Pectin Lyase Activity in a Penicillium italicum Strain

An extracellular pectin lyase (PNL) [poly-(methoxygalacturonide)lyase; EC 4.2.2.10] produced by Penicillium italicum CECT 2294 grown on a surface bran (natural medium) or in a submerged (synthetic medium) culture was investigated. Both culture filtrates showed macerating activity at low pH on cucumber, potato, and orange tissues. The physicochemical properties of the enzyme obtained from both culture methods were identical, as well as its catalytic properties, which were assayed by different methods. The molecular mass of the PNL obtained by gel filtration chromatography was 22 kDa; the isoelectric point was 8.6, as determined by chromatofocusing; and the enzyme was able to catalyze the eliminative cleavage of pectins with low (37%) and high (from 54 to 82%) degrees of esterification. The PNL produced in liquid medium showed a K_m for pectin (degree of esterification, 70%) of 3.2 mg/ml, and the optimum pH was 6.0 to 7.0. This enzyme was stable at 50°C and at pH 8.0. The ability of this PNL to macerate plant tissues in acidic environmental conditions, its stability at low pH and temperatures up to 50°C (thus preventing mesophilic microbial growth), and the absence of pectinesterase make this preparation useful for the food industry.     

Molecular and morphological analyses of solitary forms of brackish Thalassiosiroid diatoms (Coscinodiscophyceae), with emphasis on their phenotypic plasticity

Blooms of centric diatoms are a common feature in the Bilbao estuary during summer when river flow is at its lowest and water temperature is above 20ºC. To gain insight into the specific composition of these diatom blooms, net samples and cultures of estuarine isolates were analysed under the scanning electron microscope (SEM) and by molecular analyses of the Internal Transcribed Spacers 1 and 2 plus the coding region 5.8S (ITS region) and the 28S rRNA gene. Seven species of solitary centric diatoms belonging to four genera were found in the estuary including: Conticribra weissflogii, Cyclotella atomus var. atomus, Cyclotella cryptica, Cyclotella marina, Cyclotella meneghiniana, Discostella pseudostelligera and Thalassiosira pseudonana. Dominant species during blooms were C. meneghiniana and Co. weissflogii in the upper estuary and D. pseudostelligera and T. pseudonana in the middle estuary. The morphological traits used to differentiate between species pairs of similar morphology (C. meneghiniana/C. cryptica or D. pseudostelligera/D. woltereckii) were observed to vary with environmental conditions, denoting a great deal of phenotypic plasticity which would hinder accurate identification of the species when using morphological approaches alone.     

Interspecific Gene Flow Shaped the Evolution of the Genus Canis

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Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

Hopf bifurcation in the presomitic mesoderm during the mouse segmentation

Vertebrae and ribs arise from embryonic tissues called somites. Somites arise sequentially from the unsegmented embryo tail, called presomitic mesoderm (PSM). The pace of somite formation is controlled by gene products such as hairy and enhancer of split 7 (Hes7) whose expression oscillates in the PSM. In addition to the cyclic genes, there is a gradient of fibroblast growth factor 8 (Fgf8) mRNA from posterior to anterior PSM. Recent experiments have shown that in the absence of Fgf signaling, Hes7 oscillations in the anterior and posterior PSM are lost. On the other hand, Notch mutants reduce the amplitude of posterior Hes7 oscillations and abolish anterior Hes7 oscillations. To understand these phenotypes, we delineated and simulated a logical and a delay differential equation (DDE) model with similar network topology in wild-type and mutant situations. Both models reproduced most wild-type and mutant phenotypes suggesting that the chosen topology is robust to explain these phenotypes. Numerical continuation of the model showed that even in the wild-type situation, the system changed from sustained to damped, i.e. a Hopf bifurcation occurred, when the Fgf concentration decreased in the PSM. This numerical continuation analysis further indicated that the most sensitive parameters for the oscillations are the parameters of Hes7 followed by those of Lunatic fringe (Lfng) and Notch1. In the wild-type, the damping of Hes7 oscillations was not so strong so that cells reached the new somites before they lose Hes7 oscillations. By contrast, in the fibroblast growth factor receptor 1 (Fgfr1) conditional knock-out (cKO) mutant simulation, Notch signaling was not able to maintain sustained Hes7 oscillations. Our analysis suggests that Fgf signaling makes cells enter an oscillatory state of Hes7 expression. After moving to the anterior PSM, where Fgf signaling is missing, Notch signaling compensates the damping of Hes7 oscillations in the anterior PSM.     

Origin of the Proton-transfer Step in the Cofactor-free (1H)-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase: EFFECT OF THE BASICITY OF AN ACTIVE SITE HIS RESIDUE*

Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavorable reaction without any cofactor. In the quinaldine metabolic pathway, HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His-251/Asp-126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their k_cat and k_cat/K_m constants, with a decrease in k_cat/K_m of 5500- and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate-limiting and yield a pK_a value of ∼7.2 for WT HOD. A large solvent isotope effect was found, and the pK_a value was shifted to ∼8.3 in D₂O. Crystallographic and computational studies reveal that the mutations have a minor effect on substrate positioning. Computational work shows that both His-251 and Asp-126 are essential for the proton transfer driving force of the initial reaction. This multidisciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation.     

Genomes of Alteromonas australica,a world apart

Background

Alteromonas is a genus of marine bacteria that is very easy to isolate and grow in the laboratory. There are genomes available of the species Alteromonas macleodii from different locations around the world and an Alteromonas sp. isolated from a sediment in Korea. We have analyzed the genomes of two strains classified by 16S rRNA (>99% similarity) as the recently described species Alteromonas australica, and isolated from opposite ends of the world; A. australica DE170 was isolated in the South Adriatic (Mediterranean) at 1000 m depth while A. australica H17^T was isolated from a sea water sample collected in St Kilda Beach, Tasman Sea.

Results

Although these two strains belong to a clearly different species from A. macleodii, the overall synteny is well preserved and the flexible genomic islands seem to code for equivalent functions and be located at similar positions. Actually the genomes of all the Alteromonas species known to date seem to preserve synteny quite well with the only exception of the sediment isolate SN2. Among the specific metabolic features found for the A. australica isolates there is the degradation of xylan and production of cellulose as extracellular polymeric substance by DE170 or the potential ethanol/methanol degradation by H17^T.

Conclusions

The genomes of the two A. australica isolates are not more different than those of strains of A. macleodii isolated from the same sample. Actually the recruitment from metagenomes indicates that all the available genomes are found in most tropical-temperate marine samples analyzed and that they live in consortia of several species and multiple clones within each. Overall the hydrolytic activities of the Alteromonas genus as a whole are impressive and fit with its known capabilities to exploit sudden inputs of organic matter in their environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-483) contains supplementary material, which is available to authorized users.