Penicillin-binding protein 4 of Escherichia coli shows a novel type of primary structure among penicillin-interacting proteins

The nucleotide sequence of a 1884 bp DNA fragment of E. coli, carrying the gene dacB, was determined. The DNA codes for penicillin-binding protein 4 (PBP4), an enzyme of 477 amino acids, being involved as a DD-carboxypeptidase-endopeptidase in murein metabolism. The enzyme is translated with a cleavable signal peptide of 20 amino acids, which was verified by sequencing the amino-terminus of the isolated protein. The characteristic active-site fingerprints SXXK, SXN and KTG of class A beta-lactamases and penicillin-binding proteins were located in the sequence. On the basis of amino acid alignments we propose, that PBP4 and class A beta-lactamases share a common evolutionary origin but PBP4 has acquired an additional domain of 188 amino acids in the region between the SXXK and SXN elements.     

Associations of amino acids and related compounds in the seeds of forty-seven species of Vicia: their taxonomic and nutritional significance

1. The genus Vicia may be subdivided into groups of species characterized by associations of ninhydrin-positive compounds occurring in high concentrations in their seeds. Despite these subdivisions the overall distribution pattern emphasizes the unity of the genus and the possible value of such studies in establishing degrees of relationship between species within the genus. 2. Canavanine is a major constituent in the seeds of 17 species. 3. gamma-Hydroxyarginine, an amino acid that has not been found in other plant genera, occurs in 18 species. 4. gamma-Hydroxyornithine, a new natural amino acid, is found in two species. 5. A new naturally occurring ureido amino acid tentatively identified as ;gamma-hydroxycitrulline' is found in two species. 6. High concentrations of alphabeta-diaminopropionic acid are found in seed of V. baicalensis and of alphagamma-diaminobutyric acid in seed of V. aurantica. 7. The neurotoxic amino acid beta-cyanoalanine and its gamma-glutamyl peptide are found together in high concentrations in the seeds of 16 species of this agriculturally important genus. 8. Seven unidentified ninhydrin-positive compounds occur in high concentration (about 1% of the dry weight or more) in the seed of various species. Details of their R(F) values, ionic mobilities and colour reactions are given. 9. The total concentration of extractable ninhydrin-positive compounds varies little between seeds of different species and these compounds may, as has been suggested for those in Lathyrus, constitute a readily available form of storage nitrogen. 10. The nature and distribution, as opposed to the total concentration, of the amino acids and related compounds accumulated in the seeds of Vicia are different from those accumulated in the seeds of the related genus, Lathyrus. One particularly interesting difference is the accumulation of C(6) guanidino amino acids (arginine and gamma-hydroxyarginine) by Vicia and the accumulation of C(7) guanidino amino acids (homoarginine, gamma-hydroxyhomoarginine and the related amino acid lathyrine) by Lathyrus. Such differences afford a method for distinguishing between species of these genera.     

Amino acids in seeds and seedlings of the genus Lens

The amino acid content of seeds and 4-day-old seedlings were studied in five species of lentil: Lens culinaris, L. orientalis, L. ervoides, L. nigricans and L. odemensis. Free amino acid and also total protein amino acid content after HCl hydrolysis were determined by HPLC. The nonprotein UV-absorbing amino acids were determined by capillary zone electrophoresis (CZE). The content of free protein amino acids in seeds varied among species and increased dramatically after germination. Asparagine is quantitatively most important in both seed and seedling. The content of free nonprotein amino acids is variable in seeds and seedlings. gamma-Hydroxyarginine, gamma-hydroxyornithine, alpha-aminobutyric acid and taurine were found in both seeds and seedlings. Homoarginine was found in four species but not in L. orientalis while gamma-aminobutyric acid (GABA), alpha-aminoadipic acid (alpha-aaa) and three isoxazolinone derivatives: beta-(isoxazolin-5-on-2-yl)-alanine (BIA), gamma-glutamyl-BIA (gamma-glu-BIA) and 2-carboxymethyl-isoxazolin-5-one (CMI) were found exclusively in the seedlings. CMI was identified for the first time in lentil species. Lathyrine, beta-(2-amino-pyrimidine-4-yl)-alanine, which was reported to be in the seeds of some Lathyrus species was confirmed to be present also in the seedling of L. culinaris (trace amount), L. nigricans and L. odemensis. Trigonelline (N-methyl-nicotinic acid), a plant hormone, is present both in seeds and seedlings in different concentrations except in L. ervoides. The different combination of nonprotein amino acids among the species gives indication of their genetic relationship and might partly explain the varying compatibility for interspecies crossing.     

