Exposure to alcohol vapours reduces chilling-induced injury of excised cucumber cotyledons, but not of seedlings or excised hypocotyl segments

Cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings withfully expanded cotyledons, and excised cotyledons, first trueleaves, hypocotyl segments and fruit mesocarp discs were exposedto vapours from a series of aqueous alcohol solutions of 0 to320 mM methanol, ethanol, n-propanol, n-butanol, and n-pentanolduring chilling at 2.5C for 5 d. Certain concentrations ofeach alcohol reduced subsequent chilling-induced ion leakagefrom the cotyledons and leaves. Exposure of cotyledons to certainmethanol or ethanol solutions also reduced chilling-inducedethylene production, but not carbon dioxide production. In contrast,exposing cucumber seedlings with fully expanded cotyledons tothe same series of alcohol concentrations that resulted in reducedchilling-induced ion leakage and ethylene production of excisedcotyledons actually increased chilling injury of the seedlings.The hypocotyl region directly below the cotyledons was the siteof chilling-induced injury and contained the most chilling-sensitivehypocotyl tissue. Exposing hypocotyl segments excised from thissensitive region to alcohol solutions did not significantlyreduce chilling-inducedion leakage. Exposing excised cucumbercotyledons or hypocotyl segments to equivalent osmotic nonvolatilesolutions of mannitol and glycerol at 2.5C or to alcohol solutionsat 12.5C had no significant effect on the rate of ion leakage.For the series of alcohols used, the relationship between thelog of the alcohol concentration that minimized chilling-inducedion leakage from cucumber cotyledon discs held at 2.5C for5 d and the log of the partition coefficient of the alcoholinto olive oil or the log of the molecular weight of the alcoholswas highly significant. The same concentrations of alcoholsthat reduced chilling-inducedion leakage also reduced stomatalaperture as measured as decreased porosity of excised cotyledons.The correlation between reduced chilling injury and stomatalconductance of cotyledons exposed to a series of ethanol solutionswas highly significant. It appears that alcohols may reducechilling injury of cucumber cotyledons by inducing stomata closure.Sufficient endogenously synthesized ethanol accumulated in discsheld in N2 at 10C for 1 d to confer tolerance to chilling at2.5C for 5 d. Key words: Anaerobic, Cucumis sativus, ethanol, ion leakage, stomatal conductance     

ERRATA

The legend to Figure 3 was incomplete. The correct legend isgiven below:Fig. 3. IF results for the Gulf of Maine and adjacentslope in the surface (top row), top of thermocline (second row),chlorophyll maximum (third row) and at the base of the thermocline(fourth row). Along the x-axis, stations are plotted in orderof approximate distance from the shore (see Figure 1). The y-axescorrespond to: (A) total eukaryotic algae counts; (B) % of totalcounts labeled by antiserum to E.huxleyi (clone BT6); (C) %of total counts labeled by antiserum to P.provasolii (48-23);(D) % of total counts labeled by antiserum to P.subviridis (clonePELA CL2); (E) % of total counts labeled by antiserum to T.oceanka(clone 13-1); (F) % of total counts labeled by antiserum tochlorophyte clone B6125. * = not available. The last sentence of page 46 should read: We attribute this to a loss of eukaryotic algal cells such thatin stored samples the percentage labeled may be overestimated.     

