PCR detection of pirimicarb resistance in Australian field isolates of Aphis gossypii Glover (Aphididae: Hemiptera)

Aphis gossypii Glover (cotton aphid) is a major secondary pest of Australian cotton that readily develops resistance to the carbamate insecticide pirimicarb (Pirimor®) and to organophosphates generally. To test the pirimicarb resistance status of Australian strains of A . gossypii , a polymerase chain reaction (PCR) assay followed by restriction enzyme assay (REA) was designed to identify the Ace I polymorphism S431F known to be responsible for resistance. The method was tested against reference and 33 field strains collected over two consecutive seasons. Both methods confirmed pirimicarb resistance in two field strains, one from each cotton season, giving credence to the molecular technique described. The PCR assay proved specific for the Ace I gene. This PCR REA assay has the potential to replace bioassay for the routine pirimicarb resistance monitoring in A . gossypii. With the molecular assay providing results in 48 h, compared with 4–8 weeks for bioassay, such an assay could be used before insecticide control.     

Replisome trafficking in growing vegetative hyphae of Streptomyces coelicolor A3(2)

We observed movies of replisome trafficking during Streptomyces coelicolor growth. A replisome(s) in the spore served as a replication center(s) until hyphae reached a certain length, when a tip-proximal replisome formed and moved at a fixed distance behind the tip at a speed equivalent to the extension rate of the tip.     

The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology

The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.     

Sciurid phylogeny and the paraphyly of Holarctic ground squirrels (Spermophilus)

The squirrel family, Sciuridae, is one of the largest and most widely dispersed families of mammals. In spite of the wide distribution and conspicuousness of this group, phylogenetic relationships remain poorly understood. We used DNA sequence data from the mitochondrial cytochrome b gene of 114 species in 21 genera to infer phylogenetic relationships among sciurids based on maximum parsimony and Bayesian phylogenetic methods. Although we evaluated more complex alternative models of nucleotide substitution to reconstruct Bayesian phylogenies, none provided a better fit to the data than the GTR+G+I model. We used the reconstructed phylogenies to evaluate the current taxonomy of the Sciuridae. At essentially all levels of relationships, we found the phylogeny of squirrels to be in substantial conflict with the current taxonomy. At the highest level, the flying squirrels do not represent a basal divergence, and the current division of Sciuridae into two subfamilies is therefore not phylogenetically informative. At the tribal level, the Neotropical pygmy squirrel, Sciurillus, represents a basal divergence and is not closely related to the other members of the tribe Sciurini. At the genus level, the sciurine genus Sciurus is paraphyletic with respect to the dwarf squirrels (Microsciurus), and the Holarctic ground squirrels (Spermophilus) are paraphyletic with respect to antelope squirrels (Ammospermophilus), prairie dogs (Cynomys), and marmots (Marmota). Finally, several species of chipmunks and Holarctic ground squirrels do not appear monophyletic, indicating a need for reevaluation of alpha taxonomy.     

B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.      

Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.     

Quantitative high-pressure liquid chromatographic analysis of methylated purines in DNA of rats treated with chemical carcinogens

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O⁶-methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.     

Tebufenpyrad (Pyranica®) resistance detected in two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) from apples in Western Australia

A strain (WA) of Tetranychus urticae Koch was collected from apples in Western Australia following a reported field-control failure of tebufenpyrad. The WA strain had been exposed to five tebufenpyrad applications over four seasons. The tebufenpyrad resistance in the WA strain was confirmed at 63.29× which is the first reported instance of tebufenpyrad resistance in T. urticae. The tebufenpyrad resistance in T. urticae conferred a >210× cross-resistance to pryidaben, a 24.60× cross-resistance to fenpyroximate and a 2.20× cross-resistance to chlorfenapyr.Exp Appl Acarol 22: 633–641 © 1998 Kluwer Academic Publishers