Free amino acids in Crotalaria seeds

The free amino acids of seeds of 163 species of Crotalaria have been identified. Their pattern of distribution is compared with recent classifications of the genus and the possible ecological significance of their presence is discussed. Attention is drawn to the occurrence of toxic amino acids.     

The presence of two types of β-cyanoalanine synthase in germinating seeds and their responses to ethylene

Germinating seeds of many species contain two types of β-cyanoalanine synthase (CAS, EC 4.4.1.9) that convert HCN to β-cyanoalanine. One is cytoplasmic CAS (cyt-CAS), which is precipitated by 50 to 60% (NH₄)₂SO₄ and has a pH optimum of 10.5. Cytoplasmic CAS is present at high levels in dry seed and its activity does not increase during imbibition. The activity of cyt-CAS is not affected by exogenously applied ethylene (C₂H₄), except in rice ( Oryza sativa cv. Sasanishiki). The second type of CAS found in seed is mitochondrial CAS (mit-CAS), which is precipitated by 60 to 70% (NH₄)₂SO₄ and has a pH optimum of 9.5. Mitochondrial CAS is present at low levels in dry seed, and its activity increases greatly during imbibition in the seeds of all species tested. Exposure to C₂H₄ stimulated mit-CAS activity in seeds of rice, barley ( Hordeum vulgare cv. Hadakamugi). cucumber ( Cucumis sativus cv. Kagafushinari) and cocklebur ( Xanthium pennsylvanicum ). The increase in the mit-CAS activity in cocklebur in response to C₂H₄ commenced alter a lag period of 2 to 3 h when the duration of soaking was short (16 h), but commenced without a lag period when the seeds were soaked for three months. Application of both chloramphenicol and cycloheximide to the axial and cotyledonary tissues of cocklebur seeds strongly inhibited growth as well as the increase in mit-CAS activity. It is postulated that the mit-CAS is synthesized de novo during imbibition and that its activity is regulated by C₂H₄, CO₂ which also promotes seed germination in some species, was ineffective m stimulating mit-CAS activity in cocklebur seeds.     

Valeur trophique des acides aminés dans les graines de légumineuses et développement larvaire de Callosobruchus maculatus

Interspecific variation of protein amino acids between peanut, horse bean, french bean, lentil, cowpea and chick-pea was undertaken in relation to larval development of C. maculatus. Twenty-one groups of amino acids were identified and quantified. In all seeds, free amino acids exist in low quantities; but they are more numerous than bound amino acids. Essential amino acids for man are sufficient in the six species, except for the sulphur amino acids in all seeds, and for lysine in peanut. Utilization of those pulses, after cooking, in human food complements cereals. Larval development possibilities of C. maculatus upon the used seeds shows up that: (a) with chick-pea and cowpea, the bruchid thrives normally. However, although amino acids are higher in cowpea than in chick-pea, the better performances were observed on the latter; (b) peanut, french bean, horse bean and lentil are unsuitable for larval development of the insect. Although they are rich in amino acids, all larvae die at first in star within these pulses. We conclude that larval development of C. maculatus does not depend only on the amounts of amino acids in the seeds but also on other trophic factors. These could consist of the balance between different nutritive compounds and the presence of allelochemical substances in the tested seeds which bruchid larvae are unable to utilize.     

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a `hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.     

