scholarly journals Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host

2002 ◽  
Vol 184 (17) ◽  
pp. 4666-4671 ◽  
Author(s):  
Aaron J. Tooley ◽  
Alexander N. Glazer
Keyword(s):  
Ternary Complexes ◽  
Covalent Attachment ◽  
Heme Oxygenase 1 ◽  
Α Subunit ◽  
Ionization Time ◽  
E Coli ◽  
Metal Affinity ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin α subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Identification of Sulfur Activation Relevant Protein Genes of Extremely Thermophilic Acidianus manzaensis

2017 ◽  
Vol 262 ◽  
pp. 461-465 ◽  
Author(s):  
Hong Chang Liu ◽  
Jin Lan Xia ◽  
Zhen Yuan Nie ◽  
Ya Long Ma ◽  
Yun Yang ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Elemental Sulfur ◽  
Extracellular Proteins ◽  
Laser Desorption Ionization ◽  
Two Dimensional Gel Electrophoresis ◽  
Ionization Time ◽  
Real Time Quantitative Pcr ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Acidianus Manzaensis

The sulfur activation by extracellular proteins is considered as the crucial stage during biooxidation of elemental sulfur (S0). In order to study genes encoding sulfur-activation related extracellular proteins of extremely thermophilic Acidianus manzaensis, the extracellular proteins with higher abundance for the strain grown on S0 allotropes than that on Fe2+ were first screened by two-dimensional gel electrophoresis, and then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Nine genes amplified with PCR were satisfactory according to their agarose gel electrophoresis. The differential expression of these nine genes when the strain grown on S0 allotropes and Fe2+ were analyzed with real-time quantitative PCR (RT-qPCR). Results showed that seven of them were higher expressed when the strain grown on S0 allotropes than on Fe2+, indicating they may be related with sulfur activation by A. manzaensis.

scholarly journals Analysis of Vancomycin-Resistant Enterococci in Hemato-Oncological Patients

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 785
Author(s):  
Kristýna Hricová ◽  
Taťána Štosová ◽  
Pavla Kučová ◽  
Kateřina Fišerová ◽  
Jan Bardoň ◽  
...  
Keyword(s):  
Surface Protein ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Vancomycin Resistant Enterococci ◽  
Oncological Patients ◽  
Genes Encoding ◽  
Unique Restriction ◽  
Vancomycin Resistant ◽  
Relationship Of ◽  
The Relationship

Enterococci are important bacterial pathogens, and their significance is even greater in the case of vancomycin-resistant enterococci (VRE). The study analyzed the presence of VRE in the gastrointestinal tract (GIT) of hemato-oncological patients. Active screening using selective agars yielded VRE for phenotypic and genotypic analyses. Isolated strains were identified with MALDI-TOF MS, (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) their susceptibility to antibiotics was tested, and resistance genes (vanA, vanB, vanC-1, vanC2-C3) and genes encoding virulence factors (asa1, gelE, cylA, esp, hyl) were detected. Pulsed-field gel electrophoresis was used to assess the relationship of the isolated strains. Over a period of three years, 103 VanA-type VRE were identified in 1405 hemato-oncological patients. The most frequently detected virulence factor was extracellular surface protein (84%), followed by hyaluronidase (40%). Unique restriction profiles were observed in 33% of strains; clonality was detected in 67% of isolates. The study found that 7% of hemato-oncological patients carried VRE in their GIT. In all cases, the species identified was Enterococcus faecium. No clone persisted for the entire 3-year study period. However, genetically different clusters were observed for shorter periods of time, no longer than eight months, with identical VRE spreading among patients.

scholarly journals Carbapenem and colistin-resistant bacteria in North Lebanon: Coexistence of mcr-1 and NDM-4 genes in Escherichia coli

2021 ◽  
Vol 15 (07) ◽  
pp. 934-342
Author(s):  
Charbel Al-Bayssari ◽  
Tania Nawfal Dagher ◽  
Samar El Hamoui ◽  
Fadi Fenianos ◽  
Nehman Makdissy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Ionization Time ◽  
E Coli ◽  
Flight Mass Spectrometry ◽  
Carbapenem Resistant ◽  
The Matrix

