Domain Structure of the Selenocysteine-specific Translation Factor SelB in Prokaryotes

Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNASec, guanine nucleotides and a secondary structure of the selenoprotein mRNA following the UGA at the 3' side. A comparison of the amino acid sequences of SelB species from E. coli,Desulfomicrobium baculatum, Clostridium thermoaceticum and Haemophilus influenzae showed that the proteins consist of at least four structural domains from which the Nterminal three are well conserved and share homology with elongation factor Tu whereas the C-terminal one is more variable and displays no similarity to any protein known. With the aid of the coordinates of EF-Tu the N-terminal part has been modelled into a 3D structure which exhibits intriguing features concerning its interaction with guanine nucleotides and other components of the translational apparatus. Cloning and expression of fragments of SelB and biochemical analysis of the purified truncated proteins showed that the C-terminal 19 kDa protein fragment is able to specifically bind to the selenoprotein mRNA. SelB, thus, is a translation factor functionally homologous to EF-Tu hooked up to the mRNA with its C-terminal end. The formation by SelB of a quaternary complex in vivo has been proven by overexpression of truncated genes of SelB and by demonstration that fragments comprising the mRNA or the tRNA binding domain inhibit selenocysteine insertion

Adaptive strategies of high and low nucleic acid prokaryotes in response to declining resource availability and selective grazing by protozoa

Prokaryotes play a fundamental role in global ocean biogeochemical cycles.However,how the abundance and metabolic activity of ecologically distinct subgroups(i.e.,high nucleic acid(HNA)and low nucleic acid(LNA)cells),and their regulating factors,change in response to changing marine environmental conditions remains poorly understood.Here,we delved into the time-evolving dynamic responses of the HNA and LNA prokaryotic subgroups to declining resource availability and selective grazing by protozoa by conducting a 73-day incubation experiment in a large-volume(117,000 L)macrocosm that facilitates community-level exploration.We found that the metabolic activity of the HNA subgroup was higher than that of the LNA subgroup when the macrocosm was resource replete but that the HNA subgroup declined more rapidly than the LNA subgroup as the resources became increasingly scarce,leading to a steadily increasing contribution of LNA cells to prokaryotic activity.Meanwhile,as resources in the macrocosm became limited,protozoan grazing preference shifted from the HNA to the LNA subgroup and the contributions of the LNA subgroup to the carbon flow within the macrocosm increased.The findings highlight the resilience of LNA cells in resource-limited environments,illuminate the critical role of selective grazing by protozoa in balancing distinct prokaryotic subgroups under changing resource conditions,and demonstrate the complex and adaptive interactions between protozoa and prokaryotes across diverse environmental contexts.

Environmental Mn(Ⅱ) enhances the activity of dissimilatory arsenate-respiring prokaryotes from arsenic-contaminated soils

Many investigations suggest that dissimilatory arsenate-respiring prokaryotes (DARPs) play a key role in stimulating reductive mobilization of As from solid phase into groundwater,but it is not clear how environmental Mn(Ⅱ) affects the DARPs-mediated reductive mobilization of arsenic.To resolve this issue,we collected soil samples from a realgar tailingsaffected area.We found that there were diverse arsenate-respiratory reductase (arr) genes in the soils.The microbial communities had high arsenate-respiring activity,and were able to efficiently stimulate the reductive mobilization of As.Compared to the microcosms without Mn(Ⅱ),addition of 10 mmol/L Mn(Ⅱ) to the microcosms led to 23.99%-251.79% increases in the microbial mobilization of As,and led to 133.3%-239.2% increases in the abundances of arr genes.We further isolated a new cultivable DARP,Bacillus sp.F11,from the arseniccontaminated soils.It completely reduced 1 mmol/L As(V) in 5 days under the optimal reaction conditions.We further found that it was able to efficiently catalyze the reductive mobilization and release of As from the solid phase;the addition of 2 mmol/L Mn(Ⅱ) led to 98.49%-248.78% increases in the F11 cells-mediated reductive mobilization of As,and70.6%-104.4% increases in the arr gene abundances.These data suggest that environmental Mn(Ⅱ) markedly increased the DARPs-mediated reductive mobilization of As in arseniccontaminated soils.This work provided a new insight into the close association between the biogeochemical cycles of arsenic and manganese.

