The firefly (Photinus pyralis) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.
Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP S210A protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP S210A was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP S210A also fractionated. These results suggested that the DegP S210A -mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.
In order to develop a PCR assay for Escherichia coli O157:H7, a portion of the 60-MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then designed by employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxins I (SLT-I) and II (SLT-II), and the 60-MDa plasmid. PCR products of 1,087 bp (eaeA), 227 and/or 224 bp (SLT-I and/or SLT-II), and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC of serogroup O157.
5‐Enol‐pyruvylshikimate‐3‐phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate (marketed under the trade name Roundup1) when sufficiently expressed in transgenic plants. Dual CP4 EPSPS transgene cassettes were transformed into corn (Zea mays L.) under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P‐Ract1) and the enhanced Cauliflower mosaic virus 35S (P‐e35S) promoters, respectively, to impart fully constitutive expression in corn. Resulting events were tested for lack of chlorosis and malformation injury after two sequential applications of 1.68 kg acid equivalents (a.e.) ha−1 glyphosate. Agronomic parameters, male fertility, appropriate Mendelian segregation of the trait, plus characteristics of the transgenic integration site were also evaluated. From this selection process, the NK603 event was chosen for commercialization as the event that embodied the most optimal profile of tolerance, agronomics, and molecular characteristics. The NK603 event exhibited high glyphosate tolerance from one transgenic locus bearing a single copy of the dual cassettes integrated into the corn genome with a minimum of target sequence disruption. Trait expression in the NK603 event has remained stable over more than eight generations as shown through tolerance testing, western blots of CP4 EPSPS accumulation, and Southern blot analysis of the transgene.
asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipopolysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.
Abstract. In February and March 2009, approximately 1,500 backyard pigs of variable age became sick, and approximately 700 of them died or were euthanized in the Lower Artibonite Valley and the Lower Plateau of the Republic of Haiti. The main clinical sign was posterior ataxia followed by paresis and/or paralysis on the second or third day of illness. No gross lesions were observed at postmortem examinations. The morbidity and mortality were approximately 60% and 40%, respectively. Diagnostic samples (whole blood, brain, tonsil, lymph nodes, spleen, and lung) were negative for Classical swine fever virus and African swine fever virus. Porcine teschovirus type 1 was detected by reverse transcription polymerase chain reactions in brain samples. Results of virus isolation, electron microscopy of virus particles, histopathological analysis on brain tissues, nucleic acid sequencing, and phylogenetic analysis of the viral isolate supported the diagnosis of teschovirus encephalomyelitis. The outbreak of the disease in Haiti is the first appearance of the severe form of teschovirus encephalomyelitis in the Americas. This disease poses a potential threat to the swine industries in other Caribbean countries, as well as to Central and North American countries.
Abstract. Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription-PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding -actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.Key words: Classical swine fever virus; reverse transcription-PCR; RNA extraction. The reverse transcription-PCR (RT-PCR) is a valuabletechnique that is increasingly being used for diagnosis of animal diseases caused by RNA viruses, including classical swine fever virus (CSFV). 8,12,13,16,18 CSFV is a member of the genus Pestivirus, which belongs to the family Flaviviridae. 7 The virus is highly infectious for swine and causes losses in swine production throughout much of the world. 9 Rapid and accurate detection of CSFV is critical for disease containment. 16 A prerequisite for the performance of RT-PCR is an efficient method for RNA extraction. Currently there are numerous methods that can be used to isolate and purify RNA, but there are few studies comparing extraction methods for samples of animal origin. The present study was designed to evaluate 6 commercially available methods for extraction of total RNA from blood and tissue samples collected from a CSFV-infected pig. These methods were based on RNAqueous kit a (abbreviated as the RNAqueous in this report), Micro-to-midi total RNA purification system b (the Micro-tomidi), NucleoSpin RNA II c (the NucleoSpin), RNeasy mini kit d (the RNeasy), GenElute mammalian total RNA kit e (the GenElute), and TRIzol LS reagent b (the TRIzol). This article describes the results of the evaluation focusing mainly on the yield and purity of RNA extracted by these methods and the performance of the RNA in a real-time RT-PCR and a conventional RT-PCR for the CSFV genome and a conventional RT-PCR for the endogenous mammalian gene encoding cytoskeletal -actin. A young pig (about 50 pounds) was inoculated intranasally with 1 ml of a highly vir...
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