Effects of FIS overexpression on cell growth, rRNA synthesis, and ribosome content in Escherichia coli.

The Escherichia coli DNA binding protein FIS is a transcriptional modulator involved in the regulation of many cellular processes, including the activation of rRNA synthesis. High-level overproduction of FIS in early, mid, or late log cultures resulted in growth-phase- and media-specific variations in cell growth, rRNA synthesis, and ribosome content. FIS overproduction caused a pronounced increase in rRNA synthesis for late-exponential cultures but a substantial reduction in cell growth and ribosome content. The addition of simple sugars such as glucose or fructose reversed these phenomena, consistent with the functional role of FIS in carbon metabolism.

Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes.

Bifunctional fusion proteins consisting of organophosphate hydrolase (OPH) moieties linked to a Clostridium-derived cellulose-binding domain (CBD) were shown to be highly effective in degrading organophosphate nerve agents, enabling purification and immobilization onto different cellulose materials in essentially a single step. Enzyme kinetics studies were performed for the CBD-OPH fusions using paraoxon as the substrate. The kinetics values of the unbound fusion enzymes were similar to OPH with a modest increase in K(m). Immobilization of the enzymes onto microcrystalline cellulose resulted in a further increase in the K(m) values of approximately twofold. The pH profile of the cellulose-immobilized enzymes was also only minimally affected. The CBD-OPH fusion proteins could be immobilized onto a variety of cellulose matrixes, and retained up to 85% of their original activity for 30 days. The durability of the bound fusions increased with the amount of Avicel used, suggesting that protein/cellulose interactions may have a dramatic stabilizing effect. Repeated hydrolysis of paraoxon was achieved in an immobilized enzyme reactor with 100% degradation efficiency over 45 days. These fusion proteins should prove to be invaluable tools for the development of low cost, OPH-based cellulose materials for the simultaneous adsorption and degradation of stored or spilled organophosphate wastes.

Engineering of improved microbes and enzymes for bioremediation.

Bioremediation with microorganisms is an attractive alternative to conventional techniques, such as incineration and chemical treatment, for disposing of pollutants. Recent progress in molecular biology, microbiology, and genetics is providing the driving force towards engineering improved microbes and enzymes for bioremediation. A number of genetic engineering approaches have been developed in the past several years that have proven useful in introducing/evolving the desired properties for different biodegradative pathways or enzymes. The initial excitement generated in this area should continue to pave the way for rational or irrational design of microbes or enzymes with novel remedial properties.

Gene expression regulated by gene VI of caulimovirus: transactivation of downstream genes of transcripts by gene VI of peanut chlorotic streak virus in transgenic tobacco.

Here we document that the gene VI product of peanut chlorotic streak virus (PClSV), a newly characterized member of the group, transactivates the translation of dicistronic transcripts. Dicistronic expression units have been analyzed both in protoplast transient expression experiments and in transgenic tobacco plants. Transgenic plants containing a dicistronic transcription unit (PClSV-gene VII-GUS) under the control of PClSV full-length transcript promoter with its long leader sequence show a relatively high abundance of the expected transcript but very little, or no, GUS activity. However, high GUS activity is found when gene VI protein is then provided by subsequent infection with PClSV. The efficient translation of polycistronic mRNAs mediated by gene VI of caulimovirus has potential value in product engineering of plants.

Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) was displayed and anchored onto the surface of Escherichia coli using an Lpp-OmpA fusion system. Production of the fusion proteins in membrane fractions was verified by immunoblotting with OmpA antisera. Inclusion of the organophosphorus hydrolase signal sequence was necessary for achieving enzymatic activity on the surface. More than 80% of the OPH activity was located on the cell surface as determined by protease accessibility experiments. Whole cells expressing OPH on the cell surface degraded parathion and paraoxon very effectively without any diffusional limitation, resulting in sevenfold higher rates of parathion degradation compared with whole cells with similar levels of intracellular OPH. Immobilization of these live biocatalysts onto solid supports could provide an attractive means for pesticide detoxification in place of immobilized enzymes, affording a reduced diffusional barrier.

Improvement in recombinant protein production in ppGpp-deficient Escherichia coli.

Maintaining a metabolically productive state for recombinant Escherichia coli remains a central problem for a wide variety of growth-dependent biosynthesis. This problem becomes particularly acute under conditions of minimal cell growth such as fed-batch fermentations. In this, we investigated the possibility of manipulating the protein synthesis machinery of E. coli whereby synthesis of foreign proteins might be decoupled from cell growth. In particular, the effects of eliminating intracellular ppGpp on the synthesis of foreign proteins were studied in both batch and fed-batch operations. A significant increase in CAT production was observed from the ppGpp-deficient strain during both exponential and fed-batch phases. The increase in CAT production during exponential growth was accompanied by a simultaneous increase in CAT mRNA levels. Interestingly, CAT production was increased five-fold, while the level of CAT-specific mRNA increased only three-fold. Thus, eliminating intracellular ppGpp appears to have increase the production of recombinant protein by increasing not only the pool sizes of CAT mRNA but also possible alternations in the post-transcriptional processes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 379-386, 1997.

Elevated Fis expression enhances recombinant protein production in Escherichia coli.

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.

Molecular analysis of the essential and nonessential genetic elements in the genome of peanut chlorotic streak caulimovirus.