Prokaryote phylogeny meets taxonomy: An exhaustive comparison of composition vector trees with systematic bacteriology

We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacte-riologists' taxonomy-primarily the latest online Outline of the Bergey's Manual of Systematic Bacteri-ology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.

Prokaryote phylogeny based on ribosomal proteins and aminoacyl tRNA synthetases by using the compositional distance approach

In order to show that the newly developed K-string composition distance method, based on counting oligopeptide frequencies, for inferring phylogenetic relations of prokaryotes works equally well without requiring the whole proteome data, we used all ribosomal proteins and the set of aminoacyl tRNA synthetases for each species. The latter group has been known to yield inconsistent trees if used individually. Our trees are obtained without making any sequence alignment. Altogether 16 Archaea, 105 Bacteria and 2 Eucarya are represented on the tree. Most of the lower branchings agree well with the latest, 2003, Outline of the second edition of the Bergeys Manual of Systematic Bacteriology and the trees also suggest some relationships among higher taxa.

Effects of Biostimulation-Bioaugmentation on Coastal Microbial Community in an in situ Mesocosm System

Globally,various types of pollution affect coastal waters as a result of human activities.Bioaugmentation and biostimulation are effective methods for treating water pollution.However,few studies have explored the response of coastal prokaryotic and eukaryotic communities to bioaugmentation and biostimulation.Here,a 28-day outdoor mesocosm experiment with two treatments(bioaugmentation-A and combined treatment of bioaugmentation and biostimulation-AS)and a control(untreated-C)were carried out.The experiment was conducted in Meishan Bay to explore the composition,dynamics,and co-occurrence patterns of prokaryotic and eukaryotic communities in response to the A and AS using 16S rRNA and 18S rRNA gene amplicon sequencing.After treatment,Gammaproteobacteria and Epsilonproteobacteria were significantly increased in group AS compared to group C,while Flavobacteriia and Saprospirae were significantly reduced.Dinoflagellata was significantly reduced in AS compared to C,while Chrysophyta was significantly reduced in both AS and A.Compared to C,the principal response curve analyses of the prokaryotic and eukaryotic communities both showed an increasing trend followed by a decreasing trend for AS.Furthermore,the trends of prokaryotic and eukaryotic communities in group A were similar to those in group AS compared with group C,but AS changed them more than A did.According to the species weight table on principal response curves,a significant increase was observed in beneficial bacteria in prokaryotic communities,such as Rhodobacterales and Oceanospirillales,along with a decrease in autotrophs in eukaryotic communities,such as Chrysophyta and Diatom.Topological properties of network analysis reveal that A and AS complicate the interactions between the prokaryotic and eukaryotic communities.Overall,these findings expand our understanding of the response pattern of the bioaugmentation and biostimulation on coastal prokaryotic and eukaryotic communities.

TAAPP: Tiling Array Analysis Pipeline for Prokaryotes

High-density tiling arrays provide closer view of transcription than regular microarrays and can also be used for annotating functional elements in genomes. The identified transcripts usually have a complex overlapping architecture when compared to the existing genome annotation. Therefore, there is a need for customized tiling array data analysis tools. Since most of the initial tiling arrays were conducted in eukaryotes, data analysis methods are well suited for eukaryofic genomes. For using whole-genome tiling arrays to identify previously unknown transcriptional elements like small RNA and antisense RNA in prokaryotes, existing data analysis tools need to be tailored for prokaryotic genome architecture. Furthermore, automation of such custom data analysis workflow is necessary for biologists to apply this powerful platform for knowledge discovery. Here we describe TAAPP, a web-based package that consists of two modules for prokaryotic tiling array data analysis. The transcript generation module works on normalized data to generate transcriptionally active regions （TARs）. The feature extraction and annotation module then maps TARs to existing genome annotation. This module further categorizes the transcription profile into potential novel non-coding RNA, antisense RNA, gene expression and operon structures. The implemented workflow is microarray platform independent and is presented as a web-based service. The web interface is freely available for acedemic use at http：//lims.lshi.mafes.msstate.edu/TAAPP-HTML/.

