The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

Spatial components of foraging behavior in an African Ponerine ant,Paltothyreus tarsatus

Colonies of the African stink ant Paltothyreus tarsatuslocated in the forest have nests with shorter horizontal galleries and a smaller total foraging surface than colonies located in open areas. Each solitary worker specializes on the same central or peripheral hunting zone but she does not specialize on a particular sector during group-retrieving. The search for prey is characterized by a wandering walk with spatial parameters varying in two ways. Capture of a termite releases a path characterized by sinuosity and a decrease in speed of movement. In contrast, a failure in the course of an attempted capture releases an increase in both sinuosity and speed of movement corresponding to a socalled reserve behavior. Each worker shortens her retrieving trip in comparison with her search trip and the straightness of the homing paths depends on the size and shape of the prey. Our data show that behavioral flexibility at the individual level in P. tarsatusis important in determining spatial foraging strategy at the colony level.     

Factors influencing the effectiveness of an attracticide formulation against the Oriental fruit moth, Grapholita molesta

An attracticide formulation, LastCall?OFM, was tested against the Oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae) in replicated small plot field trials in apple, Malus domestica (Borkhausen), orchards in South‐eastern Pennsylvania, USA. Attracticide treatments were applied using a calibrated hand pump, and treated plots were compared to similar untreated plots. Male moth activity was monitored using virgin female‐baited traps, and the potential for reduction in mating activity was assessed using sentinel virgin females. A comparison of application rates showed that 1500 droplets per ha of the attracticide formulation was as effective as 3000 droplets per ha, and both application rates reduced captures in synthetic pheromone‐baited traps for prolonged periods. Droplets placed either at high or low positions within the canopy significantly reduced trap capture and mating with sentinel females. In addition, the only sentinel females that mated in the treated plots were located in the untreated portion of the tree canopy. Mate finding behaviour was equally disrupted by formulations with and without insecticide. Therefore, under the test conditions, the mechanism by which the attracticide formulation worked was by disruption of male orientation, and not by the removal of males due to insecticide poisoning. Two field cage experiments tested the impact of population density on the competitiveness of the attracticide formulation compared to virgin females. A significant proportion of males were captured in female‐baited traps at the highest female‐to‐droplet ratio tested. Equal proportions of males were captured in attracticide‐baited traps at male moth densities of 10, 20, 40, and 80 males per cage. These results clarify some of the factors influencing the effectiveness and possible mechanisms of an attracticide management tactic against the Oriental fruit moth.     

Field observations quantifying attraction of four tortricid moths to high-dosage pheromone dispensers in untreated and pheromone-treated orchards

Orientational responses of four species of feral tortricid moths (Lepidoptera: Tortricidae) to polyethylene tube dispensers of pheromone were observed in a 0.8 ha apple orchard treated with such pheromone dispensers and in an untreated 0.8 ha orchard. Male oblique‐banded leafrollers, Choristoneura rosaceana (Walker) (mean 7.2 ± 0.4 moths/night during 21 nights), Oriental fruit moths, Grapholita molesta (Busck) (mean 10.5 ± 2.1 during 20 evenings), and the redbanded leafrollers, Argyrotaenia velutinana (Walker) (mean 2.0 ± 1.1 during 14 nights) were observed approaching within 100 cm of their respective polyethylene‐tube pheromone dispensers in the untreated orchard. Furthermore, C. rosaceana (mean 2.0 ± 0.7 during 17 nights) and G. molesta (mean 1.5 ± 0.4 over 20 evenings) came within 100 cm of their respective polyethylene‐tube pheromone dispensers in the pheromone‐treated orchard. Most visits lasted less than 10 s, after which the majority of moths departed by flying upwind. In the untreated orchard, the number of C. rosaceana observed orienting to polyethylene tube dispensers was greater than the number captured in optimized monitoring traps (1.9 ± 0.4) per night of observation. The numbers of A. velutinana (2.0 ± 1.1) or G. molesta (10.5 ± 2.1) attracted to polyethylene‐tube dispensers in the untreated orchard did not differ statistically from the numbers captured in optimized monitoring traps per night of observation. In the pheromone‐treated orchard, the number of C. rosaceana (2.0 ± 0.4) or G. molesta (1.2 ± 0.2) observed orienting to polyethylene‐tube dispensers did not differ statistically from the numbers of male moths of these species captured in optimized monitoring traps per night of observation. No codling moths, Cydia pomonella L. were observed orienting to, or landing near, their respective polyethylene‐tube dispensers in either the untreated or pheromone‐treated orchards, although substantial numbers were captured in monitoring traps per night of observation (6.0 ± 1.7) in the untreated orchard. The attraction of male moths to polyethylene tube dispensers thus occurred in three of the four species observed. These results provide support for the idea that false‐plume‐following is an important component of the mechanisms mediating communicational disruption in moths by polyethylene‐tube dispensers.     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^– );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5and 3truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.     