Physiology of Post-Pollination Exudate Production in Acacia

Marginson, R., Sedgley, M. and Knox, R. B. 1985. Physiologyof post-pollination exudate production in Acacia—J. exp.Bot. 36: 1660–1668. Stigmas of A. wattsiana produced 1260 nl of secretion by 90min after pollination. The optimum temperature for post-pollinationsecretion in A. baileyana, A. brownii and A. iteaphylla was20°C. Both self and cross intraspecific, interspecific andintergeneric pollinations produced a similar positive responsein A. iteaphylla and A. baileyana. In all cases aged pollenwas as effective as fresh pollen despite reduced fluorescencein the fluorescein diacetate test. Live yeast cells, fixed chickenerythrocytes, glass beads, talc and Biogel P150 did not stimulatesecretion, nor did pollen which had been washed in water orethanol despite high retention of viability in some cases Pollendiffusates dried on to glass beads produced a positive responsein A. iteaphylla and A. gracifolia. Intraspecific and interspecificpollinations involving fresh Acacia pollen resulted in pollengermination on the stigma and pollen tube growth in the styleof A. iteaphylla and A. baileyana. In contrast washed pollenfailed to germinate and pollen germination and tube growth werereduced at 35 °C as compared with 20 °C. Key words: Acacia, pollination, secretion     

In vivo chlorophyll forms in higher plants and algae sensitive to treatment with lutein

Lamella preparations of spinach, Chlorella, Phaeodactylum, Anabatnaand Porphyra were treated with a hydrophobic reagent, lutein,and the absorption and fluorescence spectra in the red regionbefore and after treatment were compared for changes causedby the treatment. Absorption spectra of all these preparationsunderwent the same spectral change, transformation of a bandat 684 nm into a band at 666 nm. The longer the maximum wavelengthof the red peak, the greater was the fractional absorbance decreaseat 684 nm. The content of C684 (the chlorophyll form responsiblefor the 684 nm band) in the lamellae was estimated from thefractional decreases as being progressively higher in the orderof Phaeodactylum, Porphyra, Anabatna, Chlorella and spinach.The fluorescence spectra at liquid nitrogen temperature beforetreatment showed two bands. The longer wavelength band was transformedby the treatment into a shorter wavelength band(s), as describedbelow, according to the maximum wavelengths: spinach, F735F695(or F686); Chlorella, F715F700 (or F686); Phaeodactylum, anunidentified componentF690; Anabaena, F732F685 (or F695); Porphyra,F726F683. These chlorophyll forms with fluorescence maxima between715 and 735 nm were, therefore, designated C684 based on absorptionspectrophotometry, and are considered to play a role in photosystemII. (Received August 15, 1972; )     

Control of microplankton size structure in contrasting water columns of the Celtic Sea

Water column structure, microphytoplankton size spectra andnutrient concentration were analysed at six sampling stationsin the Celtic Sea. Three types of stations were distinguished:(i) where the upper mixed layer (UML) reaches the total depth(TD), (ii) where the UML is about half of TD and (iii) wherethe UML is considerably less than half the TD. The UML was nutrientrich at type A stations and was nutrient depleted at type Bstations. At type C stations, the UML was nitrate depleted andsilicate rich. Two groups of microplankton size-abundance spectra(SAS) were found: (i) a typical linear SAS and (ii) a more complex‘atypical’ SAS, with a linear part up to 160 µmand a dome at 300 µm caused by a Coscinodiscus wailesiibloom. The dome was observed at all depths at type A stationsand above the pycnocline and at the seafloor of type B stations.Combining intrinsic growth rate, sinking rate and mixing layerdepth, the C. wailesii dome persists only at type A stationsbut settles out of the UML at the remaining stations. This suggeststhat a large part of the perturbation at the right extreme ofphytoplankton SAS does not propagate along the planktonic SASbut sinks to the seafloor. This paper was presented in a session on "Size Structure ofPlankton Communities", at the ASLO Summer International Meeting,held in Santiago de Compostela, Spain, between 19 and 24 June,and coordinated by Xabier Irigoien, Roger Harris and Angel Lopez-Urrutia.     

