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Biomedical subjects

G H Lacy

Publications and source records attributed to G H Lacy.

13 recordsLinked to original sources
PubMed1998

Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers.

Male broilers (n = 416) were used to compare the efficacy of providing dietary phytase either as a commercial supplement or as a recombinant protein in transformed soybean. From 7 to 21 d of age, broilers were fed a basal diet containing 0.20% nonphytate P (nP) with additional supplementation by fungal phytase as Natuphos or as raw transformed soybeans expressing recombinant phytase at 400, 800, or 1,200 U/kg. For comparison, broilers were also fed the basal diet containing 0.08, 0.16, or 0.24 added nP. The basal diet was fed as the negative control. Diets were consumed ad libitum as a mash. All excreta were collected from each pen from 18 through 20 d of age, and the birds were killed at 21 d of age. Supplementing the basal diet with nP linearly increased body weight gain, feed efficiency, feed intake, toe ash weight and percentage, and tibia shear force and energy. Phosphorus digestibility decreased linearly as nP level increased, but P excretion increased. Dietary phytase linearly increased growth rate, feed intake, toe ash weight and percentage, tibia shear force and energy, and P digestibility, whereas excretion was decreased. Except for P digestibility, there was no difference in efficacy of responses for performance, bone mineralization, and P excretion between the two sources of phytase. It appears from this study that phytase can improve growth performance of broilers fed low nP diets when provided either as a commercial supplement or in the form of transformed seeds.

6-Phytase↗
PubMed1997

Secretion of active recombinant phytase from soybean cell-suspension cultures.

Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds. The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence. Suspension cultures were used to confirm phytase expression in soybean cells. Phytase mRNA was observed in cultures containing constitutively expressed constructs. Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway. Secretion also facilitated characterization of the biochemical properties of recombinant phytase. Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme. However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD). Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase. Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing.

6-Phytase↗
PubMed1991

Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.

Arachidonic Acids↗
PubMed1991

Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene.

The prt1 gene encoding extracellular protease from Erwinia carotovora subsp. carotovora EC14 in cosmid pCA7 was subcloned to create plasmid pSK1. The partial nucleotide sequence of the insert in pSK1 (1,878 bp) revealed a 1,041-bp open reading frame (ORF1) that correlated with protease activity in deletion mutants. ORF1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 Da. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produces a protease (Prt1) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prt1 activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prt1 promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prt1 transcription start site 205 bp upstream of ORF1. The deduced amino acid sequence of ORF1 showed significant sequence identity to metalloproteases from Bacillus thermoproteolyticus (thermolysin), B. subtilis (neutral protease), Legionella pneumophila (metalloprotease), and Pseudomonas aeruginosa (elastase). It has less sequence similarity to metalloproteases from Serratia marcescens and Erwinia chrysanthemi. Locations for three zinc ligands and the active site for E. carotovora subsp. carotovora protease were predicted from thermolysin.

Amino Acid Sequence↗
PubMed1990

Enumerating low densities of genetically engineered Erwinia carotovora in soil.

An inexpensive, quantitative, and sensitive technique was developed for detection of genetically engineered Erwinia carotovora in soil samples. Enrichment media, antibiotic resistance, and most probable number (MPN) analysis were used to enumerate as few as 1 to 10 target cells/10 g soil. The MPN technique recovered significantly higher cell densities than plating; however, densities estimated by the two techniques were strongly correlated. After inoculation of soil microcosms with genetically engineered E. carotovora, a decline rate of 1.2 log units/g soil/10 days and then subsequent disappearance was observed using the MPN technique.

Colony Count, Microbial↗
PubMed1990

Genetically Engineered Erwinia carotovora: Survival, Intraspecific Competition, and Effects upon Selected Bacterial Genera.

Environmental use of genetically engineered microorganisms has raised concerns about potential ecological impact. This research evaluated the survival, competitiveness, and effects upon selected bacterial genera of wild-type and genetically engineered Erwinia carotovora subsp. carotovora to ascertain if differences between the wild-type and genetically engineered strains exist in soil microcosms. The engineered strain contained a chromosomally inserted gene for kanamycin resistance. No significant differences in survival in nonsterile soil over 2 months or in the competitiveness of either strain were observed when the strains were added concurrently to microcosms. For reasons that remain unclear, the engineered strain did survive longer in sterilized soil. The effects of both strains on total bacteria, Pseudomonas and Staphylococcus strains, and actinomycetes were observed. While some apparent differences were observed, they were not statistically significant. A better understanding of the microbial ecology of engineered bacteria, especially pathogens genetically altered for use as biological control agents, is essential before commercial applications can be accomplished.