A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet. Project supported by the National Natural Science Foundation of China.     

Cortical hypometabolism in injured brain: New correlations with the noradrenergic and serotonergic systems and with behavioral deficits

Changes with time after injury in behavioral deficits, as determined by the Morris swim test, and the in vivo specific binding of HEAT, a selective ₁-adrenoreceptor ligand, were compared with the time-course of development of cortical hypometabolism in rats with focal freezing lesions. In our trauma model, cortical hypometabolism was detectable in the lesioned hemisphere at 4 hr, became maximal (50% of normal) at 3 days and diminished towards normal on days 5 and 10 post-injury. Progressive impairment of acquisition of the Morris water maze task was demonstrated up to day 3 post-lesion with improvement thereafter. On day 3 the latency to reach criterion was 60% longer in lesioned animals than in corresponding sham-operated ones. An increase in the volume of distribution of HEAT, limited to cortical areas of the lesioned hemisphere, was demonstrable at 4 hr post-lesion and reached its maximum on day 3 (200% of normal) with subsequent return toward normal on days 5 and 10. Several types of drugs were shown previously to modify the cortical hypometabolism associated with cerebral injury. The present data indicate that the same drugs also modify the in vivo binding of HEAT and the behavioral deficits induced by brain lesions. Ibuprofen, a non-steroidal anti-inflammatory drug, p-chlorophenylalanine, an inhibitor of serotonin synthesis, ketanserin, a specific 5HT₂-receptor antagonist, and prazosin, an ₁-adrenergic receptor blocker all normalized the in vivo binding of HEAT in the cortical areas of the lesioned hemisphere. All groups of animals treated with these drugs also showed subtle, but statistically highly significant improvements in latency to locate the platform in the Morris water maze. Taken together these results show good correlation between behavioral deficits, changes in ₁-noradrenergic receptor binding and cortical hypometabolism in injured brain. This supports the hypothesis that post-injury cortical hypometabolism is a reflection of cortical functional depression in which both the serotonergic and noradrenergic neurotransmitter systems play a role, compatible with their inhibitory effects in the cortex and their postulated involvement in cortical information processing.Special issue dedicated to Dr. Leon S. Wolfe.     

Behavioral adaptations of two phytophagous insects feeding on two species of phototoxic Asteraceae

Two phototoxic plants of the Asteraceae family were studied in relation to species of phytophagous insects for which they are hosts:Argyrotaenia velutinana Wlk. feeding onChrysanthemum leucanthemum L. andChlorochlamys chloroleucaria (Guenée) colonizingRudbeckia hirta L. The toxicity of these two plants is related to the presence of acetylenes and thiophenes that induce a light-mediated production of deleterious singlet oxygen and other free radicals (phototoxicity). Results showed that females ofA. velutinana laid their eggs preferentially in the shade and the larvae adopted hiding behaviors, such as bending of ligulate corollas and silk spinning to build opaque shelters. By avoiding direct exposure to the sun, both behaviors may reduce phototoxicity associated with ingested plant materials. Furthermore, larvae ofC. chloroleucaria demonstrated a preference in the field for pollen, which constitutes a nonphototoxic tissue of their host plant. Experimental alterations of these specific behaviors induced important biological consequences for larvae of both insects such as mortality or reduction of larval growth rate. These results reinforce the idea that behavior may constitute an efficient adaptation to avoid phototoxicity.     

Behavioral development in okapis and giraffes

Observations on the behavioral development of two okapi calves and one giraffe calf were made at Brookfield Zoo. The following behaviors were monitored for 4 to 6 mo after birth; nursing duration and nursing attempts, mother-infant distance, bunting the mother's udder, lying, moving, maternal grooming, mother and infant autogrooming, object licking, tail chewing, and contact by others in the herd. Behaviors in both species showed oscillating patterns with high levels of mother-infant contact behaviors at 3–4 wk, 9–11 wk, and 14–15 wk in okapis. Giraffe infants showed similar oscillations with high periods of contact about 2–5 wk later than those in okapis. Other behaviors oscillated in concert with these, with specific correlations occurring between nursing behaviors and grooming behaviors. A main difference between okapi and giraffe development centered around maternal motivation during the high contact (regressive) periods. In okapis, after 10–12 wk there was a low rate of nursing success, whereas in giraffes the percentage of success in nursing rose with later behavioral oscillations. The regressive periods became conflict periods in okapis, whereas in the giraffe, the mother initiated the periods. This difference was in accordance with the unique strategy of infant rearing in wild giraffes in which there is an extended “hider” period when older calves are left together in shaded areas with an adult sentry. Field studies also indicated probable oscillations of mother-infant contact and a prolonged period of the mother initiating contact with her calf.