Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Antigenic nature of the chloride-stimulated cellobiosidase and other cellulases of Fibrobacter succinogenes subsp. succinogenes S85 and related fresh isolates.

Polyclonal and monoclonal antibodies to the Cl-stimulated cellobiosidase of Fibrobacter succinogenes subsp. succinogenes S85 reacted with numerous proteins of both higher and lower molecular weights from F. succinogenes subsp. succinogenes S85, but not with Escherichia coli proteins, and only one protein each from Butyrivibrio fibrisolvens and Ruminococcus albus. Different profiles were observed for Western blots (immunoblots) of peptide digests of both the purified enzyme from F. succinogenes and immunoreactive proteins of higher and lower molecular weights, demonstrating that they were different proteins. Therefore, F. succinogenes appeared to produce numerous proteins with one or more common antigenic determinants. However, with the exception of Cl-stimulated cellobiosidase, none were cellulases that have been characterized. An affinity-purified polyclonal antibody to Cl-stimulated cellobiosidase reacted with numerous proteins in cells of each of three fresh isolates of F. succinogenes subsp. succinogenes and one of F. succinogenes subsp. elongata when analyzed by Western blotting. Antibodies to periplasmic cellodextrinase, endoglucanase 2 (EG2), and EG3, when reacted in Western blots with the various cellulases, including Cl-stimulated cellobiosidase, revealed limited antigenic similarity among the different proteins and none with either B. fibrisolvens or R. albus proteins. The periplasmic cellodextrinase antibody reacted with an antigen with a size corresponding to cellodextrinase in each of the three F. succinogenes subsp. succinogenes isolates but not with any antigens from the F. succinogenes subsp. elongata isolate. The anti-EG2 antibody reacted with single antigens in each of the four isolates, while the anti-EG3 antibody reacted with only one of the four isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of indolepyruvic acid as an intermediate of rebeccamycin biosynthesis

Summary Experimental evidence is presented to demonstrate that indolepyruvic acid is an intermediate in the rebeccamycin biosynthetic pathway. [3-¹⁴C]Indolepyruvic acid was prepared and efficiently incorporated (8%) into rebeccamycin bySaccharothrix aerocolonigenes.     

Aggregation and Metal-Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala²¹ to Gly or Glu²² to Gln were compared. At pH 7.4 and 37°C the Gln²² peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly²¹ peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln²² sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly²¹ peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope ⁶⁵Zn²⁺. Of several competing metal ions, tested at 20× the concentration of Zn²⁺, Cu²⁺ displaced >95% of the radioactivity from all three peptides and Ni²⁺ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe²⁺ and Al³⁺ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn²⁺ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly²¹ mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.     

The differentiation of Staphylococcus aureus from other Micrococcaceae isolated from bovine mammary glands

Haemolysin production, the slide coagulase test and the tube coagulase test were assessed for their capability to differentiate Staphylococcus aureus among other Micrococcaceae in 199 isolates from udders of cows in herds with a low bulk milk somatic cell count. The API-Staph test was used as a reference.
Haemolysin production was less effective in identifying Staph. aureus among Micrococcaceae than a combination of other tests. Differences were found in the predictive values of results from diagnostic protocols in which the slide coagulase test was performed on all Micrococcaceae, or on β-haemolysin-negative Micrococcaceae only. Diagnostic protocols in which haemolysin production was combined with the results of the other tests resulted in excellent diagnostic performance and a reduction in diagnostic procedures. Recommendations for routine Staph. aureus identification in bovine mastitis bacteriology are given.     

Korkormicins,novel depsipeptide antitumor antibiotics fromMicromonospora sp C39500: Fermentation,precursor directed biosynthesis and biological activities

Micromonospora sp C39500, isolated in our laboratory from a soil sample, produced a complex of seven novel depsipeptide antitumor antibiotics, designated korkormicins. The major component of the complex, korkormicin A, has a MW of 1452 and a molecular formula of C₆₆H₈₄N₁₆O₂₂. Korkormicin A exhibits potentin vivo antitumor activity against P388 leukemia and M109 lung carcinoma implanted intraperitoneally (ip) in mice, with effective doses of 0.05–0.20 mg kg^–1 injection^–1, for five or three ip injections, respectively. It is also active against Gram-positive bacteria but inactive against Gram-negative bacteria. The production of korkormicin A was enhanced by 3-fold when 0.1%l-valine was added to the production culture at 48h. A titer of 401.0 g ml^–1 was achieved in the fermenter culture supplemented with 0.1%l-valine.     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.     

Coordinated Activation of Programmed Cell Death and Defense Mechanisms in Transgenic Tobacco Plants Expressing a Bacterial Proton Pump

In plants, programmed cell death is thought to be activated during the hypersensitive response to certain avirulent pathogens and in the course of several differentiation processes. We describe a transgenic model system that mimics the activation of programmed cell death in higher plants. In this system, expression of a bacterial proton pump in transgenic tobacco plants activates a cell death pathway that may be similar to that triggered by recognition of an incompatible pathogen. Thus, spontaneous lesions that resemble hypersensitive response lesions are formed, multiple defense mechanisms are apparently activated, and systemic resistance is induced in the absence of a pathogen. Interestingly, mutation of a single amino acid in the putative channel of this proton pump renders it inactive with respect to lesion formation and induction of resistance to pathogen challenge. This transgenic model system may provide insights into the mechanisms involved in mediating cell death in higher plants. In addition, it may also be used as a general agronomic tool to enhance disease protection.     

Removal and recovery of Cu(II) from industrial effluent by immobilized cells of Pseudomonas putida II-11

A copper [Cu(II)]-accumulating strain, Pseudomonas putida II-11, isolated from electroplating effluent removed a significantly high amount of Cu(II) from growth medium and buffer. A laboratory-scale fixed bed reactor with cells of P. putida II-11 immobilized in polyacrylamide gel was constructed. The adsorption of Cu(II) by the immobilized cells was pH-dependent. Maximum removal of Cu(II) by the immobilized cells was at pH 8.0. The presence of Cr(IV), Ni(II) and Zn(II) did not significantly inhibit Cu(II) uptake whereas the presence of Pb(II) reduced Cu(II) uptake by fivefold. The presence of borate, carbonate, chloride and sulphate did not significantly inhibit Cu(II) uptake. The Cu(II) removal capacity of the bioreactor with immobilized cells did not change significantly when operated at retention times greater than 3 min. More than 90% of Cu(II) adsorbed on immobilized cells could be recovered by eluting with 0.1 m HCl. The bioreactor could be used for at least five loading-elution cycles without loss of Cu(II) removal capacity. The feasibility of using this bioreactor to remove and recover Cu(II) from electroplating effluent is discussed. Correspondence to: P. K. Wong