Introduction: The increasing incidence of infections caused by multidrug-resistant bacteria is considered a global health problem. This study aimed to investigate this resistance in Gram-negative bacteria isolated from patients hospitalized in North-Lebanon. Methodology: All isolates were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility testing was achieved using disk diffusion, E-test and Broth microdilution methods. Phenotypic detection of carbapenemase was carried out using the CarbaNP test. RT-PCR, standard-PCR and sequencing were performed to detect resistance genes and oprD gene. Conjugal transfer was carried out between our isolates and Escherichia coli J53 to detect the genetic localization of resistance genes. MLST was conducted to determine the genotype of each isolate. Results: Twenty-three carbapenem-resistant Enterobacterales of which eight colistin-resistant Escherichia coli, and Twenty carbapenem-resistant Pseudomonas aeruginosa were isolated. All isolates showed an imipenem MIC greater than 32 mg/mL with MICs for colistin greater than 2 mg/L for E. coli isolates. All the Enterobacterales isolates had at least one carbapenemase-encoding gene, with E. coli isolates coharboring blaNDM-4 and mcr-1 genes. Moreover, 16/20 Pseudomonas aeruginosa harbored the blaVIM-2 gene and 18/20 had mutations in the oprD gene. MLST revealed that the isolates belonged to several clones. Conclusions: We report here the first description in the world of clinical E. coli isolates coharboring blaNDM-4 and mcr-1 genes, and K. pneumoniae isolates producing NDM-6 and OXA-48 carbapenemases. Also, we describe the emergence of NDM-1-producing E. cloacae in Lebanon. Screening for these isolates is necessary to limit the spread of resistant microorganisms in hospitals.

Determining the Potential Link between Irrigation Water Quality and the Microbiological Quality of Onions by Phenotypic and Genotypic Characterization of Escherichia coli Isolates

2015 ◽  
Vol 78 (4) ◽  
pp. 643-651 ◽  
Author(s):  
ERIKA M. du PLESSIS ◽  
FRANCOIS DUVENAGE ◽  
LISE KORSTEN
Keyword(s):  
South Africa ◽  
Irrigation Water ◽  
Microbiological Quality ◽  
Virulence Gene ◽  
Aerobic Bacteria ◽  
Ionization Time ◽  
E Coli ◽  
Flight Mass Spectrometry ◽  
Potential Link

The potential transfer of human pathogenic bacteria present in irrigation water onto fresh produce was investigated, because surface water sources used for irrigation purposes in South Africa have increasingly been reported to be contaminated with enteric bacterial pathogens. A microbiological analysis was performed of a selected river in Limpopo Province, South Africa, that is often contaminated with raw sewage from municipal sewage works and overhead irrigated onions produced on a commercial farm. Counts of Escherichia coli, coliforms, aerobic bacteria, fungi, and yeasts and the prevalence of E. coli O157:H7, Salmonella, and Listeria monocytogenes were determined. Identities of bacterial isolates from irrigation water and onions were confirmed using matrix-assisted laser desorption ionization–time of flight mass spectrometry, PCR, and biochemical tests. To establish a potential link between the microbiological quality of the irrigation source and the onions, the E. coli isolates from both were subjected to antibiotic resistance, virulence gene, and enterobacterial repetitive intergenic consensus PCR analyses. River water E. coli counts exceeded South African Department of Water Affairs and World Health Organization irrigation water guidelines. Counts of aerobic bacteria, coliforms, fungi, and yeasts of onions from the market were acceptable according to Department of Health Directorate, Food Control, South Africa, microbiological guidelines for ready-to-eat fresh fruits and vegetables. E. coli O157:H7, Salmonella, and L. monocytogenes were not detected in onions, whereas only Salmonella was detected in 22% of water samples. Matrix-assisted laser desorption ionization–time of flight mass spectrometry and PCR identification of E. coli isolates from water and onions correlated. Of the 45 E. coli isolates from water and onions, 42.2% were resistant to multiple antibiotics. Virulence genes eae, stx1, and stx2 were detected in 2.2, 6.6, and 2.2% of the E. coli isolates, respectively. Phenotypic (antimicrobial) and genotypic (virulence gene prevalence, DNA fingerprinting) analyses showed a link between river, dam, irrigation pivot point, and onion E. coli isolates.

