Genetic analysis of a novel plasmid pZMX101 from Halorubrum saccharovorum: determination of the minimal replicon and comparison with the related haloarchaeal plasmid pSCM201

The DNA sequence of a novel haloarchaeal plasmid pZMX101 (3918 bp) from Halorubrum saccharovorum was determined and six ORFs were predicted. The largest ORF encodes a putative replication initiation protein RepA, which shares 40% sequence similarity with the Rep201 of a theta-replication plasmid pSCM201 recently isolated from Haloarcula, suggesting that pZMX101 might replicate via a theta-type mechanism. Using pZMX101 as the only haloarchaeal replicon, a shuttle vector pZMX108 was constructed and successfully transformed into Haloferax volcanii DS70. Based on this in vivo system, the minimal replicon (1978 bp) of pZMX101 was determined. It is composed of the repA gene plus c. 400-bp upstream and 300-bp downstream sequences. Significantly, the putative replication origin of pZMX101 and that of pSCM201 contain different types of sequence motifs, and these two plasmids exhibit distinct host preference for Haloferax and Haloarcula, respectively.     

Detailed identification of desert-originated bacteria carried by Asian dust storms to Japan

Several halotolerant bacteria were isolated from dust allowed to settle passively on saline medium in Higashi-Hiroshima, Japan during Asia dust events in 2005–2006. The primary identification, based on the sequence similarity of the 16S rRNA gene, revealed that these isolates were strains of Bacillus subtilis, B. licheniformis, Staphylococcus epidermidis, Gracillibacillus sp., and Halomonas venusta. A parallel investigation carried out on desert sand collected directly from sand dunes in Dunhuang, Gobi Desert, China resulted in the revivification of seven bacterial strains that were highly identical to the B. subtilis and B. licheniformis strains obtained in Higashi-Hiroshima (99.7 and 100% of 16S rDNA sequence similarity, respectively). A subsequent genetic analysis on the group of B. licheniformis isolates based on the universally house-keeping genes, gyrB and parE, revealed high sequence similarities in both genes among the strains of both locations (99.0–99.4%), which clustered them in a monophyletic line. Phenotype characterized by numerical taxonomy for 150 physiological tests confirmed the close relatedness between strains (similarity coefficient S _SM = 96.0%). The remarkable agreement between phenotype and genotype of the bacterial isolates allows us to conclude that there may have been an aerosolized dispersion of a Gobi Desert B. licheniformis by dust storms to Japan. This study provides evidence of microbial transport by yellow dust events in North-East Asia.     

A statistical method for chromatographic alignment of LC-MS data

Integrated liquid-chromatography mass-spectrometry (LC-MS) is becoming a widely used approach for quantifying the protein composition of complex samples. The output of the LC-MS system measures the intensity of a peptide with a specific mass-charge ratio and retention time. In the last few years, this technology has been used to compare complex biological samples across multiple conditions. One challenge for comparative proteomic profiling with LC-MS is to match corresponding peptide features from different experiments. In this paper, we propose a new method--Peptide Element Alignment (PETAL) that uses raw spectrum data and detected peak to simultaneously align features from multiple LC-MS experiments. PETAL creates spectrum elements, each of which represents the mass spectrum of a single peptide in a single scan. Peptides detected in different LC-MS data are aligned if they can be represented by the same elements. By considering each peptide separately, PETAL enjoys greater flexibility than time warping methods. While most existing methods process multiple data sets by sequentially aligning each data set to an arbitrarily chosen template data set, PETAL treats all experiments symmetrically and can analyze all experiments simultaneously. We illustrate the performance of PETAL on example data sets.     

Isolation and characterization of N98-1272 A, B and C, selective acetylcholinesterase inhibitors from metabolites of an actinomycete strain

A high throughput screening was carried out in order to search for inhibitors of acetylcholinesterase (AChE) from microorganism metabolites. An actinomycete strain was found to produce active compounds named N98-1272 A, B and C with IC50 of 15.0, 11.5, 12.5 microM, respectively. Structural studies revealed that the three compounds are identical to the known antibiotics, Manumycin C, B and A. Kinetic analyses showed that N98-1272 C (Manumycin A) acted as a reversible noncompetitive inhibitor of acetylcholinesterase, with a Ki value of 7.2 microM. The cyclohexenone epoxide part of the structure plays a crucial role in the inhibitory activity against AChE. Compared with Tacrine, N98-1272 A, B, and C exhibit much better selectivity toward AChE over BuChE.     

Validation of computational methods in genomics

High-throughput technologies for genomics provide tens of thousands of genetic measurements, for instance, gene-expression measurements on microarrays, and the availability of these measurements has motivated the use of machine learning (inference) methods for classification, clustering, and gene networks. Generally, a design method will yield a model that satisfies some model constraints and fits the data in some manner. On the other hand, a scientific theory consists of two parts: (1) a mathematical model to characterize relations between variables, and (2) a set of relations between model variables and observables that are used to validate the model via predictive experiments. Although machine learning algorithms are constructed to hopefully produce valid scientific models, they do not ipso facto do so. In some cases, such as classifier estimation, there is a well-developed error theory that relates to model validity according to various statistical theorems, but in others such as clustering, there is a lack of understanding of the relationship between the learning algorithms and validation. The issue of validation is especially problematic in situations where the sample size is small in comparison with the dimensionality (number of variables), which is commonplace in genomics, because the convergence theory of learning algorithms is typically asymptotic and the algorithms often perform in counter-intuitive ways when used with samples that are small in relation to the number of variables. For translational genomics, validation is perhaps the most critical issue, because it is imperative that we understand the performance of a diagnostic or therapeutic procedure to be used in the clinic, and this performance relates directly to the validity of the model behind the procedure. This paper treats the validation issue as it appears in two classes of inference algorithms relating to genomics - classification and clustering. It formulates the problem and reviews salient results.     

Mitochondrial proteomic analysis and characterization of the intracellular mechanisms of bis(7)-tacrine in protecting against glutamate-induced excitotoxicity in primary cultured neurons

Increasing evidence supports that the mitochondrial dysfunction, mainly caused by abnormal changes in mitochondrial proteins, plays a pivotal role in glutamate-induced excitotoxicity, which is closely associated with the pathogenesis of acute and chronic neurodegenerative disorders, such as stroke and Alzheimer's disease. In this study, post-treatment of cerebellar granule neurons with bis(7)-tacrine significantly reversed declines in mitochondrial membrane potential, ATP production, and neuronal cell death induced by glutamate. Moreover, this reversal was independent of NMDA antagonism, acetylcholinesterase inhibition, and cholinergic pathways. Using two-dimensional differential in-gel electrophoresis, we conducted a comparative analysis of mitochondrial protein patterns. In all, 29 proteins exhibiting significant differences in their abundances were identified in the glutamate-treated group when compared with the control. The expression patterns in 22 out of these proteins could be reversed by post-treatment with bis(7)-tacrine. Most of the differentially expressed proteins are involved in energy metabolism, oxidative stress, and apoptosis. In particular, the altered patterns of four of these proteins were further validated by Western blot analysis. Our findings suggest that multiple signaling pathways initiated by the altered mitochondrial proteins may mediate glutamate-induced excitotoxicity and also offer potentially useful intracellular targets for the neuroprotection provided by bis(7)-tacrine.     

A Novel DNA-Binding Protein,PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.