LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O : 3 gene expression in vivo

It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-P-oc::luxAB and YeO3-P-op1::luxAB, respectively. In vitro, at 37 degrees C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22 degrees C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice.Peer reviewe

Bioprospecting of hot springs and compost in West Anatolia regarding phytase producing thermophilic fungi

Phytase is commonly used as feed supplement for poultry and catalyses the hydrolysis of phytate into inorganic phosphates and myo-inositol phosphates. Extreme environments, especially warm habitats constitute an important resource for the discovery of microorganisms with unique enzymes. Therefore, we aimed to investigate the culturable thermophilic and thermotolerant fungal biodiversity of hot springs and compost samples in Western Anatolia and their extracellular phytase production capacities for the first time. A total of 43 environmental samples (26 soils and 17 sediments) were collected from 17 different hot springs and 1 compost sample was taken from a mushroom farm. A total of 48 filamentous fungal strains were isolated. Fourteen (29 %) strains were classified as thermophilic and 34 (71 %) strains as thermotolerant regarding to their heat requirements. Of the 48 isolates, 33 (69 %) were Aspergillus species. All isolates were quantitatively screened for their extracellular phytase activities and 42 (88 %) of the 48 isolates produces phytase in a range of 8.82 - 331.22 (U/mg). This study demonstrates that hot springs in West Anatolia harbour a rich thermophilic/thermotolerant fungal diversity possessing phytase producing potential and mushroom farming selectively enhances thermophilic fungi.Ege University Scientific Projects Foundations [17-Bil-005]; Scientific and Technological Research Council of Turkey (TUBITAK) [116Z114]We gratefully acknowledge the support for this research by the Ege University Scientific Projects Foundations, Project No: 17-Bil-005. This work was also financed by the Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 116Z114

LEGIONELLA PNEUMOPHILA: LEGIONNARIES' DISEASE

WOS: 000278550900002Legionella pneumophila was recognized as an important human pathogen after the first discovery during an investigation of a pneumonia outbreak among American Legion convention in 1976 in Philadelphia, USA. L. pneumophila is a gram-negative, mesophilic, facultative intracellular parasitic and nonspore-forming rod-shaped bacterium belonging to the gamma-subgroup of proteobacteria. L. pneumophila inhabits natural freshwater environments at low concentration. Along with the transfer from natural aquatic habitats into man-made water systems such as cooling towers, evaporative condensers, water distribution systems, whirlpool spas and hot water tanks, L. pneumophila reaches high cell density and can cause Legionnaires' disease (pneumonic legionellosis) or Pontiac fever (severe influenza-like illness). Infection occurs primarily via the inhalation of L. pneumophila-contaminated aerosols. In aquatic habitats, L. pneumophila cells are intracellular parasites of freshwater protozoa and use a similar mechanism to multiply within mammalian cells. L. pneumophila can also multiply extracellularly within biofilms and can persist within these microbial communities for years. Transmission to human primarily occurs via the inhalation of L. pneumophila containing aerosols. The bacterium enters to human phagocytic cells by coiling or conventional phagocytosis then inhibits phagosome-lysosome fusion and multiplies in the phagosome. A number of virulence factors have been described for L. pneumophila such as surface proteins, secreted factors and putative virulence factors. L. pneumophila can be identified by using cultural, serologic and various molecular techniques such as DNA sequencing and DNA-DNA hybridization. Diagnosis can be made by culture, direct fluorescent antibody staining, serological tests, urinary antigen detection or nucleic acid detection and various subtyping techniques. In order to eradicate L. pneumophila from contaminated water systems several methods are available; Thermal or chemical shock \disinfection, UV irradiation, ozone treatment, silver-copper ionization, anodic oxidation and chlorine dioxide application