Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co²⁺, Zn²⁺, and Mn²⁺. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Construction and Analysis of Photolyase Mutants of Pseudomonas aeruginosa and Pseudomonas syringae: Contribution of Photoreactivation, Nucleotide Excision Repair, and Mutagenic DNA Repair to Cell Survival and Mutability following Exposure to UV-B Radiation

Based on nucleotide sequence homology with the Escherichia coli photolyase gene (phr), the phr sequence of Pseudomonas aeruginosa PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known phr mutation following expression in Escherichia coli SY2. Stable, insertional phr mutants containing a tetracycline resistance gene cassette were constructed in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These mutants showed a decrease in survival compared to the wild type of as much as 19-fold after irradiation at UV-B doses of 1,000 to 1,550 J m⁻² followed by a recovery period under photoreactivating conditions. A phr uvrA mutant of P. aeruginosa PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 250 J m⁻². Complementation of the phr mutations in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 using the cloned phr gene from strain PAO1 resulted in a restoration of survival following UV-B irradiation and recovery under photoreactivating conditions. The UV-B survival of the phr mutants could also be complemented by the P. syringae mutagenic DNA repair determinant rulAB. Assays for increases in the frequency of spontaneous rifampin-resistant mutants in UV-B-irradiated strains containing rulAB indicated that significant UV-B mutability (up to a 51-fold increase compared to a nonirradiated control strain) occurred even in the wild-type PAO1 background in which rulAB only enhanced the UV-B survival by 2-fold under photoreactivating conditions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1 uvrA and uvrA phr backgrounds complemented with rulAB were 3.8 × 10⁻⁵ and 2.1 × 10⁻³, respectively, following a UV-B dose of 1,550 J m⁻². The construction and characterization of phr mutants in the present study will facilitate the determination of the roles of light and dark repair systems in organisms exposed to solar radiation in their natural habitats.     

Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7

Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10⁻⁸ ml/min and 1.91 × 10⁻⁸ ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10⁻² CFU/ml for S. Typhimurium and 4.58 × 10⁻⁵ CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.     

Pseudomonas linyingensis sp. nov.: A Novel Bacterium Isolated from Wheat Soil Subjected to Long-term Herbicides Application

A strain of genus Pseudomonas, LYBRD3-7^T was isolated from long-term sulfonylurea herbicides applied wheat-field soil in Linying located in Henan province of China. This strain is a strictly aerobic and Gram-negative short rod-shaped bacterium with single flagellum. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this isolate as a member of Pseudomonas, and most closely to Pseudomonas tuomuerensis CGMCC 1.1365^T (97.1?%) and P. alcaligenes IAM12411^T (97.1?%). Morphological characters and chemotaxonomic data confirmed the affiliation of strain LYBRD3-7^T to the genus Pseudomonas. The results of phylogenetic analysis, physiological and biochemical studies, and DNA–DNA hybridization allowed the differentiation of genotype and phenotype between strain LYBRD3-7^T and the phylogenetic closest species with valid names. The name proposed for the new species is Pseudomonas linyingensis sp. nov. The type strain is LYBRD3-7^T (=CGMCC 1.10701^T? =LMG 25967^T).     

Coralslurrinella hongkonensis gen. nov., sp. nov., a novel bacterium in the family Psychromonadaceae, isolated from the coral Platygyra carnosus

A novel bacterial strain, JLT2006^T, was isolated from the scleractinian coral Platygyra carnosus, located in Hong Kong, China. Cells of this strain were Gram-negative, rod-shaped or oval-shaped and motile by the means of polar flagella. They formed faint-yellow, round colonies on marine agar medium. Phylogenetic analyses based on the 16S rRNA gene sequence indicated that the strain JLT2006^T belonged to the class Gammaproteobacteria and was most closely related to Alteromonas-like bacteria of the genera Psychromonas, Pseudoalteromonas, Moritella, Shewanella and Ferrimonas, with less than 93 % sequence similarity. The predominant fatty acids were identified as C18:1ω7c/C18:1ω6c (23.0 %), C16:1ω7c/C16:1ω6c (18.2 %) and C16:0 (16.4 %). The quinone was menaquinone-7 (100 %). The polar lipids were determined to be phosphatidylglycerol, phosphatidylethanolamine, phospholipid, glycolipid and lipid. The genomic DNA G+C content was 40.3 mol%. Based on the 16S rRNA gene sequence as well as the physiological and biochemical features that separate the strain JLT2006^T from other recognized bacteria, a novel species of a new genus with the name Coralslurrinella hongkonensis gen. nov., sp. nov. is proposed. The type strain is JLT2006^T (=JCM 18796^T = CGMCC 1.10992^T).     

