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Laboratory Strains (laboratory + strain)
Selected AbstractsA new ferrous iron-uptake transporter, EfeU (YcdN), from Escherichia coliMOLECULAR MICROBIOLOGY, Issue 1 2006Cornelia Große Summary Escherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase-dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur-dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known iron-uptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site-directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild-type protein. [source] Methoprene modulates the effect of diet on male melon fly, Bactrocera cucurbitae, performance at mating aggregationsENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 1 2010Ihsan ul Haq Abstract The effect of access to dietary protein (P) (hydrolyzed yeast) and/or treatment with a juvenile hormone analogue, methoprene (M), (in addition to sugar and water) on male aggregation (lekking) behaviour and mating success was studied in a laboratory strain of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Six-day-old males were treated with (1) protein and methoprene (M+P+), (2) only protein (M,P+), or (3) only methoprene (M+P,), and compared with 14-day-old sexually mature untreated males (M,P,). The lekking behaviour of the four groups of males when competing for virgin sexually mature females (14,,16 days old) was observed in field cages. The following parameters were measured at male aggregations: lek initiation, lek participation, males calling, male,male interaction, female acceptance index, and mating success. For all these parameters, the M+P+ males significantly outperformed the other males. Moreover, for all parameters, there was a similar trend with M+P+ > M,P+ > M,P, > M+P,. More M+P+ males called and initiated and participated in lek activities than all other types of male, which resulted in higher mating success. They had also fewer unsuccessful copulation attempts than their counterparts. Whereas treatment with methoprene alone had a negative effect in young males with only access to sugar, access to dietary protein alone significantly improved young male sexual performance; moreover, the provision of methoprene together with protein had a synergistic effect, improving further male performance at leks. The results are of great relevance for enhancing the application of the sterile insect technique (SIT) against this pest species. The fact that access to dietary protein and treatment of sterile males with methoprene improves mating success means that SIT cost-effectiveness is increased, as more released males survive to sexual maturity. [source] Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolatesENVIRONMENTAL MICROBIOLOGY, Issue 12 2002Jean-Paul Pirnay Summary Genomes are constantly evolving. Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance. Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa. The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world , from sources as diverse as patients and rhizospheres , was analysed. A microscale mosaic structure for this gene , resulting from multiple intra- and possibly interspecies recombinational events , is reported. An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before. A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains. We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads. [source] Release of monocyte chemoattractant protein (MCP)-1 by a human alveolar epithelial cell line in response to Mycobacterium aviumFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2000Savita P. Rao Abstract Clinical strains of Mycobacterium avium isolated from patients with acquired immunodeficiency syndrome, but not a non-clinical laboratory strain (ATCC 25291), were found to stimulate the human alveolar epithelial cell line A549, to produce monocyte chemoattractant protein (MCP)-1. A549 cells were also found to produce elevated levels of MCP-1 in response to sonicates of the clinical strains of M. avium, and surprisingly, the non-clinical strain as well. However, sonic extracts of the clinical strains were found to induce significantly higher levels of MCP-1 production compared to extracts of the non-clinical strain (P<0.001). These data suggest the existence of strain-related differences in antigen expression by M. avium. The clinical and non-clinical strains of M. avium were found to attach and invade, but not replicate in A549 cells indicating that MCP-1 production by A549 cells does require the presence of viable, replicating organisms. Activation of alveolar epithelial cells by exposure to M. avium resulting in the production of chemokines which recruit inflammatory cells to the site of infection may be an important regulatory pathway for the activation of pulmonary host defense. [source] Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strainsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Tecilli Cabellos-Avelar Abstract The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22 000 transposon-induced and 26 000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process. [source] Evidence of multiple