Biocatalytic asymmetric hydroxylation of hydrocarbons by free and immobilized Bacillus megaterium cells

Screening of soil bacteria with allylbenzene resulted in a Bacillus megaterium strain, which hydroxylates simple hydrocarbons in high enantiomeric excess (ee up to 99%). Benzylic and nonbenzylic hydroxylation products were obtained, without the usually observed high preference for the benzylic position. The immobilization of the B. megaterium cells in alginate gel effectively improved the stability of the cells and increased the amounts of products formed, without loss of enantioselectivity. The product ratio ( vs. β hydroxylation) was shifted towards benzylic hydroxylation, which suggests that at least two hydroxylating enzymes with distinct regioselectivity are involved. Comparison to free-cell fermentations in small- and large-scale bioreactors (up to 2000 ml) showed that the use of immobilized cells is advantageous, as they are easier to handle and yield higher amounts of oxidation products.     

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.     

Influence of FADS polymorphisms on tracking of serum glycerophospholipid fatty acid concentrations and percentage composition in children

Background

Tracking of fatty acid (FA) contribution to plasma or serum lipids over time was shown in children and adults. However, the potential role of FADS gene variants has not been investigated.

Methods and Principal Findings

Serum GP FA composition of 331 children aged 2 and 6 years, participating in an ongoing birth cohort study, was analyzed. Correlation coefficients were estimated to describe FA tracking over 4 years and to assess the influence of FADS variants on tracking. We found low to moderate tracking (r = 0.12–0.49) of FA compositions and concentration between 2 and 6 years. Concentration changes of total monounsaturated FA and total saturated FA over time correlated closely (r = 0.79) but percentage values were unrelated (r = −0.02). Tracking for n-6 long chain polyunsaturated fatty acid (LC-PUFA) concentrations was lower in subjects homozygous for the major allele of FADS variants and higher in carriers of at least one minor allele, whereas for total n-3 LC-PUFA concentrations and compositions this was vice versa. For individual n-3 PUFA inconsistent results were found.

Conclusions and Significance

Serum GP FA composition shows low to moderate tracking over 4 years with a higher tracking for LC-PUFA metabolites than for their precursor FA. Serum PUFA levels and their tracking seem to be more influenced by lipid and lipoprotein metabolism than by FA specific pathways.     

High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase

The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZalphaA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPalpha-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter lambdaP(L), yielding 600U/g or 54,000U/g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents.     

Effects of thermal acclimation and photoacclimation on lipophilic pigments in an invasive and a native cyanobacterium of temperate regions

The freshwater cyanobacterium Cylindrospermopsis raciborskii spreads from tropical to temperate regions worldwide. This entails acclimation to varied light and temperature conditions. We studied the thermal and light acclimation of the photosynthetic machinery of C. raciborskii by monitoring alteration of the chlorophyll a and carotenoid content in German strains of C. raciborskii, in African and Australian strains of C. raciborskii, and in German strains of Aphanizomenon gracile, a native cyanobacterium belonging to the same order (Nostocales). Our results showed that temperate and tropical C. raciborskii strains did not differ in pigment acclimation to light and temperature. In contrast, the ratio of photoprotective carotenoids (namely the carotenoid glycoside 4-hydroxymyxol glycoside [aphanizophyll]) to chlorophyll a increased significantly more in C. raciborskii in comparison with A. gracile (1) with decreasing temperatures from 20 to 10°C and a moderate light intensity of 80?µmol photons m^?2?s^?1 and (2) with increasing light intensities at a suboptimal temperature of 15°C, compared to 20°C. We conclude that below 20°C photoinhibition is avoided by greater photoprotection in the invasive species C. raciborskii compared to the native species A. gracile.     

General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.     

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup‐grown Rhodosporidium toruloides NCYC 921

The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 μg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds.     

Ligand-binding specificity of an invertebrate (Manduca sexta) putative cellular retinoic acid binding protein

Intracellular lipid-binding proteins (iLBPs) are small cytoplasmic proteins that specifically interact with hydrophobic ligands. Fatty acid-binding proteins (FABPs), cellular retinoic acid-binding proteins (CRABPs) and cellular retinol-binding proteins (CRBPs) belong to the iLBP family. A recently identified insect (Manduca sexta) iLBP has been reported to possibly represent an invertebrate CRABP mimicking the role of CRABPs in vertebrate organisms. The presence in this protein of the characteristic binding triad residues involved in the interaction with ligand carboxylate head groups, a feature pertaining to several FABPs and to CRABPs, and the close phylogenetic relationships with both groups of vertebrate heart-type FABPs and CRBPs/CRABPs, makes it difficult to assign it to either FABPs or CRABPs. However, its negligible interaction with retinoic acid and high affinity (K(d) values in the 10(-8) M range) for fatty acids have been established by means of direct and competitive binding assays. As shown by phylogenetic analysis, the M. sexta iLBP belongs to a wide group of invertebrate iLBPs, which, besides being closely related phylogenetically, share distinctive features, such as the conservation of chemically distinct residues in their amino acid sequences and the ability to bind fatty acids. Our results are in keeping with the lack of cellular retinoid-binding proteins in invertebrates and with their later appearance during the course of chordate evolution.