Glutamine synthesis in germinating seeds of Cucurbita moschata

During germination, an increase in glutamine and glutamine synthetase[L-glutamate: ammonia ligase (ADP), EC 6.3.1.2 [EC] ] occurred inthe cotyledons reaching a maximum at 4 to 6 days. The enzymehad a Km of 4.5 nun for L-glutamate, and 0.67 mu for hydroxylamine.Hydroxylamine exhibited substrate inhibition kinetics. The enzymewas inhibited by calcium ion, fluoride ion and p-hydroxymercuribenzoatebut not by EDTA, sodium ion or chloride ion. The sulf hydrylinhibition was reversed by dithiothreitol. In vivo synthesisof glutamine-¹⁴C from glutamate-¹⁴C was found to parallel theincrease in glutamine content and the in vitro glutamine synthetaseactivity during germination. ¹ Present address: Department of Biology, Mercyhurst College,Erie, Pennsylvania 16501, U.S.A. (Received June 12, 1971; )     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

Dry weight carbon and nitrogen content of some euphausiids from the north Atlantic Ocean and the Celtic Sea

Euphausiids were identified, measured and then dried duringa research cruise to sites in the north-eastern Atlantic Oceanand shelf waters of the Celtic Sea in late summer. The lengthsof furcilia larvae and later stages were measured in three ways,and regressions relating each pair of measures were calculatedfor different developmental stages of each species. Four hundredand ninety-three dried specimens of nine species were weighedand length–weight regressions were calculated. A subsetof 187 specimens was selected for analysis of carbon (C) andnitrogen (N) content soon after return to the laboratory. TheC and N contents of a further 122 specimens were determinedafter the specimens had been stored for 9 years. No consistentdifferences in percentage C and N content were found betweenthe two groups, indicating no systematic change during prolongedstorage in desiccators. Calyptopes of Nyctiphanes couchi containeda lower percentage of both C (mean 36.2%) and N (mean 8.6%)than later stages (means: 40.6% C, 11.0% N). Carbon contentand C:N ratios of a group of specimens of Thysanoessa longicaudata,including some juveniles, adult males and adult females, weredistinctly higher (>46% and >5.0, respectively) than thoseof the majority of specimens of the same species.     

The Effect of Desiccation on the Longevity of Seeds of Araucaria hunsteinii and A. cunninghamii

Seeds of Araucaria hunsteinii K. Schum. dried quicker at 29°Cthan at 19°C and quicker with the seed-coat removed thanwhen intact; seeds enclosed in polyethylene bags increased inmoisture content. At 15°C, seeds in a flow of air driedquicker than seeds in a box with silica gel, which in turn driedquicker than seeds in a box with no desiccant. No loss of germinationability occurred on drying fresh seed from 53 to about 32 percent moisture content (fresh weight basis); during further desiccationthe percentage germination was related to percentage moisturecontent in the form of a sigmoid curve, culminating in a completefailure to germinate at approximately 14 per cent moisture content.A consistent relationship was observed for all treatments andthe mean critical moisture content for seed death (failure togerminate) was near 23 per cent. Excised embryos grew on 1 percent agar but died if previously subjected to 14 h of desiccationat 15°C. In contrast, no relationship was found between germination andmoisture content of A. cunninghamii D. Don on desiccation from21 to 7 per cent moisture content. Possible causes for the observeddifference in response to desiccation are discussed and methodsfor seed storage are considered. Araucaria hunsteinii, Klinkii pine, Araucaria cunninghamii, Hoop pine, desiccation, seed longevity, storage of seeds     

Unsaponifiable Lipid Biosynthesis in the Seedling of Euphorbia lathyris L.: A Bypass via Alcoholic Fermentation

[1-¹⁴C]-ethanol supplied to the cotyledons of 9-d-old Euphorbialathyris seedlings was rapidly incorporated into unsaponifiablelipids, particularly into sterols, latex triterpenols and intothe triterpene ketones of the epicuticular wax. The [¹⁴C]-triterpenoidproduction from ethanol was hardly affected by sucrose in theexternal medium when sucrose uptake rates were low, but whenthe uptake rate was higher the [¹⁴C]-triterpenoid productionfrom [¹⁴C]-ethanol was greatly reduced. This observation isconsistent with the proposition that at high sucrose uptakerates, some sucrose is converted into ethanol, so that the incorporationof [¹⁴C]-ethanol into triterpenoids is reduced by competitionwith endogenously formed ethanol. A calculation based on theputative daily ethanol production in the cotyledons and thedaily triterpenoid production of seedlings indicates that about10 % of the triterpenoid synthesis in vivo may be from ethanol. Ethanol, Euphorbia lathyris, fermentation, seedling, triterpenoid biosynthesis     