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.     

Lysinibacillus tabacifolii sp. nov., a novel endophytic bacterium isolated from Nicotiana tabacum leaves

A Gram-positive, catalase- and oxidase-positive, strictly aerobic, endospore-forming rod bacterium, designated K3514^T, was isolated from the leaves of Nicotiana tabacum. The strain was able to grow at temperatures of 8–40°C, pH 5.0–10.0 and NaCl concentrations of 0–7%. The predominant quinones (>30%) of this strain were MK-7(H2) and MK-7. Phylogenetic analysis of 16S rRNA gene sequence showed that strain K3514^T was affiliated to the genus Lysinibacillus, with its closest relatives being Lysinibacillus mangiferihumi (98.3% sequence similarity), Lysinibacillus sphaericus (97.9% sequence similarity), Lysinibacillus fusiformis (97.4% sequence similarity), and Lysinibacillus xylanilyticus (97.3% sequence similarity). However, low levels of DNA-DNA relatedness values suggested that the isolate was distinct from the other closest Lysinibacillus species. Additionally, based on analysis of morphological, physiological, and biochemical characteristics, the isolate could be differentiated from the closest known relatives. Therefore, based on polyphasic taxonomic data, the novel isolate likely represents a novel species, for which the name Lysinibacillus tabacifolii sp. nov. and the type strain K3514^T (=KCTC 33042^T =CCTCC AB 2012050^T) are proposed.     

Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons

The recessive lethal amber suppressor su⁺7(UAG-1) in Escherichia coli inserts glutamine in response to the UAG codon. The genetic analysis presented in this paper shows that the su^?7 precursor allele can give rise to suppressors of the UGA codon as well as of the UAG codon. This observation suggests that the su^?7 gene normally codes for transfer RNA^Trp, a tRNA whose anticodon can be modified by single base changes to forms that can translate either UAG or UGA. The chemical findings presented in the accompanying paper (Yaniv et al., 1974) are wholly in accord with this interpretation. Thus, a single base substitution in the anticodon sequence of a tRNA can affect both the coding specificity of the molecule and also the amino acid acceptor specificity.     

Diaminobutyricibacter tongyongensis gen. nov., sp. nov. and Homoserinibacter gongjuensis gen. nov., sp. nov. Belong to the Family Microbacteriaceae

Two bacterial strains, KIS66-7^T and 5GH26-15^T, were isolated from soil samples collected in the South Korean cities of Tongyong and Gongju, respectively. Both strains were aerobic, Gram-stain-positive, mesophilic, flagellated, and rodshaped. A phylogenetic analysis revealed that both strains belonged to the family Microbacteriaceae of the phylum Actinobacteria. The 16S rRNA gene sequence of strain KIS66-7^T had the highest similarities with those of Labedella gwakjiensis KSW2-17^T (97.3%), Cryobacterium psychrophilum DSM 4854T (97.2%), Leifsonia lichenia 2Sb^T (97.2%), Leifsonia naganoensis JCM 10592^T (97.0%), and Cryobacterium mesophilum MSL-15^T (97.0%). Strain 5GH26-15^T showed the highest sequence similarities with Leifsonia psychrotolerans LI1T (97.4%) and Schumannella luteola KHIAT (97.1%). The 16S rRNA gene sequence from KIS66-7^T exhibited 96.4% similarity with that from 5GH26-15^T. Strain KIS66-7^T contained a B2γ type peptidoglycan structure with D-DAB as the diamino acid; MK-13, MK-12, and MK-14 as the respiratory quinones; ai-C_15:0, ai-C_17:0, and i-C_16:0 as the major cellular fatty acids; and diphosphatidylglycerol, phatidylglycerol, and glycolipids as the predominant polar lipids. Strain 5GH26-15T had a B2β type peptidoglycan structure with D-DAB as the diamino acid; MK-14 and MK-13 as the respiratory quinones; ai-C_15:0, i-C_16:0, and ai-C{vn17:0} as the major cellular fatty acids; and diphosphatidylglycerol, phatidylglycerol, and glycolipids as the predominant polar lipids. Both strains had low DNA-DNA hybridization values (<40%) with closely related taxa. Based on our polyphasic taxonomic characterization, we propose that strains KIS66-7^T and 5GH26-15^T represent novel genera and species, for which we propose the names Diaminobutyricibacter tongyongensis gen. nov., sp. nov. (type strain KIS66-7^T=KACC 15515^T=NBRC 108724^T) and Homoserinibacter gongjuensis gen. nov., sp. nov. (type strain 5GH26-15^T=KACC 15524^T=NBRC 108755^T) within the family Microbacteriaceae.     

