Yasuhiro Kasahara

Metaproteomic Identification of Diazotrophic Methanotrophs and Their Localization in Root Tissues of Field-Grown Rice Plants

ABSTRACT In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.

The Oligomeric States of the Photosystems and the Light-Harvesting Complexes in the Chl b-Less Mutant

The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.

An essential enzyme for phospholipid synthesis associates with the Bacillus subtilis divisome

The mechanism by which the membrane synthetic machinery might be co‐organized with the cell‐division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl–acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell‐division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ‐anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z‐ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z‐ring formation. We propose that PlsX localization is prior to Z‐ring formation in the hierarchy of septum formation events and that PlsX is important for co‐ordinating membrane synthesis with cell division in order to properly complete septum formation.

Genome-wide analytical approaches using semi-quantitative expression proteomics for aromatic hydrocarbon metabolism in Pseudomonas putida F1.

Pseudomonas putida F1 can degrade aromatic hydrocarbons to intermediate products of the tricarboxylic acid cycle. To determine key induced proteins and enzymes required for degradation of toluene, ethylbenzene, benzene, p-cymene, and p-cumate, we performed comprehensive proteome analysis using a combination of 1-D SDS-PAGE and LC-MS/MS in cells grown in the presence of each aromatic hydrocarbon. Semi-quantitative analysis using protein content calculated from the exponentially modified protein abundance index (emPAI) was performed for each proteome data set, and the resulting data were compared. Of 5250 known proteins in P. putida F1, 1733-2368 expressed proteins were identified. All of the key enzymes in the degradation pathways were identified. Additionally, the proteins induced by the aromatic hydrocarbons, regulators, and transporters were also found. Using K-means clustering analysis of the proteome data sets, substrate-specific induced proteins were characterized, ranging from 62 to 164 in number. The functions of most of these proteins were not unknown in relation to the metabolism of aromatic hydrocarbons. These results suggest that the approaches used here are ideal as a primary investigation of the various physiological characteristics of bacterial cells.

Gene expression profiling of Pseudomonas putida F1 after exposure to aromatic hydrocarbon in soil by using proteome analysis

Pseudomonas putida F1 can metabolize toluene, ethylbenzene, and benzene for growth. Previously, we identified proteins involved in the utilization of these compounds by P. putida F1 through culture in liquid media. However, it was unclear whether laboratory analysis of bacterial activity and catabolism accurately reflected the soil environment. We identified proteins involved in the degradation of toluene, ethylbenzene, and benzene growth in soil using two-dimensional gel electrophoresis (2-DE) or standard SDS-PAGE combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS). According to 2-DE/LC–MS/MS analysis, 12 of 22 key enzymes involved in the degradation of toluene, ethylbenzene, and benzene were detected. In standard SDS-PAGE/LC–MS/MS analysis of soil with ethylbenzene, approximately 1,260 cellular proteins were identified in P. putida F1. All key enzymes and transporter and sensor proteins involved in ethylbenzene degradation were up-regulated similar to that noted in liquid cultures. In P. putida F1, aromatic hydrocarbon response in soil is the same as that observed in liquid media.

Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7

Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages.

Proteome analysis of Pseudomonas putida F1 genes induced in soil environments

Knowledge of the gene expression dynamics of a single soil bacterial strain contributes to the understanding of its behaviour, physiological state and surrounding microenvironment. Genes expressed in soil environments rather than in laboratory media are considered to particularly relevant. Here, we compared genome-wide gene expression profiles of the bacterium Pseudomonas putida F1 inoculated in three different types of nonsterile soils deduced using proteome analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Proteins commonly detected in all three samples and involved with bacterial growth and fundamental metabolism were excluded. Nine proteins were identified as specifically expressed in soil including an aldehyde dehydrogenase, a nitric oxide dioxygenase and five proteins encoded by a cluster of metabolism-associated genes. Expression factor analysis revealed that the nitric oxide dioxygenase-coding gene was induced by nitric oxide and the five clustered genes were induced under phosphate starvation. The expression of these genes can be attributed to response to soil environmental stimuli surrounding the F1 cells. These results strongly suggest that our soil metaproteome approach is useful for understanding the autecology and lifestyle of a single bacterial strain in soil environments and allows the prediction of the microenvironment surrounding the bacterial cells.

Understory Dwarf Bamboo Affects Microbial Community Structures and Soil Properties in a Betula ermanii Forest in Northern Japan

In order to understand the relationships between understory bamboo and soil properties, we compared microbial community structures in the soil of a Betula ermanii boreal forest with Sasa kurilensis present and removed using high-throughput DNA sequencing. The presence of understory S. kurilensis strongly affected soil properties, including total carbon, total nitrogen, nitrate, and the C:N ratio as well as relative soil moisture. Marked differences were also noted in fungal and bacterial communities between plots. The relative abundance of the fungal phylum Ascomycota was 13.9% in the Sasa-intact plot and only 0.54% in the Sasa-removed plot. Among the Ascomycota fungi identified, the most prevalent were members of the family Pezizaceae. We found that the abundance of Pezizaceae, known to act as mycorrhizal fungi, was related to the amount of total carbon in the Sasa-intact plot. The relative abundance of Proteobacteria was significantly higher, whereas those of Planctomycetes and Actinobacteria were lower in the Sasa-intact plot than in the Sasa-removed plot. Furthermore, the results obtained suggest that some species of the phylum Planctomycetes are more likely to occur in the presence of S. kurilensis. Collectively, these results indicate that the presence of S. kurilensis affects microbial communities and soil properties in a B. ermanii boreal forest.