Near‐infrared spectroscopic evaluation of lyophilized viral vaccine formulations

This article examines the applicability of near‐infrared spectroscopy (NIRS) to evaluate the virus state in a freeze‐dried live, attenuated vaccine formulation. Therefore, this formulation was freeze‐dried using different virus volumes and after applying different pre‐freeze‐drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze‐dried product was measured directly after freeze‐drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300–4000 cm^?1 region containing the amide A/II band which might reflect information on the coated proteins of freeze‐dried live, attenuated viruses; and (ii) the C–H vibration overtone regions (10,000–7500 and 6340–5500 cm^?1) which might supply information on the lipid layer surrounding the freeze‐dried live, attenuated viruses. The different pre‐freeze‐drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS‐DA) were developed and evaluated, allowing classification of the freeze‐dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1573–1586, 2013     

Studies on the Growth in Culture of Plant Cells: XXIII ISOLATION OF NUCLEI AND HISTONES FROM CULTURED CELLS OF ACER PSEUDOPLATANUS L.

A technique is described for the isolation of purified nucleifrom suspension culture cells of Acer pseudoplatanus. This involvesa grinding medium containing 70% (v/v) glycerol, 1 mM Mg²⁺,2 mM Ca²⁺, and Tris buffer at pH 7.8, prestorage and disruptionof the cells at –20 °C in a Potter-Elvehjem homogenizer,and purification by filtration and centrifugation in the presenceof Triton X-100. The nuclear yield is c. 25% as assessed bynuclear count or DNA estimation and the nuclei are active inthe RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H₂SO₄ andprecipitated by ethanol, 113 µg histone is obtained perµg nuclear DNA and the histone fraction contains 22% basicamino acids and has a lysine: arginine ratio of 2.6. Acid-ureagel electrophoresis shows the presence of five major histones(H1, H2A, H2B, H3, and H4 in sequence from anode to cathode)having respectively molecular weights of 24 500, 13 500, 13300, 12 800, and 11 000. There is very good correspondence betweencalf thymus histones H3 (reduced form) and H4 and two of theseAcer histones. The other Acer histones differ from the calfthymus histones H1, H2A, H2B in molecular weight but can beprovisionally equated with these by a newly developed differentialstaining reaction. Calf thymus histone H2A appears to be lessrich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (¹⁴C)lysine and[³H]tryptophan is in favour of the cytoplasmic synthesis ofthe histones.     

Relation between Ultra-Violet Spectral Change and Nucleotide Binding of the Chloroplast Coupling Factor 1

Properties of the nucleotide binding sites on chloroplast couplingfactor 1 (CF₁) were studied by equilibrium dialysis and UV spectroscopy.From our direct binding studies, we identified at least fourkinds of ADP binding sites on CF₁; a barely dissociable ADPbinding site (site A), a slowly exchangeable high affinity sitewith dissociation constant (Kd) 0.021 µM (site B), anotherslowly exchangeable high affinity site with Kd 1.6 µM(site C) and several low affinity (Kd {small tilde}30 µM)sites. The Kd values for sites B and C of the other nucleotidestested were 0.5 µM and 16 µM (GDP), 8 µM and34 µM (CDP), 17 µM and 20 µM (UDP) and 1.4µM and 1.4 µM (PP₁). From a comparison of the observed UV spectral change and theamount of nucleotide bound to these sites, as calculated fromthe above Kd values, we concluded that the nucleotide bindingto site B or G induces UV spectral changes that are almost thesame in shape and magnitude. The estimated difference molarabsorption coefficient () was 3.4?10³M^–1_ADP cm^–1for ADP at 278 nm. Our conclusions were strengthened by thegood agreement between the observed spectra and the calculatedspectra (derived from the Kd and values of ADP and GDP) whenADP and GDP were added together to CF₁. The cause of the unusual behavior of GDP in the UV differencespectrum which was unexplained in our previous report was shownto be competition between the GDP added and previously boundADP at sites B and C; this distorted the real spectrum inducedby GDP. (Received October 3, 1983; Accepted February 13, 1984)     