Physical Map of the Channel Catfish Virus Genome: Location of Sites for Restriction Endonucleases EcoRI, HindIII, HpaI, and XbaI

The overall arrangement of nucleotide sequences in the DNA of channel catfish virus has been studied by cleavage with four restriction endonucleases. Physical maps have been developed for the location of sites for EcoRI, HindIII, HpaI, and XbaI. The sum of the molecular weights of fragments generated by each restriction enzyme indicates a molecular weight of approximately 86 × 10⁶ for the channel catfish virus genome. Fragments corresponding to the molecular ends of channel catfish virus DNA have been identified by their sensitivity to exonuclease treatment. The distribution of restriction sites in the genome shows that sequences included in a 12 × 10⁶-molecular weight region at one end are repeated with direct polarity at the other end, and that the overall genomic sequence order is nonpermuted.     

Azospirillum himalayense sp. nov., a nifH bacterium isolated from Himalayan valley soil,India

Gram-negative, free-living bacterial strain ptl-3^T was isolated from Himalayan valley soil, India. Polyphasic taxonomy was performed including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analyses. 16S rRNA gene sequence analysis showed that the strain ptl-3^T belonged to the genus Azospirillum and was closely related to A. brasilense (98.7 % similarity) and A. rugosum (97 % similarity). 16S rRNA gene sequence similarity (96–95 %) was shown with other members of the genus Azospirillum. Major fatty acid 18:1ω7c was also similar to the genus Azospirillum. DNA–DNA relatedness value between strain ptl-3^T and A. brasilense was found to be 47 %. Various biochemical tests showed that the strain ptl-3^T differed from its closely related species A. brasilense. On the basis of phenotypic, chemotaxonomic and molecular genetics evidence, a bacterium with the type strain ptl-3^T is proposed as a novel species of the genus Azospirillum. The name of bacterial strain ptl-3^T has been proposed as Azospirillum himalayense sp. nov. The type strain of ptl-3^T (CCUG 58760^T, KCTC 23189^T) has been submitted to two culture collection centres. The accession numbers for 16S rRNA and nifH gene are GQ 284588 and GQ 249665. respectively.     

Paludibacter jiangxiensis sp. nov., a strictly anaerobic,propionate-producing bacterium isolated from rice paddy field

A mesophilic, obligately anaerobic, propionate-producing fermentative bacterium, designated strain NM7^T, was isolated from rural rice paddy field. Cells of strain NM7^T are Gram-negative, non-motile, non-spore-forming, short rods, and negative for catalase. The strain grew optimally at 37 °C (the range for growth 15–40 °C) and pH 7.0 (pH 5.0–7.5). The strain could grow fermentatively on various sugars, including arabinose, xylose, fructose, galactose, glucose, mannose, cellobiose, lactose, maltose, sucrose, pectin and starch. The main end products of glucose fermentation were acetate and propionate. Yeast extract was not required but stimulated the growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, and Fe(III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of genomic DNA was 42.8 mol%. The major cellular fatty acids were C_15:0, anteiso-C_15:0, C_16:0, and C_17:0. The most abundant polar lipid of strain NM7^T was phosphatidylethanolamine. 16S rRNA gene sequence analysis revealed that it belongs to the family Porphyromonadaceae of the phylum Bacteroidetes. The closest recognized species was Paludibacter propionicigenes (91.4 % similarity in 16S rRNA gene sequence). A novel species, Paludibacter jiangxiensis sp. nov., is proposed to accommodate strain NM7^T (=JCM 17480^T = CGMCC 1.5150^T = KCTC 5844^T).     

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process^1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon⁶. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site^7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes⁷. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains^10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.     