Chemical differences between seeds and elaiosomes indicate an adaptation to nutritional needs of ants

Ant-dispersed plants usually produce seeds with appendages (elaiosomes) as reward for ants. Plants that produce high-quality elaiosomes benefit because ants preferentially disperse their diaspores. We therefore hypothesized that seeds and elaiosomes differ in chemical composition in ways that make elaiosomes of high nutritional quality for ants, capable of providing essential dietary components that explain the increased fitness and higher gyne production documented for colonies with elaiosome consumption. To test the hypothesis we analysed the content and composition of lipids, amino acids, soluble carbohydrates, proteins and starch in seeds and elaiosomes of 15 central European ant-dispersed plants. After separating the different fractions, total lipids were determined gravimetrically, fatty acids and soluble carbohydrates were detected by gas chromatography (GC) and GC–mass spectrometry, free amino acids by an amino acid analyser while starch and protein were analysed photometrically. Seeds accumulated high molecular weight compounds such as proteins and starch, whereas elaiosomes accumulated more easily digestible low molecular weight compounds such as amino acids and monosaccharides. Analysis of similarities and similarity percentages analysis demonstrated that the composition of fatty acids, free amino acids and carbohydrates differed markedly between elaiosomes and seeds. The most important difference was in total amino acid content, which was on average 7.5 times higher in elaiosomes than in seeds. The difference was especially marked for the nitrogen-rich amino acid histidine. The availability of essential nutrients and, in some species, the higher nitrogen content in elaiosomes suggest that their nutritional value for larvae plays a key role in this interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amino acids of Caesalpinia tinctoria and some allied species

Two previously unreported amino acids have been found to form major constituents of seed of Caesalpinia tinctoria; they have been characterized as 4-hydroxy-3-hydroxymethylphenylalanine and 3-hydroxymethylphenylalanine. The seed also contains a large amount of the imino acid baikiain. The amino acid content of seeds of this species is compared with that for 12 other species of Caesalpinia and 14 species in closely related genera.     

Insertion of a 27 amino acid viral peptide in different zones of Escherichia coliβ-galactosidase: Effects on the enzyme activity

Abstract Seven internal, putatively exposed regions of Escherichia coli β-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides. Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique Bam HI restriction sites. By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme. Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities. The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate incorporate and study biological properties of heterologous peptides in correctly folded β-galactosidase chimeric proteins.     

华南地区桑园和荒地两种生境中红火蚁觅食工蚁的食物组成

【目的】红火蚁Solenopsis invicta Buren是世界最危险的有害入侵生物之一,2004年入侵我国华南地区,并给农林业安全、生态安全带来极大危害。调查并明确田间红火蚁觅食工蚁的食物种类及数量,不仅为评估红火蚁猎食对生态环境中节肢动物群落的影响,而且为红火蚁防治饵剂的改进提供科学依据。【方法】本研究采用蚁道剖析法,对华南地区桑园和荒地两种生境中红火蚁觅食工蚁搬运的固体残片取样和鉴定;依搬运工蚁及其固体残片尺寸进行测量分级并称重,以分析工蚁多态性与固体食物大小的关系;采用薄层色谱法和氨基酸分析法测定嗉囊液体样品的营养成分及含量。【结果】在华南地区桑园和荒地中,红火蚁觅食工蚁搬运回巢的固体食物包括固体的动物残片和植物种子,其中动物残片属于3门7纲21个类群(包括14个昆虫目),昆虫纲动物所占比例为总固体食物的45.53%~46.10%。工蚁偏好搬运长1.400±0.043 mm~2.306±0.063 mm和宽0.723±0.028 mm~1.261±0.051 mm的固体残片,其重量在0.203~0.413 mg之间。红火蚁觅食工蚁嗉囊液体由多种氨基酸、果糖和葡萄糖组成,在桑园和荒地采集到的红火蚁嗉囊液体样本中的氨基酸总含量分别为1 544.31 mg/L和861.48 mg/L,氨基酸种类分别为33种和32种,其中31种氨基酸为共有的。【结论】华南地区桑园和荒地中,红火蚁固体食物组成均以昆虫纲动物为主;参与固体残片搬运觅食蚁的80%属于中型工蚁,搬运较大型固体残片的大型工蚁仅占5%;红火蚁工蚁嗉囊液体含有丰富的氨基酸和单糖。     

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.     

