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.     

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.     

Vascular endothelial growth factor and related molecules in acute lung injury.

VEGFs and their receptors have been implicated in the regulation of vascular permeability in many organ systems, including the lung. Increased permeability and interstitial and pulmonary edema are prominent features of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Extrapolating data from other organ systems and animal experiments have suggested that overexpression of VEGF functions primarily as proinjurious molecules in the lung. Recent data, from animal models as well as from patients with ARDS, have shown decreased levels of VEGF in the lung. The role of VEGF and related molecules in ALI/ARDS is, therefore, controversial: what has become clear is that there are many unique features in the regulation of pulmonary vascular permeability and in VEGF expression in the lung. In this review, we explore a growing body of literature looking at the expression and function of VEGF and related molecules in different models of ALI and in patients with ALI/ARDS. Novel evidence points to a potential role of VEGF in promoting repair of the alveolar-capillary membrane during recovery from ALI/ARDS. Understanding the role of VEGF in this disease process is crucial for developing new therapeutic strategies for ALI/ARDS.     

Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling

The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.     

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.