chromosomal inversions in Aedes aegypti formosus from SenegalINSECT MOLECULAR BIOLOGY, Issue 5 2009S. A. Bernhardt Abstract Chromosomal inversions are prevalent in mosquito species but polytene chromosomes are difficult to prepare and visualize in members of the tribe Aedinii and thus there exists only indirect evidence of inversions. We constructed an F1 intercross family using a P1 female from a laboratory strain of Aedes aegypti aegypti (Aaa) and a P1 male Aedes aegypti formosus (Aaf) from a strain collected from south-eastern Senegal. Recombination rates in the F2 offspring were severely reduced and genotype ratios suggested a deleterious recessive allele on chromosome 3. The F2 linkage map was incongruent in most respects with the established map for Aaa. Furthermore, no increased recombination was detected in F5 offspring. Recombination rates and gene order were consistent with the presence in Aaf of at least four large inversions on chromosome 1, a single small inversion on chromosome 2 and three inversions on chromosome 3. [source] Investigation of the interactions between neutrophils and endodontic isolates of Enterococcus faecalisINTERNATIONAL ENDODONTIC JOURNAL, Issue 4 2010R. Sairafi Aim, The primary aim of this study was to investigate the effect of phagocytosis by neutrophils on the antimicrobial sensitivity of Enterococcus faecalis strains. A secondary aim was to determine whether carriage of a plasmid encoding aggregation substance (AS), which has been reported to increase the survival of some strains inside neutrophils, affected the antimicrobial susceptibility of E. faecalis after phagocytosis by neutrophils. Methodology, An assay was carried out to identify isolates of E. faecalis which demonstrated pheromone-responsive clumping caused by the production of aggregation substance (AS). Four E. faecalis strains grown to both logarithmic and stationary phases were exposed to sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) to determine the minimum inhibitory concentrations of these two agents. The antimicrobial susceptibility tests were repeated with E. faecalis strains which survived phagocytosis by neutrophils for 18 h. Results, As expected a laboratory strain of E. faecalis OG1RF which was AS negative became AS positive after introduction of the pheromone responsive plasmid pCF10 into the bacterium to give strain OG1RF(pCF10). These two strains and two endodontic isolates, E08-584 which demonstrated pheromone-responsive clumping and E08-398 which did not, were selected for further study All the test E. faecalis strains were inhibited by low concentrations of sodium hypochlorite (MIC range 0.02,0.3%) and chlorhexidine gluconate (MIC range 0.0004,0.004%). Bacteria recovered from inside neutrophils after 18 h following phagocytosis were susceptible to both ¼MIC and MIC of CHX and NaOCl. Conclusions, Aggregation substance did not appear to affect the antimicrobial susceptibility of any of the strains to CHX or NaOCl. All of the E. faecalis strains examined were capable of survival for 18 h inside the neutrophils following phagocytosis; regardless of their capacity to produce aggregation substance. In addition, all strains of E. faecalis had enhanced susceptibilities to the antimicrobial agents after residence inside neutrophils for 18 h. [source] Epidemiology of enterovirus types causing neurological disease in Austria 1999,2007: Detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypesJOURNAL OF MEDICAL VIROLOGY, Issue 2 2009Birgit Ortner Abstract Between 1999 and 2007 1,388 stool specimens from patients with acute flaccid paralysis or aseptic meningitis were submitted to the Austrian reference laboratory for poliomyelitis. Samples (201) yielded non-poliovirus enterovirus in culture. One hundred eighty-one viruses were available for typing and 78 isolates which remained serologically untyped were further analyzed by CODEHOP-PCR and sequencing of the VP1 gene and the 5,-untranslated region (5,-UTR). Typing revealed an Echovirus 30 outbreak in northwestern Austria in 2000, which was in accordance with the situation in Europe, and no dramatic seasonal changes of Coxsackie viruses were observed. In 2002/2003 a small outbreak of enterovirus 71 (EV71), affected 12 patients in the province of Styria. This virus was identified as genotype C1 and appeared to be genetically distinct from the isolates observed in 2001/2002 in Vienna. In 2004 two unrelated cases occurred in Lower Austria, which were identified as genotype C4, which has been described associated with high mortality most recently in China. In contrast to the situation in Asia the detected EV71 cases were not associated with hand,foot,mouth disease, but with serous meningitis only. This was surprising as a recent publication suggested a reduced neurovirulence of C1 genotype in children in Norway, presumably due to alterations in 5,-UTR and polymerase gene. However, comparing the 5,-UTR of the Austrian isolates and established virulent reference strains to the Norwegian isolate and an attenuated EV71 laboratory strain we did not find an indication that the genotype C1 possesses a RNA structure in its 5,-UTR leading to reduced neurovirulence. J. Med. Virol. 