Norma L. Pucheu

Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds

Aims:  A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean.

Isolation and purification of reaction center from Rhodopseudomonas viridis NHTC 133 by means of LDAO.

Two different procedures are described to isolate and purify the reaction center complex from Rhodopseudomonas viridis NHTC 133 by means of the non-ionic detergent dodecyldimethylamine oxide. Both reaction center particles thus obtained were active, as shown by a photobleaching centered at 975 nm.The reaction center also contained, in addition to bacteriochlorophyll, bacteriopheophytin. Other components were also found in this particle: cytochromes C553 and C558 and a menaquinone-like substance.The SDS gel electrophoresis of reaction centers is shown. The molecular weights of the subunits forming the reaction center in 0.5% sodium dodecyl sulfate and 1% mercaptoethanol were calculated as being: 45±1.5 and 37±1.5 kdalton, 29±1.5 and 23±1.5 kdalton.The molecular weight of the complex determined by means of gel filtration (Sepharose 6-B and Bio-Gel P-300) gives a value of approximately 240 kdalton.The minimum molecular weight of the complex calculated by disc gel electrophoresis was 231 kdalton.

Assessment of the role of chemotaxis and biofilm formation as requirements for colonization of roots and seeds of soybean plants by Bacillus amyloliquefaciens BNM339.

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.

Bioenergetic factors controlling in vitro phosphorylation of LHIα (B870) polypeptides in membranes isolated from Rhodobacter capsulatus

Abstract Membranes of Rhodobacter capsulatus strain U43 (pTX35) showed qualitatively very similar phosphorylation patterns under in vitro and in vivo conditions. In vitro, it was irrelevant whether the phosphate source was orthophosphate or ATP. Inhibitors of electron transport did not inhibit light-harvesting complex I (LHIα) (B870) polypeptide phosphorylation, except for o-phenanthroline, which was strongly inhibitory. Redox conditions regulated the amount of protein phosphorylated; external redox potentials between +200 and +300 mV promoted the reaction. Phosphorylation was inhibited by uncouplers such as carbonyl cyanide m-chlorophenyl hydrazone and nigericin plus valinomycin plus potassium ions. Inhibitors of the H+-ATPase were also inhibitory when the phosphate source was [32P]Pi or [γ-32P]ATP. From these results, it was concluded that an operative reaction center, a coupled membrane, and external redox potentials higher than +200 mV are required for optimum LHIα phosphorylation. We also demonstrated that phosphorylation of LHIα polypeptide occurs before insertion into the membrane and that phosphate is preferentially incorporated into specific domains within the cytoplasmic membrane. Intracytoplasmic membranes, identified here as light membranes, were found to contain a dephosphorylated LHIα polypeptide.

Phenazine methosulfate mediated photoinactivation of some energy linked reactions in rhodospirillum rubrum

Abstract Preillumination of Rhodospirillum rubrum chromatophores in the presence of phenazine methosulfate, under non-phosphorylating conditions results in an irreversible inhibition of the energy transduction. Protection against photoinhibition was provided during the preillumination when a continous dissipation of energy is provoked by the simultaneous photoreduction of NAD+. The results are interpreted as indicating that the photoinactivation is produced by an accumulation of the eergized form of the membrane. Different conformational forms of the ATPase complex are supposed to be responsible for the reversibility or irreversibility of the inhibited state.

Phosphorylation of LHI β During Membrane Synthesis in the Photosynthetic Bacterium Rhodovulum sulfidophilum

Cells of Rhv. sulfidophilum were grown under different conditions in the presence of 32P-phosphate and the corresponding H and L membrane fractions obtained and fractionated by SDS-PAGE. Both membranes showed almost identical polypeptide composition. The bacteriochlorophyll (Bchl) specific content in H was always lower that in L. As described before, oxygen did not regulate gene expression. Under high light, an almost two- to threefold decrease of the cellular specific Bchl content was observed. Pulse and chase experiments showed that transitions from aerobiosis to light-anaerobiosis did not quantitatively affect the Bchl content of the membranes, although a turnover of the 32P-phosphate and 35S-methionine was observed. LHI β was the only polypeptidic subunit of the Bchl-binding polypeptides that was phosphorylated in vivo, and phosphotyrosine was the only phosphorylated amino acid detectable. The phosphorylated LHI β was determined to be insoluble in the organic solvent mixture of (vol/vol) 1:1 chloroform-methanol containing ammonium acetate (0.1 m final concentration). Treatment with a chaotropic agent such as Na2CO3 solubilized the phosphorylated LHI β, indicating that part of this posttranslationally modified polypeptide was not inserted in a transmembrane position. These results were used to speculate about the regulatory properties of this posttranslational modification of LHI β on membrane differentiation.