不同保藏处理的昆虫标本DNA提取及其随机扩增多态DNA反应

实验利用CTAB法对柳二十斑叶甲Chrysomela vigintipunctata (Scopoli)、异色瓢虫Harmonia axyridis Pollas、七星瓢虫Coccinella septempunctata Linnaeus、小地老虎Agrotis ypsilon (Rottemberg)、红蜻Crocothemis servilia Drury、无齿稻蝗Oxya abentata Willemse和中华稻蝗Oxya chinensis (Thunberg)等7种昆虫进行了基因组DNA提取。从自然干燥标本、烘干标本及酒精浸泡标本获得的DNA均可用于RAPD-PCR反应,且烘干标本、酒精浸泡标本提取效果优于自然干燥标本。这种提取方法简便易行,容易掌握,且耗资小于其它分子生物学方法。     

SW.B6-Soab Nontaster Congenic Strains Completed and a Sucrose Octaacetate Congenic Quartet Tested with other Bitters

Ten SW.B6 SOA nontaster strains congenic with the SWR/J SOAtaster inbred strain were bred via repeated backcross-intercrosscycles, with selection for nontasting in each cycle. Preferenceratio distributions and phenotypic proportions across cyclesat 0.1 mM SOA were consistent with monogenic predictions. TheSW.B6 mice completed a congenic quartet with the SWR/J, B6.SWSOA taster and C57BL/6J SOA nontaster strains. The Soa locuscontrolled avoidance differences within the quartet for SOA,raffinose undecaacetate, glucose pentaacetate and brucine. Backgroundgenes not linked to Soa controlled avoidance differences forL-phenylalanine and ethanol. Avoidance of bitter picric acidwas influenced by the Soa locus, but avoidance of acetic acidwas not. The quartet pattern for quinine HCl was unclear, withindications of both Soa and background effects. Two forms ofribose tetraacetate yielded different patterns. Avoidance differencescontrolled by the Soa locus were found for the pyranose form;however, all four strains avoided the furanose form. The pleiotropiceffects of Soa allele substitution within the quartet were limitedto a subset of bitter compounds. Chem. Senses 21: 507–517,1996.     

Ultrasonication assistance increases the efficiency of isoflavones extraction from kudzu (Pueraria lobata Ohwi) roots waste

This study assesses the use of ultrasonication to improve the extraction process of classical solvent extraction methods for extracting isoflavones from the kudzu roots waste. The kudzu roots waste was produced after squeezing fresh kudzu roots to make juice. The effects of extraction time, extraction temperature, ultrasonic power, and ethanol concentration in ethanol/water mixtures were investigated. The extraction yield was found to increase with extraction time and temperature. The application of ultrasonication-assisted extraction (UAE) increased the extraction yield of water/ethanol mixture (20:80) at 25°C 3 fold. A maximum amount (7.28 g) of isoflavone was obtained from 100 g of dried kudzu roots waste by UAE with water/ethanol mixture (20:80) for 6 h at 80°C. Combining the use of ultrasonication with conventional vacuum evaporation method also reduced the concentration time for extracts from 45 to 24 min.     

