Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHA0

In Pseudomonas fluorescens strain CHA0, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.     

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.     

Significant linkage on chromosome 10p in families with bulimia nervosa

Bulimia nervosa (BN) is strongly familial, and additive genetic effects appear to contribute substantially to the observed familiality. In turn, behavioral components of BN, such as self-induced vomiting, are reliably measured and heritable. To identify regions of the genome harboring genetic variants conferring susceptibility to BN, we conducted a linkage analysis of multiplex families with eating disorders that were identified through a proband with BN. Linkage analysis of the entire sample of 308 families yielded a double peak, with the highest nonparametric multipoint maximum LOD score (MLS), of 2.92, on chromosome 10. Given the high heritability of self-induced vomiting and the reliability with which it can be measured, we performed linkage analysis in a subset (n=133) of families in which at least two affected relatives reported a symptom pattern that included self-induced vomiting. The highest MLS (3.39) observed was on chromosome 10, between markers D10S1430 and D10S1423. These results provide evidence of the presence of a susceptibility locus for BN on chromosome 10p. Using simulations, we demonstrate that both of these scores, 2.92 and 3.39, meet the widely accepted criterion for genomewide significance. Another region on 14q meets the criterion for genomewide suggestive linkage, with MLSs of 1.97 (full sample) and 1.75 (subset) at 62 centimorgans from p-ter.     

Charge microheterogeneity of ovine follicle-stimulating hormone in rams and steroid-treated wethers

Chromatofocusing was utilized to separate the isohormones of ovine follicle-stimulating hormone (oFSH) in pituitary extracts from rams, wethers, and wethers receiving Silastic implants of dihydrotestosterone (DHT), 17 beta-estradiol (E2), or both DHT and E2. Pituitary extracts were prepared by homogenization and centrifugation at 100,000 x g. Extracts of ram pituitaries yielded at least nine species (isohormones) of immunoreactive oFSH having apparent isoelectric points (pIs) of greater than 7.40, 6.74, 6.52, 5.76, 5.20, 4.74, 4.44, 4.10, and less than 4.00. These isohormones were designated by the letters A-H and Z, respectively. Eighty-four percent of the immunoreactive ram oFSH recovered from the chromatofocusing column was very acidic in nature, having apparent pIs less than 5.30. The majority of the immunoreactive oFSH was focused in an area different from that of most of the immunoreactive ovine luteinizing hormone (oLH) and ovine thyroid-stimulating hormone (oTSH). Pituitary extracts from control and steroid-treated wethers also contained these nine oFSH isohormones, but significant differences were noted in the relative distribution among the isohormones. Castration resulted in a 4-fold increase in isohormone B and a concomitant reduction in isohormones E and H. DHT administration returned these levels to the values observed in the ram, whereas E2 administration produced a significant 2-fold increase in the most acidic form (isohormone Z). The implant combination produced isohormone profiles comparable to that of E2 alone. Neuraminidase treatment in vitro of both crude pituitary extracts or highly purified iodinated oFSH abolished the most acidic form and caused a marked shift in the isohormone pattern to more basic species. These results demonstrate that 1) at least nine isohormones of oFSH can be separated reproducibly from the male ovine pituitary by chromatofocusing, 2) the majority of FSH in the pituitary exists in acidic form, 3) castration and steroid administration alter the distribution of oFSH in the pituitary among its isohormones, and 4) at least a portion of oFSH-charge microheterogeneity appears to be due to the presence of sialic acid residues on the molecule.     

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.     

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.     

The effect of ionic strength on DNA-ligand unwinding angles for acridine and quinoline derivatives.

We have quantitatively examined the unwinding angles for the complexes of a related series of acridine and quinoline derivatives with DNA. Ethidium bromide was used as a control for determining superhelix densities at different ionic strengths. Relative to ethidium, 9-aminoacridine and quinacrine had an essentially constant unwinding angle of approximately 17 degrees at all ionic strengths tested. The apparent unwinding angle for chloroquine and 9-amino-1,2,3,4-tetrahydroacridine was found to be ionic strength dependent, increasing with increasing ionic strength. This suggests that competitive nonintercalative binding at low ionic strengths causes an apparent lowering of the quinoline unwinding angle. This can also explain why 4-aminoquinaldine, examined at low ionic strength, gives a quite low apparent unwinding angle. Quinacrine along with chloroquinine and 9-aminoacridine approaches a limiting value for their unwinding angle of approximately 17 degrees. 4-aminoquinaldine and 9-amino-1,2,3,4-tetrahydroacridine could not be examined at an ionic strength above 0.03 because of their very low equilibrium binding constants.     

A simple two-step purification of human and monkey alpha fetoprotein from amniotic fluid and serum.

We developed a two-step purification system to characterize alpha fetoprotein (AFP) in early gestation amniotic fluid and late gestation fetal serum or cord blood from monkey and human. It involves only two chromatographic steps, allows preparative purification using up to 12 ml of starting sample, can purify up to 350 micrograms of AFP at one time, and can be used to purify both fetal serum or amniotic fluid AFP from two different species. This procedure will allow detailed biochemical analysis of purified AFP from different stages of fetal development.     

Infectious canine hepatitis in a gray fox (Urocyon cinereoargenteus)

A free-ranging adult male gray fox (Urocyon cinereoargenteus) with moderate diarrhea and thick ocular mucus discharge was examined postmortem. Microscopically, the fox had intranuclear inclusion bodies within hepatocytes. Canine adenovirus-1 was identified by polymerase chain reaction (PCR) and nucleotide sequencing. To our knowledge, this is the first report of clinical infectious canine hepatitis in a gray fox.