Jerome J. Kukor

Acquisition of Digestive Enzymes by Siricid Woodwasps from Their Fungal Symbiont

Larvae of the woodwasp, Sirex cyaneus, contain midgut digestive enzymes that enable them to utilize the major fungal and plant polysaccharides found in their food. At least two classes of enzymes, the Cχ-cellulases and the xylanases, are not produced by the larvae. Instead, larvae acquire these enzymes while ingesting tissue of Amylostereum chailletii, the fungal symbiont that occurs in the wood on which the larvae feed.

The digestion of protein and carbohydrate by the stream detritivore, Tipula abdominalis (Diptera, Tipulidae)

SummaryThe digestive system of larvae of Tipula abdominalis (Diptera, Tipulidae), a stream detritivore, is poorly adapted for the digestion of the major polysaccharides in its diet, but well adapted for the digestion of protein. These crane fly larvae are unable to digest the major cell wall polysaccharides of higher plants, i.e., cellulose, hemicellulose and pectin. The only polysaccharides toward which the midguts of T. abdominalis exhibited any activity were α-amylose and laminarin, indicating that polysaccharide digestion is restricted to α-1,4-and β-1,3-glucans. The most concentrated source of these two classes of carbohydrates in submerged leaf litter would be associated fungal tissue. The midgut of T. abdominalis is strongly alkaline throughout, with a maximum pH near 11.5 in a narrow zone near the midpoint. Proteolytic activity in the midgut is extraordinarily high, and the pH optimum for midgut proteolytic activity is above 11. We conclude that the high alkalinity and high proteolytic activity observed in T. abdominalis larvae are manifestations of a highly efficient protein-digesting system, a system of crucial importance to a nitrogen-limited organism which must derive its nitrogen from a resource in which much of the limited nitrogen present is in a “bound” form in complexes of proteins with lignins and polyphenols.

Sequence analysis of the gene cluster encoding toluene-3-monooxygenase from Pseudomonas pickettii PKO1

The nucleotide (nt) sequence and gene organization of the locus encoding the initial step of the toluene-3-monooxygenase (Tbu) pathway from Pseudomonas pickettii PKO1 has been determined. This is the first reported nt sequence for a toluene monooxygenase which hydroxylates the C-3 position of toluene. Six tightly assembled structural genes encoding several Tbu were identified and were designated tbuA1, tbuU, tbuB, tbuV, tbuA2 and tbuC. Comparison of the deduced amino acid (aa) sequences of each open reading frame (ORF) with translated sequences from the GenBank database revealed significant overall homology to peptides from the toluene-4-monooxygenase (Tmo) from Pseudomonas mendocina KR1, the multicomponent phenol hydroxylase (Dmp) from Pseudomonas sp. strain CF600 and the methane monooxygenases (Mmo) from both Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. Similarities in both size and aa sequence between the peptides from these multicomponent oxygenases and the putative peptides from Tbu suggested roles for the TbuA1, TbuB, TbuV, TbuA2 and TbuC proteins.

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 103 to 105 bacteria ml−1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.

The role of ingested fungal enzymes in cellulose digestion in the larvae of cerambycid beetles

Ingested fungal enzymes are responsible for cellulose digestion in the xylophagous larvae of four species ofcerambycid beetles from four different subfamilies: Bellamira scalaris (Lepturinae), Graphisurusfasciatus (Lamiinae), Orthosoma brunneum (Prioninae) and Parandra brunnea (Parandrinae). When the larvae are maintained on their normal diets of fungus-infected wood, their midgut fluids are active against microcrystalline cellulose, and they are able to digest

Cellulose digestion inMonochamus marmorator Kby. (Coleoptera: Cerambycidae): Role of acquired fungal enzymes

^{14}C-cellulose

The nutritional ecology of larvae of Alsophila pometaria and Anisota senatoria feeding on early- and late-season oak foliage

. By contrast, when larvae are maintained on fungus-free, enzyme-free diets, their midgut fluids lack cellulolytic activity, and they are unable to digest labeled cellulose. We argue that ingested fungal enzymes may be responsible for cellulose digestion in many, perhaps even all, xylophagous larvae from the three coleopteran families Anobiidae, Buprestidae, and Cerambycidae.

