Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases.     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.     

Effect of the structure of phospholipid on the kinetics of intravesicle scooting of phospholipase A2

Action of pig pancreatic phospholipase A2 on vesicles of over 50 synthetic 1,2-diacylglycerol-3-phosphate derivatives and analogs is examined in the absence of any additives. In general, shorter acyl chains and small substituents on the phosphate make a better substrate, while phospholipids with large apolar substituents are not hydrolyzed. The interfacial turnover rate constant for scooting kinetics, ki, for the various phospholipids were from less than 0.1 to 1 per min. Intervesicle exchange of the bound enzyme is faster in vesicles of phospholipids with larger polar substituents, and it is promoted in the presence of anions like chloride, sulfate and thiocyanate. These factors lower the residence time of the enzyme on the bilayer and therefore effectively decrease the rate of hydrolysis. The apparent Km for the enzyme in the interface of anionic phospholipids in the presence of salts is in the 40 to 100 microM range which is 3- to 7-times larger than the dissociation constants for the bound enzyme measured by fluorescence enhancement of Trp-3. The quantum yield of the bound enzyme in vesicles of the various lipids is found to be up to 4-fold different. It is suggested that this difference is due to the E* + S to E*S equilibrium, where E*S has higher fluorescence intensity. The role of calcium in generating the enzyme binding site at the anionic interface, the role of anion anchoring site on the enzyme, and the relationship between the catalytic efficiency and the fluorescence quantum yields are discussed.     

Spatial patterns and feeding of meiobenthic harpacticoid copepods in relation to resident microbial flora

Distributional and feeding relationships of harpacticoid copepods and their microbial prey were examined in a tidal channel at Great Sippewissett Marsh. A horizontal zonation of photosynthetic microorganisms was composed of: 1) a diatom area; 2) a purple sulfur bacterial (Thiocapsa sp.) area; and 3) a clear area. Four species of harpacticoid copepods were associated with given areas. Leptocaris brevicornis occurred in very high densities in the diatom area but in relatively low densities in other areas. Mesochra lilljeborgi occurred in significantly higher densities in the purple and clear areas.Feeding experiments, using resident microbial flora labelled with NaH¹⁴CO₃ and ³H-thymidine, were conducted to determine which foods are 1) ingested but simply pass through the gut and 2) ingested, and retained. These experiments indicated that L. brevicornis ingested diatoms and the heterotrophs associated with the diatoms, but only retained the heterotrophic portion. Microscopic examination indicated that diatoms were passed out intact in feces. Oscillatoria sp. (cyanobacterium) was not ingested. Mesochra lilljeborgi ingested Spirulina sp. (cyanobacterium), Thiocapsa sp., and the heterotrophs associated with Thiocapsa but only retained the Thiocapsa label.These data for harpacticoids suggest that spatial distributions of meiofauna may be closely coupled with microbial food organisms which they consume. Also, that while several microbial foods may be ingested, only certain microbes are digested and assimilated as a food resource, further indicating the complexity of feeding relationships among the meiofauna.     

Kinetic Concepts for Measuring Microbial Rate Constants: Effects of Nutrients on Rate Constants

We investigated the effect of preincubation of environmental waters amended with inorganic nutrients (nitrogen, phosphorus, and traces of iron and magnesium) on the kinetics of the microbial transformation of phenol, propanil, propyl ester of (2,4-dichlorophenoxy)acetic acid, methyl parathion, Ronnel, and methoxychlor in pond and river waters. No effect on the second-order rate constants for these compounds was observed, although there was an increase in the bacterial populations and the pseudo-first-order rate constants. The use of nutrient-amended waters could be a useful tool for estimating second-order rate constants for an expanded number of compounds. This technique would provide a larger data base for predicting the behavior of xenobiotic compounds in the environment by using currently available mathematical models.     

Semicontinuous and Continuous Production of Chloroperoxidase by Caldariomyces fumago Immobilized in k-Carrageenan

Three strains of Caldariomyces fumago were immobilized in 4% k-carrageenan and tested for semicontinuous production of chloroperoxidase (CPO). Over an 80-day period, growing in defined medium, C. fumago strains CMI 89362 and ATCC 11925 produced enzyme concentrations of 99 and 71 mg/liter, respectively, during six production periods of 12 to 14 days, while C. fumago DAOM 137632 produced only 24 mg of CPO per liter during six growth periods of 10 days. CPO production was unaffected by various regimens of washing between transfers. Mycelial growth was primarily restricted to the head surface, and bead size increased linearly with time. Attempts to restrict growth but maintain CPO production were unsuccessful. Pigment production, fructose utilization, and pH change in the immobilized cell cultures compared closely with the growth characteristics of free cell cultures. By using an airlift tower fermentor with an external loop run with continuous medium replacement of 20 ml/h (D = 0.016), strain CMI 89362 in bead form produced CPO at 40 mg/liter for 11 days.     

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.     