The DNA genome of caulimoviruses contains a set of essential genes: I (movement gene), IV (major capsid protein gene), V (reverse transcriptase gene), and VI (gene coding for a post-transcriptional activator of the expression of other virus genes). In peanut chlorotic streak caulimovirus (PCISV), three ORFs, A, B, and C, are located between genes I and IV. They are dissimilar to other caulimovirus ORFs. ORF VII of PCISV is a homolog of ORF VII of soybean chlorotic mottle caulimovirus (SoCMV), but is not similar to the nonconserved ORF VII in other caulimoviruses. The sequence complementary to a portion of tRNA(Met), thought to be essential for the priming of minus-strand DNA synthesis in caulimoviruses, is located within the coding sequence of ORF A. To explore the functional significance of ORFs VII, A, B, and C, various mutations were engineered into an infectious DNA clone of PCISV. ORFs VII and B are shown to be dispensable, while ORFs A and C are essential. ORF C is a possible functional equivalent of gene III in other caulimoviruses. Sequences within ORF A that are required for efficient priming of minus-strand synthesis are likely to extend beyond the 12-bp tRNA-binding site. Complete deletion of ORF VII was correlated with severe symptoms, notably with the necrosis of apical meristems. Significance of these observations for the understanding of replication and pathogenesis of plant pararetroviruses and for the improvement of caulimovirus-based expression vectors is discussed.

A naturally occurring deletion mutant of figwort mosaic virus (caulimovirus) is generated by RNA splicing.

A naturally occurring deletion mutant is observed in plants infected with figwort mosaic virus (FMV), a caulimovirus. The encapsidated mutant genome is formed spontaneously in association with two different strains of FMV in four host plant species. The mutant also appears when cloned wild-type viral DNA is used as the inoculum. The deletion mutant alone is not infectious and it appears unable to replicate after its formation, even in the presence of wild-type virus. The gene for chloramphenicol acetyltransferase was inserted at different positions in the deletion mutant genome, and subsequent transient assays showed that gene expression of the mutant occurs despite the deletion. Sequence analyses of the mutant genome revealed a deletion of 1237-bp segment encompassing a major portion of the coat protein gene and the 5' end of the downstream reverse transcriptase gene. This deletion is associated with consensus signals for RNA splicing including the conserved 5' and 3' splice sites plus surrounding sequences, putative branch point(s) for lariat formation, and an extremely high adenosine content (41%) of the removed fragment. This suggests that splicing of the FMV full-length transcript has occurred prior to reverse transcription and this accounts for the presence and accumulation of encapsidated DNAs with the same deletion.

Sequence of figwort mosaic virus DNA (caulimovirus group).

The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region.

Physical maps of the genomes of dahlia mosaic virus and mirabilis mosaic virus-two members of the caulimovirus group.

The nucleic acids of dahlia mosaic virus (DaMV) and mirabilis mosaic virus (MMV) have been isolated and compared with the native DNA of cauliflower mosaic virus (CaMV). The native DNAs of these viruses separated into circular and linear molecules during gel electrophoresis to produce patterns nearly identical to those of CaMV. The DNAS of DaMV and MMV were cloned in bacteria and used for mapping the cleavage sites for 14 different restriction endonucleases. These sites were confirmed with native viral DNA. The S1 nuclease cleavage sites and the sizes of single-stranded denaturation products of the native DNA of each virus was used to determine the location of the four single-stranded interruptions present in each virus. The largest denaturation fragment of each virus migrated in gels at about the same rate as the a strand (which has one discontinuity) of CaMV. These features have been used to construct physical maps of the viral genomes.

Mapping of the coat protein gene of cauliflower mosaic virus by its expression in a prokaryotic system.

Polypeptides recognized by antisera to cauliflower mosaic virus (CaMV) coat protein were found in lysates of Escherichia coli cells that carry the complete CaMV genome on plasmid vectors. Synthesis of this cross-reacting material in E. coli was detected when the complete CaMV DNA sequence was present at either of two sites and in either orientation at one site of the cloning vector, suggesting prokaryotic promoter activity for some sequence within the viral DNA. Using an immunosorbent assay to screen subclones containing various segments of the CaMV genome, open reading region IV alone was found to be sufficient for synthesis of the antigen, thereby mapping the coat protein gene to this region.

Isolation and properties of the inclusion bodies of cauliflower mosaic virus.

In an attempt to isolate new gene products of cauliflower mosaic virus a procedure was developed for isolation of the cytoplasmic inclusion bodies induced by the virus. Two types of inclusions, the relative ratios of which vary with virus strain, were obtained. One type was the native, vacuolated inclusion encountered in sections of tissue observed with the electron microscope. It contains virus particles. A second, granular type of inclusion body is devoid of virus. It consists wholly of electron-dense material that resembles the matrix material of the native inclusion body. Granular inclusions consist of a single 55,000-dalton protein which is also a major constituent of the native vacuolated type. This protein is not antigenically related to the virus.

Total mercury in water, sediment, and selected aquatic organisms, Carson River, Nevada--1972.

A 1971-72 study of the Nevada Carson River drainage system by the Geological Survey, U.S. Department of Interior, revealed substantial amounts of mercury from pre-1900 gold and silver milling operations of the Comstock Lode. A monitoring survey was initiated to determine the extent of mercury uptake from corresponding surface water and sediments for seven aquatic species collected from five sampling stations along the watercourse. Total mercury content in fish ranged from 0.02 to 2.72 ppm; highest concentrations occurred in piscivorous white bass (0.50-2.72 ppm) sampled from Lahontan Reservoir. Residue levels appeared to be related to fish size, as demonstrated by highly significant correlations between wet weight and mercury content of five of the six species. Concentrations also appeared to be directly influenced by the species' position on the aquatic food chain. These results indicate that mercury levels in some fish from the Carson River drainage system may exceed the 0.50 ppm maximum concentration considered by the Food and Drug Administration, U.S. Department of Health, Education, and Welfare, to be safe for human consumption.