Structure and gene cluster of a tyvelose-containing O-polysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T related to Yersinia pseudotuberculosis.

An O-polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Yersinia entomophaga MH96T by mild acid hydrolysis and studied by 2D NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: where Tyv indicates 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. entomophaga are related to those of some Y. pseudotuberculosis serotypes.

Serratia entomophila bet gene induction and the impact of glycine betaine accumulation on desiccation tolerance.

AIMS: The genes involved in choline transport and oxidation to glycine betaine in the biopesticidal bacterium Serratia entomophila were characterized, and the potential of osmoprotectants, coupled with increased NaCl concentrations, to improve the desiccation tolerance of this species was investigated. METHODS AND RESULTS: Serratia entomophila carries sequences similar to the Escherichia coli betTIBA genes encoding a choline transporter and dehydrogenase, a betaine aldehyde dehydrogenase and a regulatory protein. Disruption of betA abolished the ability of Ser. entomophila to utilize choline as a carbon source. Quantitative reverse-transcriptase PCR analysis revealed that betA transcription was reduced compared to that of the upstream genes in the operon, and that NaCl and choline induced bet gene expression. Glycine betaine and choline increased the NaCl tolerance of Ser. entomophila, and osmotically preconditioned cultures survived better than control cultures following desiccation and immediately after application to agricultural soil. CONCLUSIONS: Addition of glycine betaine and NaCl to growth medium can greatly enhance the desiccation survival of Ser. entomophila, and its initial survival in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Serratia entomophila is sensitive to desiccation and does not persist under low soil moisture conditions. Techniques described here for enhancing the desiccation survival of Ser. entomophila can be used to improve formulations of this bacterium, and allow its application under a wider range of environmental conditions.

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica).

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and alpha-amylase were still detected in the midgut of diseased larvae.

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

Characterization of estrogenic compounds in water samples collected from United Kingdom estuaries.

This report describes the identification of important estrogenic compounds in surface and sediment pore-water samples collected from the Tyne and Tees estuaries (United Kingdom) through the application of toxicity identification evaluation (TIE) procedures. The Tyne and Tees estuaries represent estuaries that have been historically impacted by industrial activities and continue to receive treated domestic sewage and industrial effluent. In 1998, Dabholm Gut on the Tees received a mixture of treated and untreated effluent, while Howdon sewage treatment works (STW) discharged primary treated effluents. An estrogenically active water sample collected from Howdon STW on the Tyne was shown to contain 17beta-estradiol, androsterone, and an unknown estrogenic compound(s). Most of the activity contained in a sample collected from the Dabholm Gut combined discharges on the Tees was also due to 17beta-estradiol with additional activity from nonylphenol and (tentatively) bis(2-ethylhexyl)phthalate. The only sediment pore-water sample to demonstrate estrogenic activity was collected from Dabholm Gut.

Toxicity characterisation of organic contaminants in stormwaters from an agricultural headwater stream in south east England.

The transient movement of pesticides at biologically active concentrations during storm events is considered to be a cause of biological impoverishment in some headwater streams. The programme of work described sought to identify compounds that are the cause of toxic effects during such events. Along with targeted pesticide analysis, toxicity identification evaluation (TIE) procedures were used to identify compounds with a demonstrated toxic effect. These procedures were specifically directed towards isolating and attributing toxicity to classes of organic contaminants in samples collected from an English headwater stream during a storm event. The organic load was isolated by means of solid-phase extraction (SPE). Bioassay of the SPE extract at x 100 whole water concentrations confirmed that the samples contained substances toxic to Daphnia magna, although the raw samples were not toxic. Targeted pesticide analysis identified simazine and diuron as the major pesticides present and, using a toxicity unit (TU) approach. were shown to be responsible for a significant amount of the observed concentrate toxicity during a runoff event. However, they were not present in sufficient quantities to be totally responsible for a more toxic later event. By simplification of the SPE isolate using reverse-phase HPLC, fractions from which were tested for toxicity, the cause of concentrate toxicity in the later event was isolated to two discrete fractions. GC-MS analysis of these fractions identified nonylphenol. endosulfan sulphate and pendimethalin as present, with the majority of toxicity attributed to nonylphenol (NP). The main advantage of the TIE approach is that it allows biological active compounds with a demonstrated effect to be identified that may not be selected by more traditional techniques.

Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens.

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.

Airborne levels of isopropyl alcohol disinfectant in a laminar-airflow hood.

Airborne levels of isopropyl alcohol when used as a disinfectant in a horizontal laminar-airflow hood were measured during a 10-day period to determine whether levels conformed to the Occupational Safety and Health Administration (OSHA) guidelines. An infrared vapor analyzer was positioned in the airflow of the six-foot horizontal laminar-airflow hood for 10 days to continuously monitor the levels of isopropyl alcohol under usual procedural conditions. A continuous printout of the levels of isopropyl alcohol allowed determination of the eight-hour time-weighted average level. The mean (+/- S.D.) eight-hour time-weighted average level of isopropyl alcohol in the laminar-airflow hood was 99.6 +/- 30.6 parts per million (ppm). By OSHA standards, the eight-hour time-weighted average airborne level of isopropyl alcohol in the work place cannot exceed 400 ppm. Usual conditions in the laminar-airflow hood at this institution complied with the OSHA standard for airborne isopropyl alcohol.

An analysis of the relationship among stuttering behaviors.

The purpose of this study was to determine whether different topographies of disfluent behavior form a response class. A within-subject, repeated reversals (ABAB) design was used to analyze the stuttering behavior of three adult stuttering speakers. A single type of stuttering behavior was punished for each subject while frequencies of occurrence of other types were concurrently measured. The results showed that: (1) stuttering behaviors displayed direct behavioral covariation for all subjects, illustrating the existence of a response class; and (2) the response classes observed included both kernel and accessory features of stuttering. The results are discussed in terms of the literature on response classes and two-factor learning theory of stuttering with special emphasis on the implications of these results for our understanding of the development of stuttering.