Catabolism of the lignin substructure model compound dehydrodivanillin by a lignin-degrading Streptomyces

The lignin-degrading actinomycete Streptomyces viridosporus T7A readily degrades the lignin model compound dehydrodivanillin. Four mutants of this organism (produced by irradiation of spores with ultraviolet light) were shown to have lost the ability to catabolize dehydrodivanillin. These mutant strains retained an undiminished ability to degrade Douglas-fir lignin (¹⁴C-lignin ¹⁴CO₂) as compared to the wild-type strain. None of the strains accumulated detectable quantities of dehydrodivanillin when grown on lignocellulose. Thus it appears that the enzymes involved in dehydrodivanillin catabolism are not a part of the streptomycete's system for degrading polymeric lignin. It is concluded that dehydrodivanillin is probably not a relevant model compound for study of lignin polymer degradation by Streptomyces viridosporus. Since many stable mutants completely lacking DHDV-degrading ability were readily obtained, it is suggested that the relevant catabolic enzymes may be encoded on a plasmid.Abbreviations DHDV dehydrodivanillin     

Com Yield Increases Relative to Nonfumigant Chemical Control of Nematodes

Corn yields were measured after application of nematicides in 16 experiments, mostly in medium-to-heavily textured soil, at 12 locations in Iowa during 1973-1976. The average maximum yield increase in plots treated with nematicides was 21% over yields in untreated plots. Yields were correlated negatively with nematode numbers or nematode biomass in nearly all comparisons. Correlations of nematode numbers in the soil with yield averaged -0.56 for Helicotylenchus pseudorobustus, -0.45 for Hoplolaimus galeatus, -0.51 for Pratylenchus spp., and -0.64 for Xiphinema americanum. Correlation coefficients for numbers of nematodes in the roots and yield averaged -0.63 for Pratylenchus spp. and -0.56 H. galeatus. Correlation coefficients for yield and total number of nematodes averaged -0.65 in roots and -0.55 in soils. Negative correlations also were greater for comparisons of yield with total parasitic-nematode biomass than with numbers of individual nematodes of a species or total numbers of parasitic nematodes.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

A SURVEY FOR 1,3,6,7-TETRAHYDROXY-C-GLYCOSYLXANTHONES EMPHASIZING THE “PRIMITIVE” LEPTOSPORANGIATE FERNS AND THEIR ALLIES

A survey for 1,3,6,7-tetrahydroxy-C-glycosylxanthones of representative species within the primitive vascular plants, emphasizing the leptosporangiate ferns, has indicated a limited distribution of these compounds within three leptosporangiate families: Hymenophyllaceae, Aspleniaceae and Marsileaceae. In the Hymenophyllaceae the distribution of these compounds appears to be a useful criterion for segregating species of Mecodium from other species of Hymenophyllum (sensu lato) and suggests that the tubulate vs. the valvate indusial condition may not be an ideal character for separating all species of Hymenophyllum (s.l.) from those of Trichomanes (s.l.). These compounds appear useful for delimiting several species of Elaphoglossum section Pachyglossa and support a relationship among the Aspleniaceae, Athyriaceae, and Elaphoglossaceae. Their presence in Marsilea also raises questions as to the origin of this group of plants.     

The properties of immobilized caldolysin, a thermostable protease from an extreme thermophile

Caldolysin, the extracellular thermostable metal-chelator-sensitive lytic protease from Thermus T-351 was immobilized to Sepharose 4B, CM-cellulose, and controlled pore glass (CPG). Although protein binding efficiencies were high (96, 88, and 95%), some loss of enzyme activity occurred on immobilization (26, 69, and 89%). The pH optimum of both CM-cellulose and CPG-immobilized Caldolysin was decreased by about one pH unit. The K(m) for Sepharose-Caldolysin was unchanged with respect to the free protease, while those for CM-cellulose-Caldolysin and CPG-Caldolysin were lower by approximately one order of magnitude. Immobilization to both Sepharose and CM-cellulose increased the thermostability of Caldolysin at high temperatures, while CPG-Caldolysin was less thermostable than the free protease.     

The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein.     

Intragenic Sequences Affect the Expression of the Gene Encoding Glial Fibrillary Acidic Protein

We show that the expression of the gene encoding glial fibrillary acidic protein (GFAP) gene is affected by at least three cis-acting elements. A positive regulatory element that is located between nucleotides -1,631 and -1,479 can confer cell type-specific expression on a heterologous gene. A second regulatory element is located between nucleotides -97 and -80. The third is a negative regulatory element that is located within the first intron of the gene. Deletion of this element activates GFAP expression in HeLa cells, and affects promoter function in glioma cells.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.