High-Salinity-Induced Iron Limitation in Bacillus subtilis

Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B. subtilis cultures. Taken together, these findings strongly suggest that B. subtilis cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B. subtilis in natural settings, in which bioavailable iron is usually scarce.     

PARma: identification of microRNA target sites in AGO-PAR-CLIP data

PARma is a complete data analysis software for AGO-PAR-CLIP experiments to identify target sites of microRNAs as well as the microRNA binding to these sites. It integrates specific characteristics of the experiments into a generative model. The model and a novel pattern discovery tool are iteratively applied to data to estimate seed activity probabilities, cluster confidence scores and to assign the most probable microRNA. Based on differential PAR-CLIP analysis and comparison to RIP-Chip data, we show that PARma is more accurate than existing approaches. PARma is available from http://www.bio.ifi.lmu.de/PARma     

The isolation of viable egg cells of wheat (Triticum aestivum L.)

The isolation of viable egg cells of wheat has been achieved without enzymatic maceration of the ovules. 2,4-D applied to the stigmas resulted 3 to 7 days later in soft ovule tissues which disintegrated upon mechanical manipulation. The isolated egg cells were viable even 2 h after isolation. Their morphology corresponded to that of the in situ egg cells. The mean isolation frequency was 20% (two egg cells per ten ovules).     

Grouping Myxococci (Corallococcus) Strains by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI TOF) Mass Spectrometry: Comparison with Gene Sequence Phylogenies

Nine Corallococcus isolates and three type strains of Corallococcus species were characterized by Intact Cell Mass Spectrometry using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The resulting phenetic clustering was compared to the phylogenetic grouping based upon sequences of two housekeeping genes. The three dendrograms of relatedness resembled each other in that the isolates were highly similar to the type strains of Corallococcus exiguus and Corallococcus coralloides, while Corallococcus macrosporus and Myxococcus xanthus were more distantly related. While certain pairs of organisms were recovered by spectrometry and genes sequence analysis, others were detected by two of the three approaches. The degree of similarity determined by sequence analysis of the two genes was not higher than that revealed by MALDI-TOF analysis. The results show that the spectral profile, consisting of about 25 to 45 masses ranging between 2 and 20 kDa, have indeed taxonomic significance, confirming literature data that ribosomal proteins and certain housekeeping proteins are responsible for the masses obtained. Provided the availability of a database of type strains, MALDI-TOF analysis of unknown strains appears to be a rapid and inexpensive method to taxonomically cluster environmental isolates, expanding the spectrum to strains other than those of medical importance predominantly investigated so far.     

Fluorinated vitamin b(12) analogs are cofactors of corrinoid-dependent enzymes: a f-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions

The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B(12) analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B(12). Hence, it is possible to use F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.     

Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea electron microscopical and biochemical analyses of C-phycocyanin and allophycocyanin aggregates

C-phycocyanin and allophycocyanin from the red alga Rhodella violacea were investigated by electron microscopy and biochemical methods using samples taken from the same fractions.The molecular weights of the native biliprotein aggregates C-phycocyanin and allophycocyanin are about 139,000 (140,000) and 130,000 (145,000) as revealed by calibrated gel chromatography, gradient gel electrophoresis and morphological measurements on the basis of an average protein packing density. These molecular weights are direct evidence for a trimeric aggregation form ()₃ of these biliproteins. Independently, their monomers were determined to be about 34,400 (C-phycocyanin) and 33,900 (allophycocyanin).C-phycocyanin and allophycocyanin are ringshaped, six-membered, biliprotein aggregates with dimensions of about 10.2×3.0 nm and 10.0×3.0 nm, respectively. The aggregates are made up of six subunits, 3 and 3, which are assumed to be associated in alternating positions. They are arranged in regular hexagons in C₆ symmetry. Hexameric aggregates ()₆, so far only isolated for C-phycocyanin, originate by face to face association of two trimeric aggregates.