Algoriphagus shivajiensis sp. nov., isolated from Cochin back water,India

Novel orange-pigmented, Gram-negative, rod-shaped, non-motile bacteria, designated strains NIO-S3^T and NIO-S4, were isolated from a water sample collected from Cochin back waters, Thanneermukkom and Arookutty, Kerala, India. Both strains were positive for oxidase and catalase activities, and hydrolyzed gelatin and Tween 40. The predominant fatty acids were iso-C_15:0, anteiso-C_15:0, iso-C_17:0 3OH, C_16:1ω7c/C_16:1ω6c (summed feature 3) and iso-C_17:1ω9c/C_16:0 10-methyl (summed feature 9), whereas MK-7 was the major respiratory quinone, and phosphatidylethanolamine, two unidentified phospholipids and one unidentified lipid were the only polar lipids. The DNA G+C content of the two strains was 43.7 and 43.6 mol%, respectively. The 16S rRNA gene sequence analysis indicated that they were members of the genus Algoriphagus and closely related to Algoriphagus olei CC-Hsuan-617^T, Algoriphagus aquatilis A8-7^T, Algoriphagus aquaeductus LMG 24398^T and Algoriphagus mannitolivorans DSM 15301^T, with pairwise sequence similarities of 96.8, 96.6, 96.2 and 96.2%, respectively. DNA–DNA hybridization between strains NIO-S3^T and NIO-S4 showed a relatedness of 89%. Based on data from the current polyphasic study, the strains are proposed as a novel species of the genus Algoriphagus, for which the name Algoriphagus shivajiensis sp. nov. is proposed. The type strain of A. shivajiensis is NIO-S3^T (=JCM 17885^T = MTCC 11066^T).     

A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines

The Cdc7 protein kinase of Saccharomyces cerevisiae is a critical regulator of several aspects of DNA metabolism and cell cycle progression. We describe the isolation of a human gene encoding a Cdc7 homolog. The Cdc7^Hs protein sequence is 27% identical to that of the yeast protein, includes features unique to yeast Cdc7, and contains all conserved catalytic residues of protein kinases. The human sequence also shows significant similarity to the cyclin-dependent kinases, in accordance with evidence that yeast Cdc7 is related to the cdks. CDC7^Hs is expressed in many normal tissues, but overexpressed in certain tumor types and all transformed cell lines examined. In some of the tumors tested, CDC7^Hs expression correlates with expression of a proliferation marker, the histone H3 gene. In other cases, no such correlation was observed. This suggests that CDC7^Hs expression may be associated with hyperproliferation in some tumors and neoplastic transformation in others.     

Aliiglaciecola coringensis sp. nov., isolated from a water sample collected from mangrove forest in Coringa,Andhra Pradesh,India

A Gram-negative, rod shaped, motile, aerobic bacterium, designated as strain AK49^T was isolated from a water sample from a mangrove forest in Coringa village, Andhra Pradesh, India. Strain AK49^T was observed to form yellow coloured, smooth, circular, convex colonies on marine agar, with entire margins. Cells of strain AK49^T are 0.5–1.0 µm wide and 1.5–3.5 µm long. Growth was observed at 25–37 °C (optimum 30 °C), 2–6 % NaCl (optimum 2 %) and pH 6–8 (optimum 7). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain AK49^T is closely related to two species recently reclassified as members of the genus Aliiglaciecola: Aliiglaciecola lipolytica JCM 15139^T (sequence similarity 95.43 %) and Aliiglaciecola litoralis JCM 15896^T (sequence similarity 96.91 %). The major cellular fatty acids of strain AK49^T were found to include C_16:0, C_18:1ω7c and summed feature 3 (C_16:1ω7c/C_15:0 iso-2-OH). The polar lipid content of cell membrane was found to include phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid, an unidentified lipid and an unidentified glycolipid. The genomic DNA G+C content of strain AK49^T was determined to be 41.9 mol%. Based on the taxonomic methods, including chemotaxonomic, phenotypic and phylogenetic approaches, strain AK49^T is described here as a novel species belonging to the genus Aliiglaciecola, for which the name Aliiglaciecola coringensis sp. nov. is proposed. The type strain of Aliiglaciecola coringensis sp. nov. is AK49^T (=MTCC 12003^T = JCM19197^T).     