Journal Article↗
PubMed1989

Genetically Engineered Erwinia carotovora in Aquatic Microcosms: Survival and Effects on Functional Groups of Indigenous Bacteria.

The survival of genetically engineered Erwinia carotovora L-864, with a kanamycin resistance gene inserted in its chromosome, was monitored in the water and sediment of aquatic microcosms. The density of genetically engineered and wild-type E. carotovora strains declined at the same rate, falling in 32 days below the level of detection by viable counts. We examined the impact of the addition of genetically engineered and wild-type strains on indigenous bacteria belonging to specific functional groups important in nutrient cycling. For up to 16 days, the densities of total and proteolytic bacteria were significantly higher (P < 0.05) in microcosms inoculated with genetically engineered or wild-type E. carotovora, but by 32 days after inoculation, they had decreased to densities similar to those in control microcosms. Inoculation of genetically engineered or wild-type E. carotovora had no apparent effect on the density of amylolytic and pectolytic bacteria in water and sediment. Genetically engineered and wild-type E. carotovora did not have significantly different effects on the densities of specific functional groups of indigenous bacteria (P > 0.05).

Journal Article↗
PubMed1986

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.

Cell Wall↗
PubMed1975

Transfer of antibiotic resistance plasmid RP1 into Pseudomonas glycinea and Pseudomonas phaseolicola in vitro and in planta.

The wide host-range antibiotic resistance plasmid RP1 was transferred from Pseudomonas aeruginosa via Escherichia coli into Pseudomonas glycinea. The plasmid was then acquired by Pseudomonas phaseolicla both in vitro and in planta in Phaseolus limensis leaves and pods. This was the first step in the design of a model system to determine the possible epidemiological significance of antibiotic resistance plasmids in the control of plant disease.

Acridines↗
PubMed1975

Antimicrobial agents from marine algae.

The antimicrobial activity of five compounds extracted from marine algae was tested against Staphylococcus aureus, Salmonella choleraesuis, Mycobacterium smegmatis, Candida albicans, and Escherichia coli. Three of the compounds, cycloeudesmol, laurinterol, and debromolaurinterol, exhibited activity at concentrations approaching that of streptomycin. None of the compounds inhibited all of the organisms tested. There appeared to be selectivity for gram-positive microbes.

Anti-Bacterial Agents↗
PubMed1974

Soil ecology of Coccidioides immitis at Amerindian middens in California.

Outbreaks of coccidioidomycosis and isolation of Coccidioides immitis have been reported from Amerindian middens. This study was undertaken to determine the most important ecological component(s) for the occurrence of C. immitis at archeological sites. Soils from 10 former Indian villages with no prior history of coccidioidal infection were collected and cultured. The physicochemical properties of the midden soils were compared with nonmidden soils and positive soils. The following theories for the sporadic distribution of the pathogen in the soil of the Lower Sonoran Life Zone were considered: (i) the Larrea tridentata (creosote bush) association, (ii) the preference for saline soils, (iii) isolation near rodent burrows, and (iv) animals as possible agents of dispersal. Results showed that a high percentage of the midden soils contained C. immitis, whereas none of the adjacent, nonmidden soils yielded the fungus. Physicochemical analyses revealed that the dark color and alkaline pH of the midden soils were due to past organic contamination. Repeated isolations were made from soils with low to moderate alkalinity. Alkalinity and sandy texture were consistent features of all soils in this study. However, the lack of any reports of nonsandy infested soils possibly indicates that the sandy texture and alkalinity may be factors in the distribution of this fungus. The organic content, soil parent material, and color were not important in the soil ecology. L. tridentata was not significant in the macroflora at the infested sites surveyed. Samples collected without reference to rodent burrows yielded a high percentage of recoveries. Animals, although not the major natural reservoir, cannot be ignored as possible factors in the ecology of C. immitis.

Animals↗