Sequence diversity of beta-tubulin (tubA) gene in Phaeosphaeria nodorum and P. avenaria

Full-length coding sequences of the beta-tubulin gene (tubA) were PCR-amplified and sequenced from 42 Phaeosphaeria isolates, including 16 P. nodorum and 23 P. avenaria species from cereals, two Polish isolates from rye (Secale cereale L.), and one isolate from dallis grass (Paspalum dilatatum Poir). A tubA gene of size 1556bp was identified in wheat- and barley-biotype P. nodorum (PN-w and PN-b), P. avenaria f. sp. avenaria (Paa), homothallic P. avenaria f. sp. triticea (P.a.t.) (Pat1) and the P.a.t. isolate (Pat3) from the State of Washington. The tubA gene length polymorphisms were detected in two P.a.t. isolates (Pat2) from foxtail barley (Hordeum jubatum L.), one from dallis grass and two Polish isolates from rye. These size differences were due to the variation of intron lengths among these three Phaeosphaeria species. All Phaeosphaeria isolates have identical 1344bp exons that can be translated into a 447 amino acid beta-tubulin. Like glyceraldehyde-3-phosphate dehydrogenase, the beta-tubulin amino acid sequence was identical in all Phaeosphaeria species used in this study, with the exception of the two Pat2 isolates. Six amino acid differences were evident in the beta-tubulin of these Pat2 isolates.     

Chemical Constituents of <i>Pedicularis longiflora</i> var. <i>tubiformis</i> (Orobanchaceae), a Common Hemiparasitic Medicinal Herb from the Qinghai Lake Basin,China

Pedicularis longiflora var. tubiformis (Orobanchaceae) is an abundant parasitic herb mainly found in the Xiaopohu wetland of the Qinghai Lake Basin in Northwestern China. The species has an important local medicinal value, and in this study, we evaluated the chemical profile of its stems, leaves and seeds using mass spectrometry. Dried samples of stems, leaves and seeds were grinded, weighted, and used for a series of extractions with an ultrasonic device at room temperature. The chemical profiles for each tissue were determined using Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid ChromatographyMass Spectrometry (LC-MS). Twenty-seven amino acids and organic acids were identified and quantified from stems, leaves and seeds. The content of amino acids detected in leaves and seeds was higher than the amount found in stems. Six flavonoids were also detected, including isoorientin, orientin, luteolin-7-O-glucoside, luteolin, apigenin and tricin. The concentrations of luteolin-7-O-glucoside, luteolin and tricin were the highest and more concentrated in leaves, while that of orientin was the lowest and mainly found in stems. Soluble monosaccharides and oligosaccharides below tetramer were also examined, and our analyses detected the presence of arabitol, fructose, galacturonic acid, glucose, glucuronic acid, inositol, sucrose, and trehalose. This is the first study to identify and quantify the main components of amino acids, organic acids, flavonoids and soluble sugars from stems, leaves and seeds of P. longiflora var. tubiformis. Eight of the amino acids detected are essential for humans, highlighting the medicinal importance of this species. Results shown here can be used as a reference case to develop future studies on the chemical constituents of Pedicularis herbs and other medicinal plants from the Tibetan region.     

‘Uncommon’ amino acids in the seeds of 64 species of Caesalpinieae

Characteristic associations of free amino acids occur in the seeds of various groups of species within the Caesalpinieae. Guilandina species are distinctive in accumulating 4-ethylideneglutamic acid in their seeds, Gymnocladus and Gleditzia species in accumulating isomers of 3-hydroxy-4-methylglutamic acid, Bussea species in accumulating azetidine-2-carboxylic acid, Peltophorum species in accumulating a previously undescribed imino acid tentatively identified as a derivative of 4-hydroxypipecolic acid.     

β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

Glaucacyclopeptide A from the seeds of Annona glauca

The seeds of Annona glauca furnished two cyclopeptides one of which is novel. The structure was elucidated on the basis on mass spectrometry, 2D NMR methods and amino acids analysis.     

2-amino-4-acetylaminobutyric acid, 2,4-diaminobutyric acid and 2-amino-6N-oxalylureidopropionic acid (oxalylalbizziine) in seeds of acacia angustissima

A new amino acid previously detected in 17 species of Acacia has been isolated from seeds of Acacia angustissima and identified as oxalylalbizziine. These seeds also contain more than 6% dry weight of 2-amino-4-acetylaminobutyric acid, which has not been reported previously in a legume, and lower concentrations of 2,4-diaminobutyric acid.     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality

Beyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20-90 min for standard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.