The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse, Tracheoniscus rathkei

Larvae of the balsam fir sawyer,Monochamus marmorator Kby. (Coleoptera, Cerambycidae), contain midgut digestive enzymes active against hemicellulose and cellulose. Cellulases from larvae fed on balsam fir wood infected with the fungus,Trichoderma harzianum Rifai (Deuteromycetes, Moniliales, Moniliaceae), were found to be identical to those of the cellulase complex produced by this fungus when compared using chromatography, electrophoresis, and isofocusing. When larvae are maintained on a fungus-free diet, their midgut fluids lack cellulolytic activity, and they are unable to digest cellulose. Cellulolytic capacity can be restored by feeding the larvae wood permeated by fungi. We conclude that the enzymes which enableM. marmorator larvae to digest cellulose are not produced by the larvae. Instead, the larvae acquire the capacity to digest cellulose by ingesting active fungal cellulases while feeding in fungus-infected wood.

Enumeration and characterization of BTEX-degrading bacteria from hypoxic environments functional with mixed electron acceptors☆

The larvae of Alsophila pometaria (Harr.), feeding on the young foliage of oak, has a higher relative growth rate (RGR) and relative nitrogen accumulation rate (RNAR) than the larvae of Anisota senatoria (J. E. Smith), feeding on the mature foliage of oak. Although the young oak foliage is more efficiently digested by A. pometaria (higher ADs), it is not more efficiently assimilated and used for growth (no difference in ECIs). Thus, the higher growth rate of A. pometaria is due entirely to a higher consumption rate (RCR and RNCR). Young foliage is significantly higher in nitrogen and water than mature foliage, but phenol and tannin levels are comparable in young and old foliage. A. pometaria consumes the foliage of different oak species at the same rate, independent of nitrogen content, while A. senatoria increases its consumption rate in response to decreased nitrogen levels. As a result, the growth rate of A. pometaria is directly related to leaf nitrogen content, while the growth rate of A. senatoria is independent of leaf nitrogen. The two species of insects have digestive systems that are very similar biochemically, and that are well‐designed for effective protein digestion. Tannins and phenols do not influence the nutritional indices of either species. We suggest that the major benefit of spring feeding is the availability of succulent, high‐nitrogen foliage, and not the avoidance of hightannin foliage. The spring feeder appears to have a feeding strategy that favors rapid growth at the expense of efficiency, while the late summer feeder has a strategy that favors efficiency over rate.

The transformation of Saperda calcarata (Coleoptera: Cerambycidae) into a cellulose digester through the inclusion of fungal enzymes in its diet

SummaryThe digestive tract of the common woodlouse, Tracheoniscus rathkei Brandt (Isopoda: Oniscoidea), contains digestive enzymes active against α-1,4-glucans, which are the chief storage polysaccharides of vascular plants, algae, fungi, and animals, and β-1,3-glucans, which are present in algae and fungi. Digestive tract extracts also exhibit significant activity toward xylan and carboxymethyl-cellulose but negligible activity toward microcrystalline cellulose, substrates representative of the major structural polysaccharides of vascular plants. Low activity was detected toward pectin, and no activity was detected toward chitin. Activity toward xylan is due in part to microbial enzymes acquired from the leaf litter which was the isopods normal food. Although ingested microbial xylanases are stable and active in the gut fluid, they do not make a quantitatively significant contribution to the isopods ability to assimilate the hemicellulosic component of its diet. However, the assimilation of carbon from labeled plant fiber is enhanced in isopods which have acquired a cellulase by ingestion of leaf litter amended with a commercial preparation of the cellulase complex from the fungus, Penicillium funiculosum. This result demonstrates the potential contribution of acquired enzymes to the digestion of plant fiber in terrestrial detritivores. We urge caution, however, in assigning an important digestive function to ingested enzymes on the basis of evidence that only indicates that such enzymes are present in the gut fluid without additional evidence that their presence results in an enhancement of digestive efficiency.