Metabolic changes associated with cluster root development in white lupin (Lupinus albus L.): relationship between organic acid excretion, sucrose metabolism and energy status.

Under phosphorous deficiency, plants of white lupin (Lupinus albus L.) develop root clusters, which are also called proteoid roots due to their preferential presence in the Proteaceae. In their mature stage, these roots acidify the soil and excrete high amounts of carboxylates [up to 1.5 and 7 micromol (g FW)(-1) h(-1) of malate and citrate, respectively] enabling lupins to utilise sparingly available sources of phosphate. Using the amplified fragment length polymorphism (AFLP) technique, we identified genes predominantly expressed in juvenile and mature cluster roots. Transcripts for two enzymes involved in glycolysis, fructokinase and phosphoglucomutase, were identified in juvenile cluster roots and one, sucrose synthase, in mature cluster roots. In order to verify these observations we performed quantitative reverse transcription-polymerase chain reaction (RT-PCR) and could confirm the increased transcript level. Measurements of enzymatic activities showed that fructokinase and phosphoglucomutase activities increased in juvenile cluster roots, whereas sucrose synthase activity was maximal in mature cluster roots. These results indicate that formation of proteoid roots and citrate excretion increase sink strength locally. Production of citrate and inhibition of respiration are likely to result in an increased NADH/NAD+ ratio, which may be toxic for the plant. The fermentation pathway would allow oxidation of NADH by decarboxylation of pyruvate and subsequent reduction of the resulting acetaldehyde. Determination of alcohol dehydrogenase activity showed that this enzyme is strongly induced in mature proteoid roots. However, ethanol production was not increased, indicating that pyruvate is shunted to citrate synthesis and not to ethanol production.

The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes.

The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A(2) from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30+/-5 in RO and 40/60+/-3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42+/-3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.

Classification and identification of propionibacteria based on ribosomal RNA genes and PCR.

A rapid method was developed to differentiate the genus Propionibacterium from other genera by using a modified multiplex-PCR (MPCR) approach. Three 16S rRNA-targeted oligonucleotide primers were designed to amplify simultaneously two DNA-fragments in the MPCR assay. The universal primer pair bak11w and bak4 (corresponding to the E. coli 16S rRNA positions 8-25 and 1522-1540, respectively) was used in combination with the primer pair bak4 and gd1 (5'-TGCTTTCGATACGGGTTGAC-3'). The later sequence corresponding to a 16S rRNA motif that is unique for the genus Propionibacterium. Propionibacteria were identified by the amplification of a Propionibacterium-genus specific 900-bp fragment whereas MPCR with DNA from other bacteria generated only a DNA fragment of 1500 bp in amplifications with the two universal primers. The whole procedure including cell lysis, MPCR amplification and analysis can be performed within 1 day, detection limits are at approximately 10(3) cfu propionibacteria (or 35 pg DNA). In addition, the taxonomic situation of the genus Propionibacterium was reexamined using a cycle sequencing strategy. Based on the 16S rDNA, a phylogenetic tree of all the Propionibacterium type strains was reconstructed.

Effect of dimethyl sulfoxide on lysine production by a mutant of Bacillus subtilis with homoserine dehydrogenase activity.

Some Bacillus subtilis mutants with different levels of homoserine dehydrogenase were described. Strains that do not accumulate methionine have a high homoserine dehydrogenase activity. Low activity was detected in mutants where cell growth was completely inhibited by 0.7 mmol/L methionine. A low concentration of dimethyl sulfoxide had a stimulatory effect on lysine production by the methionine-sensitive mutant of Bacillus subtilis.

[Determination of 2-6-diaminopimelic acid in samples of bacteria isolated from the rumen of sheep using an automatic amino acid analyzer]. / Stanovenie kyseliny 2-6-diaminopimelovej vo vzorkách baktérií, izolovaných z bachora ovce, automatickým analyzátorom aminokyselín.

The method of the use of the HD 1200-E automatic amino acid analyzer for the separation of amino acids was modified for the determination of 2-6-diaminopimelic acid (DAPA) as a bacterial marker, besides the other amino acids in the acid hydrolyzates of samples of bacteria isolated from the rumen of sheep. The reproducibility of the determination of DAPA in a standard amino acid mixture found in the tests corresponded with the reproducibility of the determination of the other amino acids as indicated by the manufacturer of the apparatus. The lower limit of DAPA determination sensitivity is between 2 and 5 nmol. In samples of bacteria isolated from rumen wall, from feed particles of rumen contents and from rumen fluid, different contents of nitrogen and DAPA were obtained; this is ascribed to the different proportions of bacterial species in the bacterial populations having different functions.