81:317,324, 2009. © 2008 Wiley-Liss, Inc. [source] In vitro new dialysis protocol to assay the antiseptic properties of a quaternary ammonium compound polymerized with denture acrylic resinLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2004C. Pesci-Bardon Abstract Aims:, To develop an in vitro protocol in order to assess the antiseptic properties of a quaternary ammonium compound polymerized with acrylic denture resin base, using experimental resin discs and dialysis membranes. Methods and Results:, Experimental acrylic resin discs were polymerized with Poly 202063A, an ammonium compound (2,50%). Antiseptic properties were assayed against two reference strains (Escherichia coli, Staphylococcus aureus) and a laboratory strain (Candida albicans), using three different conditions (test A, B and C). In test A, according to classical protocols the resin discs were first soaked in large volumes of microbial inoculum (45 ml). An original dialysis protocol was then designed to recreate the small biofilm volume on the prosthetic surface. In test B, discs and bacterial inoculum (600 ,l) were introduced in a dialysis bag and dialysed against a sterile buffer. A bactericidal effect was observed against E. coli and Staph. aureus (<0·1% viable cells in initial bacterial suspension). A dose-dependent fungistatic effect was observed against C. albicans. Finally, in test C discs and sterile buffer (600 ,l) were introduced in a dialysis bag and dialysed against microbial inoculum. Reduced activity was found outside the dialysis bag, demonstrating that free ammonium was able to diffuse through the dialysis membrane, displaying antiseptic properties. Conclusions:, The present protocol demonstrated that a quaternary ammonium compound remains efficient after heat polymerization with an acrylic denture base resin, both in immediate and distant microbial environments. Significance and Impact of the Study:, Such removable prosthetic devices with intrinsic antiseptic properties would contribute to improve the long-term management of denture stomatitis. [source] Differential Flo8p-dependent regulation of FLO1 and FLO11 for cell,cell and cell,substrate adherence of S. cerevisiae S288cMOLECULAR MICROBIOLOGY, Issue 5 2007Lars Fichtner Summary Cell,cell and cell,surface adherence represents initial steps in forming multicellular aggregates or in establishing cell,surface interactions. The commonly used Saccharomyces cerevisiae laboratory strain S288c carries a flo8 mutation, and is only able to express the flocculin-encoding genes FLO1 and FLO11, when FLO8 is restored. We show here that the two flocculin genes exhibit differences in regulation to execute distinct functions under various environmental conditions. In contrast to the laboratory strain ,1278b, haploids of the S288c genetic background require FLO1 for cell,cell and cell,substrate adhesion, whereas FLO11 is required for pseudohyphae formation of diploids. In contrast to FLO11, FLO1 repression requires the Sin4p mediator tail component, but is independent of the repressor Sfl1p. FLO1 regulation also differs from FLO11, because it requires neither the KSS1 MAP kinase cascade nor the pathways which lead to the transcription factors Gcn4p or Msn1p. The protein kinase A pathway and the transcription factors Flo8p and Mss11p are the major regulators for FLO1 expression. Therefore, S. cerevisiae is prepared to simultaneously express two genes of its otherwise silenced FLO reservoir resulting in an appropriate cellular surface for different environments. [source] Evaluation of semiochemical toxicity to houseflies and stable flies (Diptera: Muscidae)PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 8 2010Rajinder S Mann Abstract BACKGROUND: The housefly, Musca domestica L., and stable fly, Stomoxys calcitrans (L.) are cosmopolitan pests of both farm and home environments. Houseflies have been shown to be resistant to a variety of insecticides, and new chemistries are slow to emerge on the market. Toxicities of selected semiochemicals with molecular structures indicative of insecticidal activity were determined against adults from an insecticide-susceptible laboratory strain of houseflies. The three most active semiochemicals were also evaluated against recently colonized housefly and stable fly strains. RESULTS: Nineteen semiochemicals classified as aliphatic alcohols, terpenoids, ketones and carboxylic esters showed toxicity to houseflies and stable flies. Rosalva (LC50 = 25.98 µg cm,2) followed by geranyl acetone and citronellol (LC50 = 49.97 and 50.02 µg cm,2) were identified as the most toxic compounds to houseflies. Permethrin was up to 144-fold more toxic than rosalva on the susceptible strain. However, it was only 35-fold more toxic to the insecticide-tolerant field strain. The compounds generated high toxicity to stable flies, with LC50 values ranging from 16.30 to 40.41 µg cm,2. CONCLUSION: Quantification of LC50 values of rosalva, citronellol and geranyl acetone against susceptible housefly and field-collected housefly and stable fly