Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide.

Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids. The amino acid sequence of the P. aeruginosa katB was approximately 65% identical to that of a catalase from a related species, Pseudomonas syringae. The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase-deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E. coli strain (HB101). The KatB protein was purified to homogeneity and determined to be a tetramer of approximately 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene. Interestingly, KatB was not produced during the normal P. aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively. In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide. KatB was localized to the cytoplasm, while KatA, the "housekeeping" enzyme, was detected in both cytoplasmic and periplasmic extracts. A P. aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeroginosa to exogenous hydrogen peroxide.     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity     

Structure and evolution of teleost mitochondrial control regions

We amplified and sequenced the mitochondrial control region from 23 species representing six families of teleost fish. The length of this segment is highly variable among even closely related species due to the presence of tandemly repeated sequences and large insertions. The position of the repetitive sequences suggests that they arise during replication both near the origin of replication and at the site of termination of the D-loop strand. Many of the conserved sequence blocks (CSBs) observed in mammals are also found among fish. In particular, the mammalian CSB-D is present in all of the fish species studied. Study of potential secondary structures of RNAs from the conserved regions provides little insight into the functional constraints on these regions. The variable structure of these control regions suggests that particular care should be taken to identify the most appropriate segment for studies of intraspecific variation. Correspondence to: T.D. Kocher     

Selection for non-diapause inAmblyseius cucumeris andAmblyseius barkeri and exploration of the effectiveness of selected strains for thrips control

In Europe and North America the western flower thrips,Frankliniella occidentalis, is an important pest in various greenhouse crops, such as sweet pepper and cucumber. Two species of predatory mite are commercially applied for biological control of this pest:Amblyseius cucumeris andA. barkeri. Thrips control is generally successful from March onwards. During winter, however, thrips control by these predatory mites is less effective. An important reason for this is that the commercially applied strains of both mite species enter reproductive diapause under short-day photoperiods, whereas the western flower thrips does not enter diapause. In this paper we report on selection experiments for non-diapause in strains of both mite species, aimed at obtaining predators that do not enter diapause under light- and temperature conditions prevailing in winter. Additional experiments were done to estimate the potential of the selected lines as control agents ofF. occidentalis. Selection for non-diapause proved highly successful in both predatory mite species. In a New Zealand strain ofA. cucumeris diapause incidence decreased from 41% to 0% in about ten generations; in a Dutch strain ofA. barkeri diapause incidence decreased from 67% to 0% in about six generations. Furthermore, selection for non-diapause had no influence on predator performance, measured as predation rate and oviposition rate on a diet of first instar thirps larvae. Rates of predation and oviposition were the same for selected and unselected lines in both species; rates of predation and oviposition were higher forA. cucumeris than forA. barkeri. After 18 months under non-diapause conditions, no less than 92% of a sample of the selected non-diapause line ofA. cucumeris did not enter diapause when tested under diapause-inducing conditions. This indicates that ‘non-diapause’ is a stable trait in these predatory mites. Finally, a small-scale greenhouse experiment in a sweet pepper crop showed that the selected non-diapause line ofA. cucumeris established successfully under diapause-inducing short-day conditions.     

Duration of the breeding season and response to reproductive manipulation in five breeds of sheep under northern prairie conditions

The breeding season was 157, 154, <126, 210 and 217 days for Rambouillet, Columbia, Suffolk, Rambouillet x Finnish Landrace and Columbia x Finnish Landrace ewes respectively. Treatment of cyclic ewes with pregnant mare serum gonadotropin (PMSG) (500 IU), following a 12-day treatment with progestin-containing intravaginal sponges, did not affect fertility, but did decrease the time from sponge removal to estrus, (control 48.0 +/- 3.1 hr; PMSG 39.4 +/- 1.8 hr) to the preovulatory surge of LH (control 52.7 +/- 2.8 hr; PMSG 39.0 +/- 1.7 hr) and FSH (control 52.3 +/- 2.9 hr; PMSG 42.8 +/- 1.6 hr) and caused an elevation of serum LH levels prior to the preovulatory surge (control 1.25 +/- 0.18 ng/ml; PMSG 2.31 +/- 0.22 ng/ml). Exposure of the purebred ewes to 18 hours of daylight in January, decreasing by 30 minutes a week subsequently, counteracted the seasonal reduction in the number of ewes lambing following induced breeding under natural daylight in May. Prolificacy was greatest in crossbred ewes and their fertility was not affected by season. Gestation period was longer for fall-bred ewes and varied with breed.     

Independent regulation of serologically distinct idiotypes in F1 hybrid mice

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRI_A and CRI_C, characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c x A/J)F₁ (CAF₁) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF₁ mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRI_C was markedly enhanced in mice suppressed for CRI_A.Abbreviations used in this paper anti-Id(A/J) idiotypic-specific antibodies against A/J serum Ar-specific antibodies - anti-Id(BALB/c) idiotypic-specific antibodies against BALB/c serum Ar-specific antibodies - Ar p-azophenylarsonate - BGG bovine -globulin - BSA bovine serum albumin - CAF₁ F(BALB/c x A/J) - CFA complete Freund's adjuvant - CRIA the major cross-reactive idiotype of A/J Ar-specific antibodies - CRI_C the major cross-reactive idiotype of BALB/c Ar-speck antibodies - CRI_m the minor cross-reactive idiotype of A/J Ar-specific antibodies - DTH delayed-type hypersensitivity - HP hybridoma product(s) - KLH keyhole limpet hemocyanin     

Comparison of gravimetry and planimetry in determining dry matter budgets for three species of phytophagous lepidopteran larvae

Gravimetric and a combination areal-gravimetric methods for determining dry matter budgets for leaf eating Lepidoptera were compared. The gravimetric method is based on dry weight/live weight ratios of the leaves fed to the larvae. In the areal-gravimetric method, the quantity of food offered to the larvae is determined from the area of leaf tracings and the dry matter content per unit area of the leaves. The areal-gravimetric method permits the use of larger leaf sprays and an open, gauze enclosed rearing chamber. There were no consistent differences in budget factors (growth, ingestion or egestion), nor were there any differences in the observed variability of the data attributable to the method used. However, the expected variability based on instrument precision for the gravimetric method is less than for the areal-gravimetric method. Experimental factors inherent in the gravimetric method introduce variability to the measurements that are not present in the areal method. Thirty to 60% of the variability in budget factors was attributed to intrinsic properties of the larvae, even though the larvae were taken from the same egg masses.

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Résumé Les budgets en matière sèche consommée par des lépidoptères ont été comparés par les méthodes gravimétrique et planimétrique. La méthode gravimétrique est basée sur le rapport poids sec/poids frais de feuilles consommées par les chenilles. Avec la méthode planimétrique, la quantité d'aliment proposée aux chenilles est déterminée par les tracés de la surface des feuilles et le contenu de matière sèche par unité de surface des feuilles. La méthode de planimétrie permet l'utilisation de plus grands rameaux de feuilles et de cages d'élevage extérieures en gaze. Il n'y avait pas de différence appréciable dans les éléments du budget (croissance, ingestion et déjection), ni aucune différence dans la variabilité observée des données attribuable à la méthode utilisée. Cependant, la variabilité attendue d'après la précision des mesures avec la méthode gravimétrique est inférieure à celle de la méthode planimétrique. est inférieure à celle de la méthode planimétrique. Des éléments expérimentaux, inhérents à la méthode gravimétrique, introduisent une variabilité dans les mesures que l'on n'a pas avec la méthode planimétrique. 30–60% de la variabilité dans la consommation ont été attribués à des paramètres internes à la chenille, même quand elles provenaient toutes de la même ooplaque.