Augusto F. Garcia

Analysis of Genomic Diversity Among Photosynthetic Stem-nodulating Rhizobial Strains from Northeast Argentina

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.

Redox-controlled, in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (32P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the α subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (32P)ATP or under conditions of photophosphorylation with ADP and (32P)Pi. The identity of the phosphorylated LHI-α subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the α subunit of the LHI antenna complex under redox control.

Isolation and characterization of a light harvesting complex II lacking the γ-polypeptide from Rhodobacter capsulatus

Abstract Extraction of membranes isolated from the wild-type strain 37b4 of Rhodobacter capsulatus with Na 2 CO 3 removed quantitatively the 14000 M r γ-polypeptide from the light-harvesting complex II (LHC II). The same procedure was used to extract the γ-polypeptide from the isolated LHC II. The LHC II free of the γ-polypeptide ( M r 14000) and containing the pigment-binding polypeptides α ( M r 10000) and β ( M r 8000) showed the same absorption spectrum as LHC II in the presence of the γ-polypeptide. Reaction center (RC) bleaching of the extracted and untreated membranes showed the same kinetics. Furthermore, kinetics of RC bleaching at different light intensities were identical in extracted and unextracted preparations, indicating that the LHC II lacking γ-polypeptide was able to transfer excitation energy efficiently to the LHC I-RC complexes. This efficient coupling was also confirmed by low temperature (77 K) fluorescence spectroscopy. The results indicate that the γ-polypeptide is not required for the function of the LHC II.

The Phosphorylation of Light-Harvesting Polypeptides LHIα (B870) and LHIIα (B800-850) of Rhodobacter capsulatus B10 Was Higher Under Chemotrophic Oxic Than Under Phototrophic Anoxic Growth Conditions

Abstract. In Rhodobacter capsulatus B10 (wild type) both α subunits of the light-harvesting complexes are phosphorylated during photosynthetic membrane synthesis. During the process of insertion of these polypeptides, there is a dephosphorylation resulting in intracytoplasmic membranes in which no radioactive phosphate could be detected. Moreover, we show that their phosphate-specific contents depends on the growth conditions, the highest being observed under oxic conditions. When photosynthetic membrane synthesis was induced under light and anaerobiosis, a decrease in the phosphate-specific contents ensued. An inverse relationship exists between specific phosphorylation levels and the degree of membrane differentiation. The phosphorylation is thus a transient phenomenon characteristic of the photosynthetic membrane synthesis governed by the external redox conditions constituting an additional post-transcriptional level of regulation.

Association of LHIα(B870) Polypeptide with Phospholipids During Insertion in the Photosynthetic Membrane of an LHII− Mutant of Rhodobacter capsulatus

Abstract. Membranes from in vivo labeled cells of Rhodobacternncapsulatus U43[pTX35] grown photosynthetically carried 60% ofnnthe [32P]-Pi in the “heavy” fraction (HM) afternnsucrose gradient sedimentation. Metal-chelating chromatography of eithernn“heavy” or “light” (LM) membrane fractions renderednnsimilar Bchl-protein complex profiles after octyl-glucoside treatment,nnincluding most of the radioactivity in the same corresponding elutionnnfraction (F II). Similar labeling distribution of pigment-protein complexesnnwas obtained for membranes of dark-grown cells induced by lowering oxygennntension. Fractions derived from HM showed highly labeled LHIα, whereas thennsame complex from LM was essentially [32P]-Pi-free, as revealednnby SDS-PAGE followed by autoradiography. Phospholipid analysis showed annsimilar pattern for membranes isolated from cells photosynthetically ornnsemiaerobically grown, being the most abundant: phosphatidylglycerol,nnphosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of thennphospholipids from HM comigrated with LHIα during SDS-PAGE and dissociatednnfrom the complexes only after solvent extraction and hydrophobicnnchromatography. However, a small amount remained always attached to LHIα,nnindicating an unusual strong interaction. These results suggest the existencennof two operationally defined membrane regions carrying LHIα complexesnndiffering in phosphorylation status and protein-phospholipid interaction.n

Adaptation of ornamental aroids to their indoor light environments. I. Spectral and anatomical characteristics

Adaptation responses are described to typical artificial-light environments of six aroid species, representing some of the most important indoor plants, for which physiological, anatomical and biochemical data are surprisingly lacking. The results indicate that: (1) they have a high chlorophyll content and no change in leaf thickness, for which changes in spectral absorbance (measured as blue-red ratio relative absorbance) would be associated with larger effects of light environment on leaf spectra than in sun-plants; (2) mature leaves fully expanded in greenhouse environments at the beginning of the low-intensity experiments could adapt in the same way as leaves expanded under fluorescent and blended-light lamps. These results show an unexpected high capacity for light adaptation; (3) there is a characteristic increase in grana stacking at low light intensity with no change in chlorophyll a/b ratio, and (4) the mechanism of plant adaptation differs significantly from that described for obligate or facult...