Effect of alcoholic extracts of wild plants on the inhibition of growth of Aspergillus flavus,Aspergillus niger,Penicillium chrysogenum,Penicillium expansum,Fusarium moniliforme and Fusarium poae moulds

Fungicidal activity of wild plants Larrea tridentata, Karwinskia humboldtiana, Ricinus communis, Eucalyptus globulus, Ambrosia ambrosioides, Nicotiana glauca, Ambrosia confertiflora, Datura discolor, Baccharis glutinosa, Proboscidea parviflora, Solanum rostratum, Jatropha cinerea, Salpianthus macrodonthus y Sarcostemma cynanchoides was evaluated against the moulds species Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum, Penicillium expansum, Fusarium poae y Fusarium moniliforme moulds species. Alcoholic extracts 6% (w/v) were prepared using six grams of dried plant powders (leaves and stems) and alcohol (70% ethanol or 70% methanol). A spore suspension (1x10(6); ufc/ml) of each mould was prepared by adding saline solution (0.85%) and 0.1% tween 80. The extracts were mixed with Czapeck yeast agar (CYA) at 45-50 degrees C in 1:10 relation on Petri dishes. Triplicate Petri dishes of each treatment and for each mould were centrally inoculated and three Petri dishes were used without treatment as controls. The inoculated dishes and controls were incubated at 25 +/- 2 degrees C for eight days. The incubated dishes were examined each 48 h and after the colony diameter (radial growth) was measured. Two mould species were controlled by L. tridentata, B. glutinosa and P. parviflora. Extracts of L. tridentata in methanol or ethanol at 41.5-100% inhibited all six species of moulds.     

Nucleotide Sequences of Genes Coding for Photosynthetic Reaction Centers and Light-Harvesting Proteins of Acidiphilium rubrum and Related Aerobic Acidophilic Bacteria

The nucleotide sequences of the puf operons of the Zn-bacteriochlorophylla(Zn-BChl a)-containing photosynthetic aerobic bacteria, Acidiphiliumrubrum and Acidiphilium angustum, were determined. The nucleotidesequences of the pufL and –M of Acidiphilium cryptum,Acidiphilium multivorum, and Acidiphilium organovorum were alsodetermined. The puf operons of A. rubrum and A. angustum containedpufB, –A,–L, –M, and –C as seen in otherpurple bacteria with an unknown gene directly upstream of pufB.Comparing the deduced amino acid sequences of the puf genesof the Acidiphilium species with those of other purple bacteriashowed that His L168, which is highly conserved in other bacteria,is replaced by a glu-tamic acid in the Acidiphilium species.The three-dimensional structures of the reaction centers ofBlastochloris (Rhodopseudomonas) viridis and Rhodobacter sphaeroidessuggest that this residue locates closely to a special pairof bacteriochlorophylls and may be involved in the stabilizationand function of "Zn-BChl a". The relative content of chargedamino acid residues in the L and M subunit is a little lowerin A. rubrum (10%of total) than in B. viridis (12%), and thetendency is more pronounced in the cyto-chrome subunit: 12.5%in A. rubrum and 18.8% in B. viridis. (Received July 24, 1997; Accepted September 9, 1997)     

Initial Characterization of the FlgE Hook High Molecular Weight Complex of Borrelia burgdorferi

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.     

Selection against glycosylation sites in potential target proteins of the general HMWC N-glycosyltransferase in Haemophilus influenzae

The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyltransferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coli showed that HMWC of NTHi was a general glycosyltransferase capable of glycosylating selected asparagines in proteins other than its HMWA substrate, including Asn78 in E. coli 30S ribosomal protein S5. The equivalent residue in S5 homologues in H. influenzae or other sequenced Pasteurellaceae genomes is not asparagine, and these organisms also showed significantly fewer than expected potential sites of glycosylation in general. Expression of active HMWC in E. coli resulted in growth inhibition compared with expression of inactive enzyme, consistent with glycosylation by HMWC detrimentally affecting the function of some E. coli proteins. Together, this supports the presence of a selective pressure in the Pasteurellaceae against glycosylation sites that would be modified by the general N-glycosyltransferase activity of HMWC.