Pulsed export of carbon in the north-western Mediterranean Sea

The short term(hourly scale)variability of heterotrophic prokaryote(HP)vertical distribution and respiratory activity,was investigated in the north-western(NW)Mediterranean Sea.HP vertical distribution was determined on board by flow cytometry analysis of seawater samples collected by series of CTD casts.Cell counts and viability were determined for all samples.HP respiratory rates were determined later in the laboratory from filtered seawater samples(23 dm^(3))from 300-1150-m depth.The average cell viability was 94.8%±2.2%(n=240).There was no accumulation of dead cells,due to quick decay of damaged cells.In the epipelagic layer,three HP groups were distinguished,two(HNA1,HNA2)who se cells exhibited a high nucleic acid content and one(LNA)with low nucleic acid content cells.HNA2 was most populated at 50 m but not detected at 90 m and below,presumably aerobic anoxygenic photoheterotrophic bacteria(AAPs).The variability in HP abundance was mainly confined in the upper 80 m.A few secondary peaks of HP abundance were observed(80-150 m)in connection with abundance troughs in the surface layer.HP cells were continuously present in a wide layer around 500 m(mean 191×10^(3)cells/cm^(3)).Below this layer,HP abundance randomly exhibited peaks,coupled to respiratory rate peaks.The HP abundance and variability in the water column was suppressed during a strong wind event.The observed sporadic variability was tentatively interpreted through a pulsed carbon-export mechanism induced by the microorganism production of dissolved poly saccharide s,followed by flocculation and rapid sinking.This mechanism would thus contribute to(ⅰ)preventing organic matter accumulation in the epipelagic layer,(ⅱ)seeding the water column with live HP cells,and(ⅲ)supplying the aphotic water column with fre sh and labile organic matter.This important vertical flux mechanism needs further observations and modelling.

Vertical microbial profiling of water column reveals prokaryotic communities and distribution features of Antarctic Peninsula

Prokaryotic diversity and community composition in the water column of eight stations(63 samples) around the Antarctic Peninsula of the Southern Ocean were investigated. Through pyrosequencing of the V3–V4hypervariable regions of the 16S ribosomal RNA gene, we characterized 4 720 089 valid reads representing 48 188operational taxonomic units(OTUs, 97% similarity). The community was dominated by the phyla Pseudomonadota(original name: Proteobacteria, 47%), Oxyphotobacteria(26%), and Bacteroidota(original name: Bacteroidetes, 18%), which comprised an average of 91% of the total OTUs in all samples. The prokaryotic community composition varied vertically within the water column. Water column prokaryotic communities exhibited a clear depth profile, with higher microbial richness and higher diversity observed with increasing water depth. Cluster analysis of the community composition of water column samples exhibited a similar trend with depth. Correlation with environmental factors suggested distinct variation in prokaryotic community composition with changes in depth, salinity, temperature and dissolved oxygen levels. Functional prediction showed presence of active nitrogen, sulphur and methane metabolic cycles along the vertical transect of the studied region. These results will improve our knowledge of prokaryotic diversity and community composition at different depth of water column for better understanding of the microbial ecology and nutrient cycles in Antarctic Peninsula region of the Southern Ocean.

Prokaryotic diversity and community composition in the surface sediments of the Changjiang River Estuary in summer

Microorganisms are fundamental for the functioning of marine ecosystems and are involved in the decomposition of organic matter, transformation of nutrients and circulation of biologically-important chemicals. Based on the complexity of the natural geographic characteristics of the Changjiang River Estuary, the geographic distribution of sedimentary microorganisms and the causes of this distribution are largely unexplored. In this work, the surface sediment samples from the adjacent sea area of the Changjiang River Estuary were collected. Their prokaryotic diversity was examined by high-throughput sequencing technology, and the environmental factors of the bacterial community were investigated. The results indicated that the distribution of prokaryotic communities in the sediments of the study areas showed obvious spatial heterogeneity. The sampling sequences divided the sample regions into three distinct clusters. Each geographic region had a unique community structure, although Proteobacteria, Bacteroidota, Desulfobacterota, Acidobacteriota, and Actinobacteriota all existed in these three branches. Canonical correspondence analysis demonstrated that prokaryotic diversity and community distribution were significantly correlated with the geographic location of sediment, seawater depth, and in particular, nutrient content(e.g., total phosphorus, total organic carbon and dissolved oxygen). Moreover, it was found for the first time that the metal ions obviously affected the composition and distribution of the prokaryotic community in this area. In general, this work provides new insights into the structural characteristics and driving factors of prokaryotic communities under the background of the ever-changing Changjiang River Estuary.

