The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.
Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.
A number of studies have demonstrated a protective effect associated with N-acetyl-l-cysteine (NAC) against toxic chemical exposure. However, the impact of long-term oral dosing on tissue pathology has not been determined. In this study, the authors assessed the impact of long-term oral NAC administration on organ histopathology and tissue glutathione (GSH) and total glutathione- S-transferase (GST) activity levels in Sprague-Dawley (SD) rats. Groups of 20 SD rats (10 males, 10 females), 8 weeks of age, were dosed daily by oral gavage with deionized H 2 O (negative controls) or NAC solution at a rate of 600 or 1200 mg/kg/day for 30 days. Animals were euthanized 6 h after treatment on study day 30. There were no significant differences in final body weights or weekly average weight gain between treatment groups. Serum alanine amino-transferase (ALT) activities were significantly elevated ( p ≤.05) in NAC-treated animals compared to controls when measured on study day 30. Histopathologic evaluation of the stomach, small intestine, liver, kidneys, spleen, thymus, and lungs revealed no lesions associated with NAC administration. When measured on study day 30, total GST activity for kidney and skin from NAC-treated animals were increased 39% to 131% as compared to controls. Tissue GSH concentrations from NAC-treated animals were increased 24% to 81% as compared with negative controls. Further studies are needed to determine if the observed increase in tissue GSH concentration and GST activity provide a degree of chemoprotection against dermal and systemic chemical toxicants.
ABSTRACT The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 μM DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.
We cloned into the structural fimbrial subunit gene from a fimbriated Haemophilus influenzae b a 1.5-kb kanamycin resistance gene. The resultant strain (RKAW5) was tested by Southern analysis, hemagglutination, and electron-micrographic examination to confirm gene inactivation. In comparison with the parent, RKAW5 exhibited a significant decrease in adherence to human buccal epithelial cells and in nasal colonization of yearling rhesus monkeys.