Distinct Palmitoylation Events at the Amino-terminal Conserved Cysteines of Env7 Direct Its Stability,Localization, and Vacuolar Fusion Regulation in S. cerevisiae

Palmitoylation at cysteine residues is the only known reversible form of lipidation and has been implicated in protein membrane association as well as function. Many palmitoylated proteins have regulatory roles in dynamic cellular processes, including membrane fusion. Recently, we identified Env7 as a conserved and palmitoylated protein kinase involved in negative regulation of membrane fusion at the lysosomal vacuole. Env7 contains a palmitoylation consensus sequence, and substitution of its three consecutive cysteines (Cys¹³–Cys¹⁵) results in a non-palmitoylated and cytoplasmic Env7. In this study, we further dissect and define the role(s) of individual cysteines of the consensus sequence in various properties of Env7 in vivo. Our results indicate that more than one of the cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane localization, and phosphorylation. Furthermore, individually, each cysteine can serve as a minimum requirement for distinct aspects of Env7 behavior and function in cells. Cys¹³ is sufficient for membrane association, Cys¹⁵ is essential for the fusion regulatory function of membrane-bound Env7, and Cys¹⁴ and Cys¹⁵ are redundantly essential for protection of membrane-bound Env7 from proteasomal degradation. A role for Cys¹⁴ and Cys¹⁵ in correct sorting at the membrane is also discussed. Thus, palmitoylation at the N-terminal cysteines of Env7 directs not only its membrane association but also its stability, phosphorylation, and cellular function.     

Maize Yield Response to Water Supply and Fertilizer Input in a Semi-Arid Environment of Northeast China

Maize grain yield varies highly with water availability as well as with fertilization and relevant agricultural management practices. With a 311-A optimized saturation design, field experiments were conducted between 2006 and 2009 to examine the yield response of spring maize (Zhengdan 958, Zea mays L) to irrigation (I), nitrogen fertilization (total nitrogen, urea-46% nitrogen,) and phosphorus fertilization (P₂O₅, calcium superphosphate-13% P₂O₅) in a semi-arid area environment of Northeast China. According to our estimated yield function, the results showed that N is the dominant factor in determining maize grain yield followed by I, while P plays a relatively minor role. The strength of interaction effects among I, N and P on maize grain yield follows the sequence N+I >P+I>N+P. Individually, the interaction effects of N+I and N+P on maize grain yield are positive, whereas that of P+I is negative. To achieve maximum grain yield (10506.0 kg·ha⁻¹) for spring maize in the study area, the optimum application rates of I, N and P are 930.4 m³·ha⁻¹, 304.9 kg·ha⁻¹ and 133.2 kg·ha⁻¹ respectively that leads to a possible economic profit (EP) of 10548.4 CNY·ha⁻¹ (CNY, Chinese Yuan). Alternately, to obtain the best EP (10827.3 CNY·ha⁻¹), the optimum application rates of I, N and P are 682.4 m³·ha⁻¹, 241.0 kg·ha⁻¹ and 111.7 kg·ha⁻¹ respectively that produces a potential grain yield of 10289.5 kg·ha⁻¹.     

Burkholderia denitrificans sp. nov., isolated from the soil of Dokdo Island, Korea

A novel, Gram-negative, bacterial strain KIS30-44^T was identified from wet forest soil collected on the Korean island of Dokdo. Growth of the strain was observed at 15?C30°C, pH 5?C9, 0?C3% NaCl, and 950 mM KNO₃. KIS30-44^T reduced nitrate to nitrogen gas. Analysis of the 16S rRNA gene sequence showed that KIS30-44^T was phylogenetically related to Burkholderia sacchari, Burkholderia mimosarum, and Burkholderia oxyphila (98.1%, 98.0%, and 98.0% sequence similarity, respectively). The genomic G+C content was 63.5 mol%. KIS30-44^T exhibited less than 52% DNA-DNA relatedness with the type strains of 9 closely related Burkholderia species. The major isoprenoid quinone was Q-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminolipids. The major fatty acids in KIS30-44^T were C_16:0, C_18:1 ??7c and summed feature 3 (iso-C_15:0 2-OH and C_16:1 ??7c), and the strain contained half the amount of C_17:0 cyclo found in the 9 closely related Burkholderia species. The results of these phenotypic, 16S rRNA gene sequence, DNA-DNA hybridization, and chemotaxonomic data indicate that KIS30-44^T represents a novel species within the genus Burkholderia, for which the name Burkholderia denitrificans (Type strain KIS30-44^T =KACC 12733^T =DSM 24336^T) is proposed.     

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 10⁶ CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10⁴ to 10⁷ CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.