A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts.

Wounding is a constant threat to plant survival throughout their lifespan; therefore, understanding the biological responses to wounds at the cellular level is important. The protoplast system is versatile for molecular biology, however, no wounding studies on this system have been reported. We established a new approach for wounding research using mechanically damaged Arabidopsis mesophyll protoplasts. Wounded protoplasts showed typical wounding responses, such as increased MPK6 kinase activity and upregulated JAZ1 expression. We also assessed expression profiles and protein stability of the basic helix-loop-helix transcription factor MYC2 in wounded protoplasts. Promoter activity, gene expression, and protein stability of MYC2 were compromised, but recovered in the early stage of wounding. In the late stage, the promoter activity and expression of MYC2 were increased, but the protein stability was not changed. According to the results of the present study, this new cell-based approach will be of use in various molecular studies on plant wounding.

Involvement of NO3- in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric Screening Method.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 DNRA bacteria were isolated from NO3--reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N2O reduction. A common prominent physiological feature observed in the isolates was NO2- accumulation before NH4+ production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO2--to-NH4+ reduction at submillimolar NO3- concentrations and downregulation of nrfA or nirB transcription when NO3- was being reduced to NO2- In batch and chemostat experiments, both isolates produced NH4+ from NO3- reduction when incubated with excess organic electron donors, while incubation with excess NO3- resulted in NO2- buildup but no substantial NH4+ production, presumably due to inhibitory NO3- concentrations. This previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil.IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3- and NO2- Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3- and NO2- to NH4+, a reactive nitrogen compound with a higher tendency to be retained in the soil matrix. Therefore, stimulation of DNRA has often been proposed as a strategy to improve fertilizer efficiency and reduce greenhouse gas emissions. Such attempts have been hampered by lack of insights into soil DNRA bacterial ecophysiology. Here, we have developed a new screening method for isolating DNRA-catalyzing organisms from agricultural soils without apparent DNRA activity. Physiological characteristics of six DNRA isolates were closely examined, disclosing a previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity, which may be a key to understanding why DNRA activity is rarely observed at substantial levels in nitrogen-rich agricultural soils.

Identification of primary effecters of N2O emissions from full-scale biological nitrogen removal systems using random forest approach.

Wastewater treatment plants (WWTPs) have long been recognized as point sources of N2O, a potent greenhouse gas and ozone-depleting agent. Multiple mechanisms, both biotic and abiotic, have been suggested to be responsible for N2O production from WWTPs, with basis on extrapolation from laboratory results and statistical analyses of metadata collected from operational full-scale plants. In this study, random forest (RF) analysis, a machine-learning approach for feature selection from highly multivariate datasets, was adopted to investigate N2O production mechanism in activated sludge tanks of WWTPs from a novel perspective. Standardized measurements of N2O effluxes coupled with exhaustive metadata collection were performed at activated sludge tanks of three biological nitrogen removal WWTPs at different times of the year. The multivariate datasets were used as inputs for RF analyses. Computation of the permutation variable importance measures returned biomass-normalized dissolved inorganic carbon concentration (DIC·VSS-1) and specific ammonia oxidation activity (sOURAOB) as the most influential parameters determining N2O emissions from the aerated zones (or phases) of activated sludge bioreactors. For the anoxic tanks, dissolved-organic-carbon-to-NO2-/NO3- ratio (DOC·(NO2--N + NO3--N)-1) was singled out as the most influential. These data analysis results clearly indicate disparate mechanisms for N2O generation in the oxic and anoxic activated sludge bioreactors, and provide evidences against significant contributions of N2O carryover across different zones or phases or niche-specific microbial reactions, with aerobic NH3/NH4+ oxidation to NO2- and anoxic denitrification predominantly responsible from aerated and anoxic zones or phases of activated sludge bioreactors, respectively.

Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria.

Novel approaches and reasons to isolate methanotrophic bacteria with biotechnological potentials: recent achievements and perspectives.

The recent drop in the price of natural gas has rekindled the interests in methanotrophs, the organisms capable of utilizing methane as the sole electron donor and carbon source, as biocatalysts for various industrial applications. As heterologous expression of the methane monooxygenases in more amenable hosts has been proven to be nearly impossible, future success in methanotroph biotechnology largely depends on securing phylogenetically and phenotypically diverse methanotrophs with relatively high growth rates. For long, isolation of methanotrophs have relied on repeated single colony picking after initial batch enrichment with methane, which is a very rigorous and time-consuming process. In this review, three unconventional isolation methods devised for facilitation of the isolation process, diversification of targeted methanotrophs, and/or screening of rapid growers are summarized. The soil substrate membrane method allowed for isolation of previously elusive methanotrophs and application of high-throughput extinction plating technique facilitated the isolation procedure. Use of a chemostat with gradually increased dilution rates proved effective in screening for the fastest-growing methanotrophs from environmental samples. Development of new isolation technologies incorporating microfluidics and single-cell techniques may lead to discovery of previously unculturable methanotrophs with unexpected metabolic potentials and thus, certainly warrant future investigation.

Niche specialization of bacteria in permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica.

Perennially ice-covered lakes in the McMurdo Dry Valleys, Antarctica, are chemically stratified with depth and have distinct biological gradients. Despite long-term research on these unique environments, data on the structure of the microbial communities in the water columns of these lakes are scarce. Here, we examined bacterial diversity in five ice-covered Antarctic lakes by 16S rRNA gene-based pyrosequencing. Distinct communities were present in each lake, reflecting the unique biogeochemical characteristics of these environments. Further, certain bacterial lineages were confined exclusively to specific depths within each lake. For example, candidate division WM88 occurred solely at a depth of 15 m in Lake Fryxell, whereas unknown lineages of Chlorobi were found only at a depth of 18 m in Lake Miers, and two distinct classes of Firmicutes inhabited East and West Lobe Bonney at depths of 30 m. Redundancy analysis revealed that community variation of bacterioplankton could be explained by the distinct conditions of each lake and depth; in particular, assemblages from layers beneath the chemocline had biogeochemical associations that differed from those in the upper layers. These patterns of community composition may represent bacterial adaptations to the extreme and unique biogeochemical gradients of ice-covered lakes in the McMurdo Dry Valleys.

Complete Genome Sequence of Psychrobacter alimentarius PAMC 27889, a Psychrophile Isolated from an Antarctic Rock Sample.

Psychrobacter alimentarius PAMC 27889, a Gram-negative, psychrophilic bacterium, was isolated from an Antarctic rock sample. Here, we report the complete genome of P. alimentarius PAMC 27889, which has the nonmevalonate methylerythritol phosphate pathway of terpenoid biosynthesis and a complete gene cluster for benzoate degradation.

Evaluation of the sensitivity and specificity of primer pairs and the efficiency of RNA extraction procedures to improve noroviral detection from oysters by nested reverse transcription-polymerase chain reaction.

Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoV-polluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoV-seeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp® Viral RNA Mini kit with a QIAshredder™ Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.