Possible initial events of photophosphorylation in membranes of Rhodopseudomonas. Viridis and Rhodopseudomonas. Capsulata Ala+r

There is still controversy as to whether the initial phosphate acceptor in oxidative phosphorylation and photophosphorylation is ADP or AMP. Ozawa et al. [ 1,2] have found evidence indicating that in mitochondria AMP may be the initial phosphate acceptor. In chloroplasts Roy et al. (31 postulated that AMP is initially phosphorylated in the l&it. On the other hand, Smith et al. [4] by studying the early kinetics of ATP and ADP formation, conclude that in chloroplasts ADP is the first nucleotide that accepts a phosphate molecule. A similar proposal was advanced recently by Aflalo et al. [5]. In photosynthetic bacteria, Yamamoto et al. [6] and Lutz et al. [7] have described membrane bound adenine nucleotides as participants, among other possibilities, in transphosphorylations which finally lead to ATP formation. We report here on some experiments carried out in membranes of both Rps. viridis and Rps. capsulutu concerned with the possible initial events of phosphate uptake in photophosphorylation.

Effect of Light and Oxygen and Adaptation to Changing Light Conditions in a Photosynthetic Mutant in Which the LHII Complex of Rhv. sulfidophilum Was Heterologously Expressed in a Strain of Rb. capsulatus Whose puc Operon Was Deleted

In this paper we show the effect of oxygen and light on the expression of the photosynthetic apparatus of a mutant heterologously expressing the puc operon. This mutant was obtained by introducing in trans an expression plasmid, bearing the puc A, B, and C genes of Rhv. sulfidophilum, as well as its own promoter, in an LHII− mutant of Rb. capsulatus. The results showed that oxygen and light repressed LHII expression. Even low-light intensities lowered the LHII content to undetectable levels by spectrophotometry or by SDS-PAGE. In high-light grown cells, where the relative ratios of LHI and LHII complexes were significantly diminished, we were able to detect LHII complexes. Under the latter condition, the absorption spectrum showed that some pigment accumulated in the membrane even in the absence of cell division. These pigments were used in a later step to assemble LHII complexes, when the high-light grown cells were transferred to semiaerobiosis in the dark. Transition of high-light grown cells to low-light conditions allowed us to study the adaptability of these heterologous mutant cells. We observed that adaptation never occurred, in part probably owing to energy limitation.

ASSESSMENT OF THE ROLE OF FLUORESCENT ROOT AND SEED EXUDATES IN CROP PLANTS

Fluorescent compounds have been reported in plant leaves, having an important biological role in the protection towards UV irradiation. It is also known that stress conditions in plants stimulate the production of some of these compounds in their aerial portion. However, less is known about their production by roots or seeds and their presence in the soil surrounding them. We report here that, soybean, sunflowers and rapeseed produced fluorescent seed and root exudates. Phosphorus deficiency in rapeseed stimulates the excretion of fluorescent compounds by roots, mainly chlorogenic acid and scopoletin. When the effect of the fluorescent fraction from soybean seed exudates on Macrophomina phaseolina was tested, an enhanced induction of sclerotia formation, hyphal squeezing and lysis in the mycelium was observed. The fluorescent compounds and perhaps other chemical molecules also present in exudates might constitute a transitory defense barrier against early attack by pathogens.

PMS photo-inhibition in Rhodospirillum rubrum membranes in the presence of permeant entities affecting either the Δψ or the Δ pH components of the protonmotive force

Preincubation of Rhodospirillum rubrum chromatophores in the light and in the presence of 50 I.~M PMS was shown [ 1,2] to produce an inhibition of photophosphorylation. The inhibition was prevented either in the presence of phosphate + ADP [l] or when a dissipation of the energized state is induced by another energy-utilizing reaction, namely NAD+ reduction [2]. More than one configuration was postulated [ 21 to be involved in the energy-dependent conformational change which accounts for the transient photoinhibition produced by PMS in the presence of NAD’ and succinate. The chemiosmotic theory [3-61 postulates that the electrochemical proton gradient generated during electron transport accounts for a coupled ATP synthesis. On the other hand it was postulated [7,8] that conformational changes are necessary steps in the phosphorylation process and are directly linked to electron transport. Both in chloroplasts [S-15] and chromatophores isolated from photosynthetic bacteria [ 161 there have been reports that connect membrane energization to conformational changes. In the presence of different permeant ions the electric (A