strains showed that semiochemicals could serve as potent insecticides for fly control programs. Copyright © 2010 Society of Chemical Industry [source] First report of cyromazine resistance in a population of UK house fly (Musca domestica) associated with intensive livestock productionPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 7 2010Howard A Bell Abstract BACKGROUND: House fly control in livestock-rearing facilities is heavily reliant on the use of the larvicide cyromazine. While extensive use of this compound has led to the development of resistance in several countries, no elevated tolerance has so far been reported from the United Kingdom. RESULTS: Tolerance to cyromazine in larvae derived from a field strain collected at an intensive pig unit was significantly elevated over that of insects taken from a susceptible laboratory strain. Resistance factors (RFs) of 2.9 and 2.4 were returned for assays initiated with eggs and neonate larvae respectively. The RF for field strain larvae exposed from neonate increased significantly to 3.9 and 5.6 following rounds of selection at 1.0 and then 1.5 mg kg,1 cyromazine. CONCLUSION: Low-level resistance to cyromazine in UK house flies is reported here for the first time. The geographic extent of this resistance is unknown but, if widespread, may lead to control failures in the future, and indicates that careful stewardship of this compound in the United Kingdom is now required. © Crown copyright 2010. Reproduced with permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd. [source] Inheritance mode and realized heritability of resistance to imidacloprid in the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae)PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 6 2009Yan Hua Wang Abstract BACKGROUND: The brown planthopper, Nilaparvata lugens (Stål), is a serious pest that causes enormous losses to the rice crop in Asia. The genetic basis of imidacloprid resistance was investigated in N. lugens. RESULTS: The resistant strain, selected for imidacloprid resistance from a field population of N. lugens collected from Nanjing, Jiangsu Province, China, showed a 964-fold resistance compared with the laboratory strain. Progenies of reciprocal crosses (F1 and F1,) showed similar dose,mortality responses (LC50) to imidacloprid, and also exhibited a similar degree of dominance (D), 0.58 for F1 and 0.63 for F1,. Chi-square analyses of self-bred and backcross progenies (F2, F2, and BC respectively) rejected the hypothesis for a single gene control of the resistance. The estimated realized heritability (h2) of imidacloprid resistance was 0.1141 in the resistant strain of N. lugens. CONCLUSION: The results showed that imidacloprid resistance in N. lugens was autosomal and was expressed as an incompletely dominant trait, probably controlled by multiple genes. Copyright © 2009 Society of Chemical Industry [source] Resistance to cyfluthrin and tetrachlorvinphos in the lesser mealworm, Alphitobius diaperinus, collected from the eastern United StatesPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 7 2006Ronda L Hamm Abstract The lesser mealworm, Alphitobius diaperinus (Panzer), is an important pest in poultry facilities. The toxicity of cyfluthrin and tetrachlorvinphos to five strains of the lesser mealworm was compared with the toxicity to a susceptible laboratory strain. Bioassays were carried out with both larvae and adults. For the susceptible strain, cyfluthrin and tetrachlorvinphos had similar toxicity to adults, but cyfluthrin was 5 times more toxic to larvae when compared with tetrachlorvinphos. High levels of resistance to tetrachlorvinphos in two beetle strains were detected in both larvae and adults, although these strains were heterogeneous and still contained susceptible individuals. Resistance to cyfluthrin ranged from 1.7- to 9.5-fold for adults and from 0.5- to 29-fold for larvae at the LC95. Overall, the patterns of resistance did not mirror the insecticide use patterns reported at these facilities. The implications of these results to management of the lesser mealworms are discussed. Copyright © 2006 Society of Chemical Industry [source] Complete Nucleotide Sequence of a Coxsackievirus B4 Strain that Establishes Infection in ICR Mice Pancreas and Induces Glucose IntoleranceTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 5 2008Mi Zhou Abstract Some coxsackievirus B serotypes are potentially diabetogenic. Previous studies revealed that the virulence and the tissue damage varied with the genetics of the virus strain as well as with the genetics of the mice. A single amino acid variation can alter virulence and tropism in both murine and in vitro models. However, the genetic determinants of this phenomenon have not been determined. In this study, infections with a laboratory strain of coxsackievirus B4 resulted in a diabetes-like syndrome in ICR mice, characterized by chronic pancreatic inflammation together with dysregulation in glucose metabolism, loss of pancreatic acinar tissue and persistent infection in islets. To characterize the genetic determinants involved in the mouse pancreas