Genome Structure of the Legume, Lotus japonicus

The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.Key words: Lotus japonicus, genome structure, Fabaceae, comparative analysis     

From the sequence to cell modeling: comprehensive functional genomics in Escherichia coli

As a result of the enormous amount of information that has been collected with E. coli over the past half century (e.g. genome sequence, mutant phenotypes, metabolic and regulatory networks, etc.), we now have detailed knowledge about gene regulation, protein activity, several hundred enzyme reactions, metabolic pathways, macromolecular machines, and regulatory interactions for this model organism. However, understanding how all these processes interact to form a living cell will require further characterization, quantification, data integration, and mathematical modeling, systems biology. No organism can rival E. coli with respect to the amount of available basic information and experimental tractability for the technologies needed for this undertaking. A focused, systematic effort to understand the E. coli cell will accelerate the development of new post-genomic technologies, including both experimental and computational tools. It will also lead to new technologies that will be applicable to other organisms, from microbes to plants, animals, and humans. E. coli is not only the best studied free-living model organism, but is also an extensively used microbe for industrial applications, especially for the production of small molecules of interest. It is an excellent representative of Gram-negative commensal bacteria. E. coli may represent a perfect model organism for systems biology that is aimed at elucidating both its free-living and commensal life-styles, which should open the door to whole-cell modeling and simulation.     

Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.     

Plastidial fatty acid signaling modulates salicylic acid- and jasmonic acid-mediated defense pathways in the Arabidopsis ssi2 mutant

A mutation in the Arabidopsis gene ssi2/fab2, which encodes stearoyl–acyl carrier protein desaturase (S-ACP-DES), results in the reduction of oleic acid (18:1) levels in the mutant plants and also leads to the constitutive activation of NPR1-dependent and -independent defense responses. By contrast, ssi2 plants are compromised in the induction of the jasmonic acid (JA)–responsive gene PDF1.2 and in resistance to the necrotrophic pathogen Botrytis cinerea. Although S-ACP-DES catalyzes the initial desaturation step required for JA biosynthesis, a mutation in ssi2 does not alter the levels of the JA precursor linolenic acid (18:3), the perception of JA or ethylene, or the induced endogenous levels of JA. This finding led us to postulate that the S-ACP-DES–derived fatty acid (FA) 18:1 or its derivative is required for the activation of certain JA-mediated responses and the repression of the salicylic acid (SA) signaling pathway. Here, we report that alteration of the prokaryotic FA signaling pathway in plastids, leading to increased levels of 18:1, is required for the rescue of ssi2-triggered phenotypes. 18:1 levels in ssi2 plants were increased by performing epistatic analyses between ssi2 and several mutants in FA pathways that cause an increase in the levels of 18:1 in specific compartments of the cell. A loss-of-function mutation in the soluble chloroplastic enzyme glycerol-3-phosphate acyltransferase (ACT1) completely reverses SA- and JA-mediated phenotypes in ssi2. In contrast to the act1 mutation, a loss-of-function mutation in the endoplasmic reticulum–localized ω6 oleate desaturase (FAD2) does not alter SA- or JA-related phenotypes of ssi2. However, a mutation in the plastidial membrane–localized ω6 desaturase (FAD6) mediates a partial rescue of ssi2-mediated phenotypes. Although ssi2 fad6 plants are rescued in their morphological phenotypes, including larger size, absence of visible lesions, and straight leaves, these plants continue to exhibit microscopic cell death and express the PR-1 gene constitutively. In addition, these plants are unable to induce the expression of PDF1.2 in response to the exogenous application of JA. Because the act1 mutation rescues all of these phenotypes in ssi2 fad6 act1 triple-mutant plants, act1-mediated reversion may be mediated largely by an increase in the free 18:1 content within the chloroplasts. The reversion of JA responsiveness in ssi2 act1 plants is abolished in the ssi2 act1 coi1 triple-mutant background, suggesting that both JA- and act1-generated signals are required for the expression of the JA-inducible PDF1.2 gene. Our conclusion that FA signaling in plastids plays an essential role in the regulation of SSI2-mediated defense signaling is further substantiated by the fact that overexpression of the N-terminal–deleted SSI2, which lacks the putative plastid-localizing transit peptide, is unable to rescue ssi2-triggered phenotypes, as opposed to overexpression of the full-length protein.     

Functional analysis of 1440 Escherichia coli genes using the combination of knock-out library and phenotype microarrays

Escherichia coli is one of the best elucidated organisms. However, about 40% of E. coli genes have not been assigned to their function yet. We analyzed 1440 single gene knock-out mutants using the GN2-MicroPlate, which permits assay of 95 carbon-source utilizations simultaneously. In the knock-out library there are 1044 of so called y-genes with no apparent function. The raw dataset was analyzed and genes were interrelated by the clustering method of the GeneSpring software. In the resulted dendrogram of genes, a group of genes with known and related function tended to be assembled into a cluster. Our clustering method would be useful for functional assignment of so called y-genes with no apparent function, since the resulted dendrogram could connect y-genes to phenotype and function of well-studied genes.     

Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12

We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems. A high correlation coefficient of expression profile was found between several two-component mutants. Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems. The compiled data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/ 2_components).     

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.