Acyl-CoA reductase and acyl-CoA: fatty alcohol acyl transferase in the microsomal preparation from the bovine meibomian gland

Biosynthesis of wax esters, one of the two major products of the meibomian gland, was found to be catalyzed mainly by the microsomes of the bovine meibomian gland. The microsomal preparation catalyzed hexadecanoyl-CoA reduction to hexadecanol without any accumulation of the aldehyde intermediate. Maximal rates of reduction occurred at pH 6.5 and required both NADH and NADPH; the latter alone gave considerable rates whereas NADH alone was ineffective. Exogenous hexadecanal reduction catalyzed by the same preparation showed a preference for NADH. The hexadecanoyl-CoA saturation pattern was slightly sigmoidal and concentrations higher than 125 microM inhibited reduction. The fatty alcohol generated from hexadecanoyl-CoA was found as free alcohol and as wax esters. Esterification of hexadecanol to wax esters catalyzed by the meibomian gland microsomal preparation required exogenous acyl-CoA or ATP and CoA and was not affected by exogenous cholesterol. Maximal rates of esterification were observed at neutral pH. Hexadecanoyl-CoA concentrations higher than 125 microM inhibited esterification. Hexadecanol showed a typical substrate saturation pattern with an apparent Km of 125 microM. Radio gas-liquid chromatography showed that, in the presence of exogenous hexadecanoyl-CoA, hexadecanol gave hexadecyl hexadecanoate whereas in the presence of ATP and CoA both C16 and C18 endogenous acids were used to esterify the alcohol. Consistent with the composition of the meibomian gland secretion, exogenous acyl-CoA longer than C14 and shorter than C20 gave maximal rates of esterification of hexadecanol.     

EFFECT OF TREE CANOPY REMOVAL BY GYPSY MOTH LARVAE ON THE MACROALGAE OF A RHODE ISLAND HEADWATER STREAM1

A spring-fed, headwater stream in central Rhode Island was examined during the period from June to October, 1979 to 1982. In the first two summers, a dense riparian canopy reduced the light penetration at the stream surface to a range of 5 to 18% of incident radiation. The lotic macroalgal community during this period was limited to 1 to 4 species covering < 1 to 35% of the stream bottom. However, in June and July, 1981, the surrounding leaf canopy was removed by a massive gypsy moth larval outbreak. Light penetration to the stream during this summer increased to 73% by early July, thereby resulting in a rise in water temperatures by 3.7°C. Even though there was a partial regrowth of leaves in late July and August of 1981, macroalgal cover values continued to rise to an early August peak of 80%. During the third summer, 88% of the macroalgal abundance could be attributed to illumination and water temperature. The filamentous diatom Funotia pectinalis ( O.F. Müll.) Rabh. was the predominant species in the midsummer of all four years, accounting for at least 60% of the total cover. In 1981. an important taxon was the desmid Hyalotheca dissiliens (S. Smith) Bréb., a species which was not seen in other years. A less severe gypsy moth defoliation occurred in 1982 but did not produce significant differences in light, temperature or macroalgal cover from 1979 and 1980. The results indicate that light and temperature can be limiting during the summer in spring-fed, headwater streams and that seed populations of some species are present in undetect-able levels during these periods of suboptimal growth conditions. In addition, it appears that stream macroalgal communities can be quite resilient, recovering rapidly following a major perturbation .     

Relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules

The relationship between the mitogenic activity of influenza type A viruses for murine B lymphocytes and the receptor-binding specificity of their hemagglutinin was examined. Receptor-binding specificity was determined by the ability of the virus to agglutinate erythrocytes that had been sialidase treated and then enzymatically resialylated to contain sialyloligosaccharides with defined sequences. Distinct differences in receptor-binding specificity were observed between strongly and weakly mitogenic viruses of the H3 subtype, with strong mitogenic activity correlating with the ability of the virus to recognize the sequence N-glycolylneuraminic acid alpha 2,6 galactose (NeuGc alpha 2,6Gal). Viruses isolated early in the evolution of the H3 subtype (from 1968 to 1971) are relatively weak mitogens and recognize the sequence N-acetylneuraminic acid alpha 2,6 galactose (NeuAc alpha 2,6Gal) but not NeuGc alpha 2,6Gal. H3 viruses isolated since 1972 are strongly mitogenic, and these viruses recognize both NeuGc alpha 2,6Gal and NeuAc alpha 2,6Gal. The amino acid substitution of Tyr for Thr at residue 155 of HA1 may be critical to this change in receptor-binding specificity and mitogenic activity of the later H3 viruses. Horse serum-resistant variants of H3 viruses, which bind preferentially to the sequence NeuAc alpha 2,3Gal, are poorly mitogenic. Differences were also observed between the receptor-binding specificity of the strongly mitogenic H3 viruses and viruses of the H2 and H6 subtypes, the mitogenic activity of which is limited to strains of mice that express the class II major histocompatibility complex glycoprotein I-E. The results indicate that the receptor-binding specificity of the hemagglutinin plays a critical role in determining the mitogenic activity of influenza viruses.