adaptation, the laboratory strain of coxsackievirus B4 was cloned for molecular characterization. Comparing the whole genome sequence of this virus strain with the other coxsackievirus B4 strains revealed some differences. Altogether 15 nucleotides were changed, resulting in 10 amino acid substitutions, which might be responsible for the pathogenic phenotype of this strain in mice. Anat Rec, 291:601,609, 2008. © 2008 Wiley-Liss, Inc. [source] Functional males in pair-mating outcrossing hermaphroditesBIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY, Issue 2 2010VALERIA DI BONA In the mating system of simultaneously hermaphroditic animals, sexual allocation is predicted to vary as a function of the number of potential mates. According to the Hermaphrodite's Dilemma, sexual conflict over the preferred sexual role in hermaphroditic animals is resolved by reciprocity (i.e. by alternating sexual roles), accompanied by the animals' occasional cheating in the preferred role at a relatively low frequency. In a 350-generation-old laboratory strain of the pair-mating outcrossing hermaphroditic polychaete worm Ophryotrocha diadema, we show that 9% of the individuals mated only in the male role over long periods, indicating a male-role preference (temporary functional males). Furthermore, 2% of the individuals mated for their whole lifetime exclusively as males (permanent functional males). These findings indicate that the sex allocation of some individuals may vary from the predicted optimal sex allocation for the population. Morphologically, functional males exhibited a hermaphroditic phenotype (i.e. they matured a single batch of oocytes that they never laid and acted as functional males). We show that temporary functional males appeared in hermaphroditic populations under promiscuous mating regimes significantly more often than under monogamous ones. Indeed, under promiscuity, there are many mating opportunities and O. diadema hermaphrodites compete for mates, whereas, under monogamy, the two partners regularly take turns in laying cocoons and fertilizing their partner's cocoon. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 451,456. [source] Escherichia coli,-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocationCELLULAR MICROBIOLOGY, Issue 10 2007Hanno Troeger Summary Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (Rt) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an Rt decrease (36 ± 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-, or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased Rt and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli,-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines. [source] Improved 2-DE of microorganisms after acidic extractionELECTROPHORESIS, Issue 8 2006Ben R. Herbert Professor Abstract 2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100,kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH,3 by 80,mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. [source] Subchronic exposure of BALB/c and C57BL/6 strains of Mus musculus to the radioactive environment of the Chornobyl, Ukraine exclusion zoneENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2001Brenda E. Rodgers Abstract Environmental contamination resulting from the Chornobyl, Ukraine, disaster offers a unique opportunity to examine the in vivo biological effects of chronic, low-dose exposure to radiation. Laboratory studies of acute exposure to ionizing radiation have been used to estimate risk and potential human health effects by the extrapolation of laboratory data to situations of low-dose environmental radiation exposure. Few studies, however, have explored the biological consequences of low-dose exposure via in situ environmental radiation in a sentinel species. In the present study, laboratory strains of Mus musculus (BALB/c and 57BL/ 6) were placed in environmental enclosures in the Red Forest region of the Chornobyl exclusion zone. Blood samples were obtained every 10 d, and the micronucleus (MN) test was employed to assess the potential for cytogenetic damage from exposure to Chornobyl radiation. Radionuclide uptake was monitored throughout the study, and dose was estimated for each individual as well as for their offspring. Total dose for the mice experimentally exposed to this environment averaged 1162 mGy for BALB/c (30 d) and 1629 mGy for C57BL/6 (40 d). A higher MN frequency for both strains was observed at day 10, although this change was only statistically significant in the C57BL/6 mice (,23 = 13.41, p = 0.003). Subsequent samples from C57BL/6 resulted in values at or less than the initial frequencies. In BALB/c mice, an increase in MN was also evident at day 30 (,22 = 10.38, p = 0.006). The experimental design employed here allows for the incorporation of traditional laboratory strains, as well as transgenic strains of Mus, as sentinels of environmental radiation contamination. [source] Microevolutionary analysis of the nematode genus Pristionchus suggests a recent evolution of redundant developmental mechanisms during vulva formationEVOLUTION AND DEVELOPMENT, Issue 4 2001Jagan Srinivasan SUMMARY