scholarly journals Strain Diversity of CTX-M-Producing Enterobacteriaceae in Individual Pigs: Insights into the Dynamics of Shedding during the Production Cycle

2014 ◽  
Vol 80 (21) ◽  
pp. 6620-6626 ◽  
Author(s):  
Katrine Hartung Hansen ◽  
Valeria Bortolaia ◽  
Peter Damborg ◽  
Luca Guardabassi
Keyword(s):  
Fragment Length Polymorphism ◽  
Epidemiological Studies ◽  
Production Cycle ◽  
Ionization Time ◽  
Content Type ◽  
E Coli ◽  
Strain Diversity ◽  
Flight Mass Spectrometry ◽  
Additional Resistance ◽  
M 14

ABSTRACTThe aim of this study was to evaluate the population dynamics of CTX-M-producingEnterobacteriaceaein individual pigs on a farm positive for CTX-M-14-producingEscherichia coli. Fecal samples were collected once around the farrowing time from five sows and four times along the production cycle from two of their respective offspring. Multiple colonies per sample were isolated on cefotaxime-supplemented MacConkey agar with or without prior enrichment, resulting in 98 isolates identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and tested forblaCTX-M. CTX-M-positive isolates (n= 86) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harboringblaCTX-Mwere characterized in 22 representative isolates by replicon typing and restriction fragment length polymorphism. Based on the PFGE results, all individuals shed unrelated CTX-M-14-producingE. colistrains during the course of life. Concomitant shedding of CTX-M-2/97-producingProteus mirabilisorProvidencia rettgeriwas observed in two sows and two offspring. At least two genetically unrelated CTX-M-producingE. colistrains were isolated from approximately one-fourth of the samples, with remarkable differences between isolates obtained by enrichment and direct plating. A clear decrease in strain diversity was observed after weaning. Dissemination ofblaCTX-M-14within the farm was attributed to horizontal transfer of an IncK plasmid that did not carry additional resistance genes and persisted in the absence of antimicrobial selective pressure. Assessment of strain diversity was shown to be influenced by the production stage from which samples were collected, as well as by the isolation method, providing useful information for the design and interpretation of future epidemiological studies of CTX-M-producingEnterobacteriaceaein pig farms.

scholarly journals OXA-244-Producing Escherichia coli Isolates, a Challenge for Clinical Microbiology Laboratories

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Yannick Hoyos-Mallecot ◽  
Thierry Naas ◽  
Rémy A. Bonnin ◽  
Rafael Patino ◽  
Philippe Glaser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Microbiology ◽  
Single Point ◽  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Content Type ◽  
E Coli ◽  
Flight Mass Spectrometry ◽  
Mutant Derivative

ABSTRACT OXA-244 is a single-point-mutant derivative of OXA-48 displaying reduced carbapenemase activity. Here, we report the microbiological features of seven OXA-244-producing Escherichia coli isolates. Only one isolate grew on ChromID Carba Smart medium (bioMérieux), but six of the seven isolates grew on ChromID extended-spectrum-β-lactamase (ESBL) medium (bioMérieux), as they coproduced an ESBL and/or a plasmid-encoded cephalosporinase. The production of a carbapenemase was detected in 57.1%, 71.4%, 71.4%, and 100% of the E. coli isolates using the Carba NP test, the Rapidec Carba NP test (bioMérieux), a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) hydrolysis assay (Bruker), and the OXA-48 K-SeT assay (Coris BioConcept), respectively. Our results indicate that OXA-244-producing E. coli isolates are difficult to detect, which may lead to their silent spread.