Molecular analysis and identification of the radC gene from the phototrophic bacterium Rhodobacter capsulatus B10

The radC gene, whose product plays a role in the prokaryotic repair of DNA damage after UV and X-ray irradiation, was cloned and sequenced from the phototrophic bacterium Rhodobacter capsulatus B10. The gene codes for a protein of 214 amino acids with a molecular mass of 23,792 Da. The deduced amino acid sequence showed significant homology with the RadC proteins of Escherichia coli, Bacillus subtilis and Haemophilus influenzae. Northern blot analysis indicated that under both chemotrophic and phototrophic growth conditions the radC gene was relatively highly expressed and was induced about five-fold after UV-irradiation. Primer extension analysis revealed that transcription was initiated from the same position before and after UV treatment. Mutants (radC negative) have a low survival rate and a slower growth rate than the wild type.

A simulation study comparing the impact of experimental error on the performance of experimental designs and artificial neural networks used for process screening

Many variables and their interactions can affect a biotechnological process. Testing a large number of variables and all their possible interactions is a cumbersome task and its cost can be prohibitive. Several screening strategies, with a relatively low number of experiments, can be used to find which variables have the largest impact on the process and estimate the magnitude of their effect. One approach for process screening is the use of experimental designs, among which fractional factorial and Plackett–Burman designs are frequent choices. Other screening strategies involve the use of artificial neural networks (ANNs). The advantage of ANNs is that they have fewer assumptions than experimental designs, but they render black-box models (i.e., little information can be extracted about the process mechanics). In this paper, we simulate a biotechnological process (fed-batch growth of baker’s yeast) to analyze and compare the effect of random experimental errors of different magnitudes and statistical distributions on experimental designs and ANNs. Except for the situation in which the error has a normal distribution and the standard deviation is constant, it was not possible to determine a clear-cut rule for favoring one screening strategy over the other. Instead, we found that the data can be better analyzed using both strategies simultaneously.

The LHIα and LHIIα complexes in association with phospholipids are able to be inserted in heavy membranes of Rhodobacter capsulatus B10

Abstract. We show in this paper that a complex constituted by phospholipids and LHI and LHII α polypeptides was inserted in a heavy membrane fraction in a nonextractable form, indicating a transmembrane localization. The best accepting membranes originated from aerobically grown cells. Addition of ATP during the insertion inhibited this reaction 25 to 30% in heavy membranes isolated from aerobically grown cells (HMaer) and a higher inhibition (60 to 65%) was detected when using heavy membranes isolated from photosynthetically grown cells (HMpho). Purification by gel filtration of a crude Na2CO3 extract yielded three phosphate-labeled fractions. Two of them contained protein and phospholipids in a stable association. However, only fractions containing phosphatidylethanolamine were shown to be reconstituted. The third radioactive fraction contained labeled ATP and protein, but no phospholipids and could not be reassociated to the heavy membranes of any origin. A model for the insertion of the LH polypeptides is presented in which the recently synthesized polypeptides are phosphorylated and become associated to anionic phospholipids. The interaction of this complex to the membrane spontaneously leads to stable insertion.

Oxygen and light effects on the expression of the photosynthetic apparatus in Bradyrhizobium sp. C7T1 strain

Photosynthetic bradyrhizobia are nitrogen-fixing symbionts colonizing the stem and roots of some leguminous plants like Aeschynomene. The effect of oxygen and light on the formation of the photosynthetic apparatus of Bradyrhizobium sp. C7T1 strain is described here. Oxygen is required for growth, but at high concentration inhibits the synthesis of bacteriochlorophyll (BChl) and of the photosynthetic apparatus. However, we show that in vitro, aerobic photosynthetic electron transport occurred leading to ADP photophosphorylation. The expression of the photosynthetic apparatus was regulated by oxygen in a manner which did not agree with earlier results in other photosynthetic bradyrhizobia since BChl accumulation was the highest under microaerobic conditions. This strain produces photosynthetic pigments when grown under cyclic illumination or darkness. However, under continuous white light illumination, a Northern blot analysis of the puf operon showed that, the expression of the photosynthetic genes of the antenna was considerable. Under latter conditions BChl accumulation in the cells was dependent on the oxygen concentration. It was not detectable at high oxygen tensions but became accumulated under low oxygen (microaerobiosis). It is known that in photosynthetic bradyrhizobia bacteriophytochrome photoreceptor (BphP) partially controls the synthesis of the photosystem in response to light. In C7T1 strain far-red light illumination did not stimulate the synthesis of the photosynthetic apparatus suggesting the presence of a non-functional BphP-mediated light regulatory mechanism.