To identify the mechanisms by which molecular variation is introduced into developmental systems, microevolutionary approaches to evolutionary developmental biology have to be taken. Here, we describe the molecular and developmental characterization of laboratory strains of the nematode genus Pristionchus, which lays a foundation for a microevolutionary analysis of vulva development. We describe 13 laboratory strains of the Pristionchus genus that are derived from natural isolates from around the world. Mating experiments and ITS sequence analysis indicated that these 13 strains represent four different species: the gonochoristic species P. lheritieri and three hermaphroditic species, P. pacificus, P. maupasi, and an as yet undescribed species Pristionchus sp., respectively. P. pacificus is represented by five different strains isolated from California, Washington, Hawaii, Ontario, and Poland. Developmental differences during vulva formation are observed between strains from different species but also between strains of P. pacificus, like the strains from California and Poland. In particular, redundant developmental mechanisms present during vulva formation in P. pacificus var. California are absent in other strains. Amplified restriction fragment length polymorphism (AFLP) analyses of the P. pacificus strains revealed that the American strains are highly polymorphic. In contrast, the developmentally distinct strain from Poland is identical to the Californian strain, suggesting that the developmental differences rely on a small number of changes in developmental control genes rather than the accumulation of changes at multiple loci. [source] Effects of clinical isolates of Pseudomonas aeruginosa on Staphylococcus epidermidis biofilm formationFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2010Maria Pihl Abstract Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonization by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA FISH and confocal laser scanning microscopy. Among the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect, and even after 6 h, S. epidermidis levels in dual-species biofilms were reduced by >85% compared with those without P. aeruginosa. Interestingly, strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa, which may develop over time in chronic infections, could counteract colonization by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon. [source] Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strainsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Tecilli Cabellos-Avelar Abstract The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22 000 transposon-induced and 26 000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process. [source] Phenotypic diversity of Flo protein family-mediated adhesion in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2009Sebastiaan E. Van Mulders Abstract The Saccharomyces cerevisiae genome encodes a Flo (flocculin) adhesin family responsible for cell,cell and cell,surface adherence. In commonly used laboratory strains, these FLO genes are transcriptionally silent, because of a nonsense mutation in the transcriptional activator FLO8, concealing the potential phenotypic diversity of fungal adhesion. Here, we analyse the distinct adhesion characteristics conferred by each of the five FLO genes in the S288C strain and compare these phenotypes with a strain containing a functional copy of FLO8. Our results show that four FLO genes confer flocculation, but with divergent characteristics such as binding strength, carbohydrate recognition and floc size. Adhesion to agar surfaces, on the other hand, largely depended on two adhesins, Flo10 and Flo11. Expression of any FLO gene caused a significant increase in cell wall hydrophobicity. Nevertheless, the capacity to adhere to plastic surfaces, which is believed to depend on hydrophobic interactions, differed strongly between the adhesins. Restoring Flo8 yielded both flocculation and cell,surface adherence, such as invasive growth, a phenotype not observed when any of the single FLO genes was overexpressed. Taken together, this study reveals how S. cerevisiae carries a small reservoir of FLO genes that allows cells to display a wide variety of adhesive properties. [source] Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrolFEMS YEAST RESEARCH, Issue 1 2003John V.W. Becker Abstract The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p -coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p -coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p -coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following ,-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol ,-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol. [source] Distribution of the C1473G polymorphism in tryptophan hydroxylase 2 gene in laboratory and wild miceGENES, BRAIN AND BEHAVIOR, Issue 5 2010D. V. Osipova The neurotransmitter serotonin is implicated in the regulation of various forms of behavior, including aggression, sexual behavior and stress response. The rate of brain serotonin synthesis is determined by the activity of neuronal-specific