铜绿假单胞菌脂肪酶Lipase基因的原核表达(英文)

[Objective] The aim of this study was to investigate the prokaryotic expression of pseudomonas aeruginosa Lipase gene.[Method]Lipase gene was amplified by PCR from the genome DNA of pseudomonas aeruginosa,and its nucleotide sequence was determined.The prokaryotic expression vector of Lipase gene was constructed by the gene recombination technique.The protein expression was induced for 4 hours by IPTG with the final concentration of 1.0 mmol/L,and then SDS-PAGE electrophoresis was analyzed.[Result]The sequence of mature peptides in Lipase gene cloned from pseudomonas aeruginosa had a 99.36% homology with that of pseudomonas aeruginosa lipase submitted in NCBI,so the prokaryotic expression vector of Lipase gene pET32a-Lip was successfully constructed.Furthermore,the results of SDS-PAGE electrophoresis showed that the target gene was expressed highly and effectively.[Conclusion]The cloned pseudomonas aeruginosa lipase with its signal peptide could be normally expressed in E.coli and also used for further study.

金黄色葡萄球菌FnBP配体结合区基因的克隆及其原核表达(英文)

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

牛瑟氏泰勒虫P23主要表面蛋白基因的克隆及原核表达(摘要)(英文)

[目的]研究牛瑟氏泰勒虫P23表面蛋白基因的克隆及原核表达。[方法]采用PCR方法扩增牛瑟氏泰勒虫中国延边株P23基因片段,将扩增产物克隆入pMD18-T载体构建重组质粒pMD18-P23经PCR、双酶切鉴定后测序;将目的基因片段亚克隆入表达载体pGEX-4T-1构建重组表达质粒pGEX-4T-P23,转化宿主菌BL21获得重组菌。通过对诱导条件的优化,根据SDS-PAGE确定表达蛋白的最佳表达条件;Western-blotting检测表达蛋白的反应原性。[结果]所克隆的牛瑟氏泰勒虫P23基因片段长507bp,与牛瑟氏泰勒虫日本株P23基因的核苷酸同源性达99.4%,表达的融合蛋白大小约为46ku;诱导时机以接种培养后2h为最佳,诱导时间以6h为最佳,诱导温度以34℃为最佳,0.008～1.000mmol/L的IPTG对表达量的影响不大。Western blotting检测表明该蛋白具有较好的抗原性。[结论]为牛瑟氏泰勒虫病的免疫学诊断和预防等研究奠定了基础。

The effects of soil properties, cropping systems and geographic location on soil prokaryotic communities in four maize production regions across China

The diversity of prokaryotic communities in soil is shaped by both biotic and abiotic factors.However,little is known about the major factors shaping soil prokaryotic communities at a large scale in agroecosystems.To this end,we undertook a study to investigate the impact of maize production cropping systems,soil properties and geographic location(latitude and longitude)on soil prokaryotic communities using metagenomic techniques,across four distinct maize production regions in China.Across all study sites,the dominant prokaryotes in soil were Alphaproteobacteria,Gammaproteobacteria,Betaproteobacteria,Gemmatimonadetes,Acidobacteria,and Actinobacteria.Non-metric multidimensional scaling revealed that prokaryotic communities clustered into the respective maize cropping systems in which they resided.Redundancy analysis(RDA)showed that soil properties especially pH,geographic location and cropping system jointly determined the diversity of the prokaryotic communities.The functional genes of soil prokaryotes from these samples were chiefly influenced by latitude,soil pH and cropping system,as revealed by RDA analysis.The abundance of genes in some metabolic pathways,such as genes involved in microbe–microbe interactions,degradation of aromatic compounds,carbon fixation pathways in prokaryotes and microbial metabolism were markedly different across the four maize production regions.Our study indicated that the combination of soil pH,cropping system and geographic location significantly influenced the prokaryotic community and the functional genes of these microbes.This work contributes to a deeper understanding of the composition and function of the soil prokaryotic community across large-scale production systems such as maize.