scholarly journals An antifungal protein from Escherichia coli

2007 ◽  
Vol 56 (5) ◽  
pp. 637-644 ◽  
Author(s):  
V. Yadav ◽  
R. Mandhan ◽  
Q. Pasha ◽  
S. Pasha ◽  
A. Katyal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fungal Infections ◽  
Sequence Similarity ◽  
Human Erythrocytes ◽  
Antifungal Protein ◽  
Toxicity Tests ◽  
Ionization Time ◽  
E Coli ◽  
Flight Mass Spectrometry

A cytosolic protein was purified from Escherichia coli BL21 that demonstrated potent antifungal activity against pathogenic strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Candida albicans. The MIC of purified protein from E. coli BL21 (PPEBL21) against Aspergillus species and C. albicans was 1.95–3.98 and 15.62 μg ml−1, respectively. In vitro toxicity tests demonstrated no cytotoxicity of PPEBL21 to human erythrocytes up to the tested concentrations of 1250 μg ml−1. Amphotericin B was lethal to 100 % of human erythrocytes at a concentration of 37.5 μg ml−1. The N-terminal amino acid sequence of PPEBL21 was found to be DLAEVASR, which showed 75 % sequence similarity with alcohol dehydrogenase of yeast. Mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry also substantiated these observations. The results suggested that E. coli BL21 might be an important bioresource of lead molecules for developing new peptide-based therapies for treating fungal infections.

scholarly journals MALDI-TOF MS Based Typing for Rapid Screening of Multiple Antibiotic Resistance E. coli and Virulent Non-O157 Shiga Toxin-Producing E. coli Isolated from the Slaughterhouse Settings and Beef Carcasses

Foods ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 820
Author(s):  
Mohamed Tharwat Elabbasy ◽  
Mohamed A. Hussein ◽  
Fahad Dhafer Algahtani ◽  
Ghada I. Abd El-Rahman ◽  
Alaa Eldin Morshdy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Laser Desorption ◽  
Rapid Screening ◽  
Laser Desorption Ionization ◽  
Multiple Antibiotic Resistance ◽  
Ionization Time ◽  
E Coli ◽  
Flight Mass Spectrometry ◽  
Beef Carcasses

Background: The emergence of multiple antibiotic resistance (MAR) Escherichia coli (E. coli) and virulent non-O157 Shiga toxin-producing Escherichia coli (STEC) poses a growing concern to the meat industry. Non-O157 STEC strains including O26, O45, O103, O111, O121, and O145 have been implicated in the occurrence of bloody diarrhea and hemolytic uremic syndrome in humans. This research assessed prevalence, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF MS) protein mass-spectra profiles, multidrug-resistance traits, polymerase chain reaction detection of virulence, and antibiotic-resistance genes of E. coli isolated from beef carcasses and slaughterhouse environments. Methods: A total of 180 convenience sponge samples were collected from two different sources-specific parts of beef carcasses and surfaces of the processing environment at the slaughterhouse of Ha′il, Saudi Arabia between September and November 2020. MALDI BioTyper and phylotype-based identification methods accurately identified and classified the samples as belonging to the genus belonging to the Escherichia coli domain of bacteria (NCBI txid: 562). Results: Expected changes were seen in the mass peak spectrum defining nine closely related isolates and four unrelated E. coli isolates. Serological typing of E. coli revealed enterotoxigenic E. coli O166 (19.10%); enteropathogenic E. coli O146 (16.36%) and O44 (18.18%); enterohemorrhagic E. coli O111 (31.18%) and O26 (14.54%). Forty-five percent of examined E. coli were resistant to seven antimicrobials; 75% of 20 selected isolates were resistant to three or more antimicrobials. phoA and blaTEM genes were detected in all selected E. coli isolates. Conclusion: This study confirmed the efficiency and validity of Matrix-assisted laser desorption/ionization time of flight mass-spectrometry in screening for multi-drug resistant E. coli isolated from slaughterhouse derived beef carcasses in Ha’il, Saudi Arabia. We contributed by revealing the distinction between related and non-related strains of E.coli in livestock. The findings in this study can inform improved policy development decision making and resource allocation related to livestock processing regarding antimicrobial use in food animals and rapid screening for effective multiple antibiotic resistance E. coli and virulent non-O157 STEC control in the slaughterhouses.

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