enzyme tryptophan hydroxylase 2. The missense C1473G substitution in mouse tryptophan hydroxylase 2 gene has been shown to lower the enzyme activity and brain serotonin level. Here, the C1473G polymorphism was investigated in 84 common laboratory inbred strains, 39 inbred and semi-inbred strains derived from wild ancestors (mostly from Eurasia) and in 75 wild mice trapped in different locations in Russia and Armenia. Among all the classical inbred strains studied, only substrains of BALB/c, A and DBA, as well as the IITES/Nga and NZW/NSlc strains were homozygous for the 1473G allele. In contrast to laboratory strains, the 1473G allele was not present in any of the samples from wild and wild-derived mice, although the wild mice varied substantially in the C1477T neutral substitution closely linked to the C1473G polymorphism. According to these results, the frequency of the 1473G allele in natural populations does not exceed 0.5%, and the C1473G polymorphism is in fact a rare mutation that is possibly eliminated by the forces of natural selection. [source] Influenza virus assays based on virus-inducible reporter cell linesINFLUENZA AND OTHER RESPIRATORY VIRUSES, Issue 5 2009Yunsheng Li Background, Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5,- and 3,-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression. Findings, Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses. Conclusions, These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers. [source] The stress response is repressed during fermentation in brewery strains of yeastJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2000M.P. Brosnan Yeast cells encounter a variety of environmental stresses during brewing and must respond to ensure cell survival. Cells can respond to stress by inducing a Heat Shock Response in which heat shock proteins (Hsps) are synthesized. In laboratory strains of Saccharomyces cerevisiae, the heat shock protein, Hsp104, plays a major role in the acquisition of tolerance to a variety of stresses such as heat, ethanol and sodium arsenite, and as such acts as an excellent stress indicator. The induction of Hsp104 in bottom-and top-fermenting brewery strains was examined when grown under laboratory and industrial fermentation conditions, and it was found that each brewing strain exhibits its own unique pattern of Hsp104 expression. During industrial fermentations, brewery strains are capable of mounting a stress response at the early stages of fermentation. However, as the fermentation proceeds, the response is repressed. The results suggest that conditions experienced in industrial brewing prevent the activation of the stress response. This study increases our understanding of alterations in gene expression patterns during the brewing process, and yields information that will aid in the definition of best practice in yeast management. [source] Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel geneMEDICAL AND VETERINARY ENTOMOLOGY, Issue 1 2003C. Brengues Abstract. Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth-instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S-transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock-down resistant (kdr)-type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage-gated sodium channel gene was obtained by RT-PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains. [source] Prion protein gene polymorphisms in Saccharomyces cerevisiaeMOLECULAR MICROBIOLOGY, Issue 4 2003Catarina G. Resende Summary The yeast Saccharomyces cerevisiae genome encodes several proteins that, in laboratory strains, can take up a stable, transmissible prion form. In each case, this requires the Asn/Gln-rich prion-forming domain (PrD) of the protein to be intact. In order to further understand the evolutionary significance of this unusual property, we have examined four different prion genes and their corresponding PrDs, from a number of naturally occurring strains of S. cerevisiae. In 4 of the 16 strains studied we identified a new allele of the SUP35 gene (SUP35,19) that contains a 19-amino-acid deletion within the N-terminal PrD, a deletion that eliminates the prion property of Sup35p. In these strains a second prion gene, RNQ1, was found to be highly polymorphic, with eight different RNQ1 alleles detected in the six diploid strains studied. In contrast, for one other prion gene (URE2) and the sequence of the NEW1 gene encoding a PrD, no significant degree of DNA polymorphism was detected. Analysis of the naturally occurring alleles of RNQ1 and SUP35 indicated that the various polymorphisms identified were associated with DNA tandem repeats (6, 12, 33, 42 or 57 bp) within the coding sequences. The expansion and contraction of DNA repeats within the RNQ1 gene may provide an evolutionary mechanism that can ensure rapid change between the [PRION+] and [prion,] states. [source] Characterization and expression of a novel Porphyromonas gingivalis outer membrane protein, Omp28MOLECULAR ORAL MICROBIOLOGY, Issue 3 2002N. Slakeski We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains. [source] | |||||||||||||||||||||||||||||||