黑白花奶牛白细胞介素-2基因的克隆和表达(英文)

[Objective] The aim of this study is to clone bovine interleukin-2 gene(IL-2)and observe its expression in prokaryotic cells.[Method]Bovine IL-2 gene was amplified from total RNA of peripheral blood lymphocytes of holstein-friesian cows by RT-PCR.Subsequently,the gene was cloned into pGEX-2T prokaryotic expression plasmid to construct recombinant,which was then transformed into Escherichia Coli BL21.After IPTG induction,SDS-PAGE analysis was conducted.[Result]A 500 bp target fragment corresponding with expectation was obtained by RT-PCR.The cloned gene successfully expressed fusion protein of about 43 kD in prokaryotic cells.[Conclusion]This study provided a theoretical and material basis for further researches on IL-2 gene.

Screening of TACE Peptide Inhibitors from Phage Display PeptideLibrary

Summary： To obtain the recombinant tumor necrosis factor-α converting enzyme （TACE） ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain （TS00） and full-length ectodomain （T1300） of TACE were amplified by RT-PCR, and the expres.sion plasmids were constructed by inserting T800 and T1300 into plasmid pET-28a and pET-28c respectively. The recombinant TS00 and T1300 were induced by IPTG, and SDS-PAGE and Western blotting analysis results revealed that TS00 and T1300 were highly expressed in the form of inclusion body. After Ni^2＋-NTA resin affinity chromatography, the recombinant proreins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence （TRWLVYFSRPYLVAT） was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells （PBMC） up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE binding peptide is an effective antagonist of TACE.

Preparation of a polyclonal antibody against immunogenic fragment of bovine intestinal peptide transporter I (<i>b</i>PepTI)

The goal of the current study is to prepare polyclonal antibody against bovine intestinal peptide transporter I (bPepTI) in order to develop assay for immunological assessment of protein levels. Antigenicity of the entire bPepTI was analyzed with DNAStar, and a fragment with high antigenicity (bPepTI ORF 1369 - 1695) was selected, cloned in pGEX-6p-1 vector, resulting in a recombinant plasmid GST-BP, which verified by double enzyme digestion and sequenced, the recombinant plasmid was introduced to BL21. Exogenous expression was induced by IPTG and validated by Western blot analysis. The recombinant protein was isolated, purified and used for production of antiserum in mice. The specificity of antiserum was evaluated with immunobloting and titer was estimated with ELISA. Results indicate that the antibody against bPepTI was produced. The optimal GST-BP antigen embedding concentration was 0.5 μg/ml. The optimal dilution was 1:400. An indirect ELISA assay indicates the effective dilution was 1:102400.

Method for Tentative Evaluation of Membrane Permeability Coefficients for Sodium and Potassium Ions in Unicellular Organisms

The membrane permeability coefficient for sodium and potassium ions in unicellular organisms can be calculated using the data for cell volume, surface and mean generation time during growth and dividing of cells by binary. Accordingly theory of proposed method, the membrane permeability coefficients for passed trough outer cell membrane sodium and potassium ions, is equal to the volume of unicellular organism divided to product between cell surface and mean generation time of cells. The calculated by this way diapason of values overlaps with experimentally measured diapason of values of permeability coefficient for sodium and potassium ions. The deviation between the theoretically calculated and experimentally measured values of permeability coefficient does not exceed one order of magnitude.

Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase （LOX, EC1.13.11.12） is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a（＋） and transformed into Escherichia coil BL21 （DE3）. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside （IPTG） and the purified recombinant protein was obtained by His.Bind Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 （acetate buffer） and the optimum temperature was 30℃ for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.