-locus variable-number tandem-repeat fingerprinting (MLVF), pulse field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. We identified four sequence types (ST) among two clonal complexes (CC): ST (CC22) known as EMRSA-15 as well as three novel STs—ST6295 (CC8), ST6293 (CC8) and ST6294. All tested MRSA were negative for sec, eta, etb, lukS-F-PV, bbp and ebpS genes. The most prevalent gene encoding toxin was sed (52.4%; n = 11/21), and adhesins were eno and fnbA (100%). Only 9.5% (n = 2/21) of MRSA were classified as multidrug-resistant. The emergence of novel MRSA with a unique virulence and the presence of epidemic clone EMRSA-15 creates challenges for controlling the spread of MRSA in NH.

Effect of Photo-Isomerization of Azo Dyes on the Photonic Bandgap in Chiral Nematics

2011 ◽  
Vol 181-182 ◽  
pp. 257-260
Author(s):  
David Statman ◽  
Andrew Jockers ◽  
Daniel Brennan
Keyword(s):  
Liquid Crystal ◽  
Liquid Crystals ◽  
Azo Dyes ◽  
Azo Dye ◽  
Photonic Bandgap ◽  
Methyl Red ◽  
Central Wavelength ◽  
Chiral Nematic ◽  
Anchoring Energy ◽  
Pitch Length

Chiral nematic liquid crystals prepared with Grandjean texture demonstrate a photonic bandgap whose central wavelength is proportional to the pitch length, P, of the liquid crystal and whose width is given by (ne – no)P. We show that methyl red doped chiral nematics undergo a shift in the photonic bandgap upon photo-isomerization. This shift is a result of (1) photo-induced change in anchoring energy on the nematic surface, and (2) change in the natural pitch length from the photo-isomerization of the azo dye.

scholarly journals Antagonism between Aminoglycosides and β-Lactams in a Methicillin-Resistant Staphylococcus aureus Isolate Involves Induction of an Aminoglycoside-Modifying Enzyme

2002 ◽  
Vol 46 (5) ◽  
pp. 1516-1521 ◽  
Author(s):  
Takashi Ida ◽  
Ryoichi Okamoto ◽  
Masato Nonoyama ◽  
Kazuhiko Irinoda ◽  
Mizuyo Kurazono ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Antimicrobial Activities ◽  
Hybrid Structure ◽  
Conjugative Plasmid ◽  
Pcr Analysis ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Methicillin Resistant ◽  
Aureus Isolate

ABSTRACT We encountered three clinical isolates of methicillin-resistant Staphylococcus aureus which were susceptible to netilmicin and arbekacin in the absence of β-lactam antibiotics but which were resistant to them in the presence of β-lactam antibiotics. One of these strains, KU5801, was used to further investigate the antagonism between aminoglycosides and β-lactam antibiotics. β-Lactam antibiotics induced bacterial synthesis of aminoglycoside-6′-N-acetyltransferase and 2"-O-phosphotransferase [AAC(6′)-APH(2")] in association with decreased antimicrobial activities of aminoglycosides. A 14.4-kb EcoRI fragment that included the genes that control for β-lactam-inducible aminoglycoside resistance was cloned from a 31-kb conjugative plasmid present in KU5801. Restriction fragment mapping and PCR analysis suggested that a Tn4001-like element containing a gene encoding AAC(6′)-APH(2") was located downstream from a truncated blaZ gene. The DNA sequence between blaR1 and a Tn4001-like element was determined. The Tn4001-IS257 hybrid structure was cointegrated into the blaZ gene, and the typical sequences for the termination of transcription were not found between these regions. We deduced that antagonism of aminoglycosides by β-lactam antibiotics in isolate KU5801 involved transcription of the aac(6′)-Ie-aph(2")-Ia gene under the influence of the system regulating penicillinase production.

scholarly journals Transketolase of Staphylococcus aureus in the Control of Master Regulators of Stress Response During Infection

2019 ◽  
Vol 220 (12) ◽  
pp. 1967-1976
Author(s):  
Xin Tan ◽  
Elodie Ramond ◽  
Anne Jamet ◽  
Jean-Philippe Barnier ◽  
Baptiste Decaux-Tramoni ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Time Lapse ◽  
Pentose Phosphate ◽  
Host Cells ◽  
Chronic Infections ◽  
Gene Encoding ◽  
Master Regulators ◽  
Sigma B ◽  
Intracellular Proliferation ◽  
Major Defect

Abstract Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. In the current study, we focused on one key PPP enzyme, transketolase (TKT). We showed that inactivation of the unique gene encoding TKT activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared with wild-type strain in early intracellular proliferation and in the ability to colonize kidneys. Transcriptional activity of the 2 master regulators sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription suggests that TKT—or a functional PPP—strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.

scholarly journals Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Transcriptional Regulation of the bla and mec Operons

2005 ◽  
Vol 187 (5) ◽  
pp. 1833-1844 ◽  
Author(s):  
Martin K. Safo ◽  
Qixun Zhao ◽  
Tzu-Ping Ko ◽  
Faik N. Musayev ◽  
Howard Robinson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Crystal Structures ◽  
Double Helix ◽  
Free Form ◽  
Binding Motif ◽  
Dimerization Domain ◽  
Gene Encoding ◽  
Dna Binding Sites ◽  
Dna Double Helix

ABSTRACT The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding β-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to β-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when β-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-Å resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix α3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.

scholarly journals Rapid Depletion of Free Vancomycin in Medium in the Presence of β-Lactam Antibiotics and Growth Restoration in Staphylococcus aureus Strains with β-Lactam-Induced Vancomycin Resistance

2008 ◽  
Vol 53 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Chie Yanagisawa ◽  
Hideaki Hanaki ◽  
Hidehito Matsui ◽  
Shinsuke Ikeda ◽  
Taiji Nakae ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Culture Medium ◽  
Pcr Amplification ◽  
Vancomycin Resistance ◽  
Gene Encoding ◽  
Vancomycin Concentration ◽  
Methicillin Resistant ◽  
Lactam Antibiotic ◽  
Absorption Level

ABSTRACT A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of β-lactam antibiotics (β-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the d-Ala-d-Ala terminal of the peptidoglycan network is replaced with d-Ala-d-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of ∼0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 μg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of β-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the β-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 μg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below ∼1.5 μg/ml at 24 h, the cells began to grow. In the culture supplemented with the β-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 μg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of ∼1.7 μg/ml or at 4 h of incubation. The gene encoding the enzyme involved in d-Ala-d-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by β-lactam antibiotics.

scholarly journals Proteophosphoglycans of Leishmania mexicana: Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes

1999 ◽  
Vol 344 (3) ◽  
pp. 787-795 ◽  
Author(s):  
Ulrich GÖPFERT ◽  
Nathan GOEHRING ◽  
Christian KLEIN ◽  
Thomas ILG
Keyword(s):  
Mrna Level ◽  
Single Copy ◽  
Gene Replacement ◽  
Mammalian Host ◽  
Cdna Expression Library ◽  
Dna Repeats ◽  
The Novel ◽  
Leishmania Mexicana ◽  
Expression Library ◽  
Gene Encoding

Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressedppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.

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