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K K Thurn

Publications and source records attributed to K K Thurn.

7 recordsLinked to original sources
PubMed1993

Absence of persistence and transfer of genetic material by recombinant Escherichia coli in conventional, antibiotic-treated mice.

Strain BST-1 is a derivative of Escherichia coli K-12 that carries a plasmid designated pURA-4 and is the expression system used by The Upjohn Company in the production of recombinant bovine somatotropin (rbSt). This plasmid also encodes an ampicillin resistance gene. The plasmidless carrier strain, BST-1C, contains a gene for tetracycline resistance which is provided by the chromosomal insertion of the transposon Tn10. Therefore, BST-1 is resistant to ampicillin and tetracycline, while BST-1C is resistant only to tetracycline. The Food and Drug Administration requested that we conduct an environmental assessment study to monitor the 'persistence of the recombinant live K-12 E. coli organism compared to the host E. coli organism'. In addition, we were requested to monitor 'the potential transfer of genetic material from (our) recombinant organism to the indigenous microflora' of the mouse gastrointestinal (GI) tract. The differences in persistence were determined by monitoring shedding of BST-1 and BST-1C in the feces of conventionally reared, outbred mice inoculated with either of the two strains. Even with antibiotic selective pressure applied (tetracycline in the water), BST-1 did not persist as well as the non-plasmid carrying parental stain, BST-1C. In the gene transfer experiments, transfer of pURA-4 was monitored by the appearance of the ampicillin resistance marker and/or by hybridization assays for the rbSt gene in indigenous, mouse-colonizing E. coli strains which had been made streptomycin resistant. At the limit of detection, no transfer of pURA-4 was detected either in vitro or in vivo. These data support an interpretation that BST-1 does not present an environmental hazard as measured by colonization/persistence in the gut of conventionally reared mammals.

Ampicillin Resistance↗
PubMed1992

Starch hydrolysis by the ruminal microflora.

The effects of grain type and processing on ruminal starch digestion are well documented but poorly understood at the biochemical and molecular levels. Waxy grains have starches high in amylopectin and are more readily digested than nonwaxy grains. However, the composition of the endosperm cell matrix and the extent to which the starch granules are embedded within it also affect starch digestion rates. Continued work is needed to determine the influence of specific cell matrix proteins, protein-starch interactions and cell wall carbohydrates on starch availability. The microbial populations that metabolize starch are diverse, differing in their capacities to hydrolyze starch granules and soluble forms of starch. Surveys show that the amylases are under regulatory control in most of these organisms, but few studies have addressed the types of amylolytic enzymes produced, their regulation and the impact of other plant polymers on their synthesis. Research in these areas, coupled with the development and use of isogeneic or near-isogeneic grain cultivars with biochemically defined endosperm characteristics, will enhance our ability to identify mechanisms to manipulate ruminal starch digestion.

Amylases↗
PubMed1985

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.

Chromosome Mapping↗
PubMed1985

Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16.

Exponentially growing cells of Erwinia chrysanthemi EC16 usually export about 98% of their pectate lyase (PL) and protease, about 40% of their polygalacturonase (PG), and about 60% of their cellulase (endoglucanase or carboxymethyl cellulase; CL). By using the R plasmid, pJB4JI (pPH1JI::Mu::Tn5), three independent Tn5 insertion mutants were obtained that exported normal levels of protease but 10% or less of PL, PG, and CL. Physical analysis revealed that single copies of Tn5 had inserted into the E. chrysanthemi chromosome, producing a similar export-defective (Out-) phenotype. The synthesis of PL, PG, and CL was not affected by the Tn5 insertions. These enzymes were released from the mutants on spheroplast formation, indicating that they were located in the periplasmic space. Tn5 insertions caused the loss of a 35-kilodalton periplasmic protein, but did not alter the outer membrane protein composition. The findings are discussed with respect to the current knowledge on protein export in gram-negative bacteria.

Cellulase↗
PubMed1985

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

Bacterial Proteins↗
PubMed1983

Tn5-Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi.

Escherichia coli (2492/pJB4JI) matings with Erwinia chrysanthemi produced kanamycin resistant (Km) transconjugants, a majority of which were gentamicin sensitive (Gm). A small proportion (about 0.8%) of the Km Gm clones were either auxotrophic or failed to catabolize galacturonate (Gtu). The R plasmid (pJB4JI) DNA was detected in the parent E. coli strain and in a Km Gm transconjugant, but not in Km GmE. chrysanthemi strains carrying Tn5-induced mutations. In Hfr crosses, Km (Tn5) was found linked with most mutations. A majority (>95%) of prototrophic recombinants were Km, except for Leu and Arg recombinants which were 30 to 50% Km. Spontaneous revertants were obtained for all markers except car, gtu, lys, thr, and trp. Prototrophic revertants, with the exception of Met, Leu, or His clones, were Km. We conclude from both genetic and physical data that Tn5 transposed from pJB4JI into different sites on the chromosome of E. chrysanthemi.

Journal Article↗