Characterization of imipenem-resistant Serratia marcescens producing IMP-type and TEM-type beta-lactamases encoded on a single plasmid

To evaluate the roles of blaIMP and blaTEM genes in the resistance of Serratia marcescens against beta-lactams and to find the spreading ways of these genes, 19 clinical isolates of imipenem-resistant Serratia marcescens were analyzed. Six strains bore blaIMP and blaTEM genes on a single plasmid, as confirmed by transferring resistance determinants via conjugation and transformation, and by detecting bla genes with PCR analysis. The six strains showed two different genomic patterns on pulsed-field gel electrophoresis. All the transconjugants and transformants gained high-level resistance to ampicillin, cephalexin, cefoxitin and cefotaxime, and showed a reduced susceptibility to imipenem, but maintained full susceptibility to aztreonam. In addition, the expressions of blaIMP and blaTEM genes were constitutive, either in Serratia marcescens clinical isolates or in their transconjugants and transformants. These findings may explain the rapid spread of the above resistance determinants among Enterobacteriaceae via transmissible plasmids in the clinical setting.     

Low levels of quantitative and molecular genetic differentiation among natural populations of Medicago ciliaris Kroch. (Fabaceae) of different Tunisian eco-geographical origin

In this paper, we analyze the genetic variability in four Tunisian natural populations of Medicago ciliaris using 19 quantitative traits and six polymorphic microsatellite loci. We investigated the amplification transferability of 30 microsatellites developed in the model legume M. truncatula to M. ciliaris. Results revealed that about 56.66% of analyzed markers are valuable genetic markers for M. ciliaris. The most genetic diversity at quantitative traits and microsatellite loci was found to occur within populations (>80%). Low differentiations among populations at quantitative traits Q _ST  = 0.146 and molecular markers F _ST  = 0.18 were found. The majority of measured traits exhibited no significant difference in the level of Q _ST and F _ST . Furthermore, significant correlations established between these traits and eco-geographical factors suggested that natural selection should be invoked to explain the level of phenotypic divergence among populations rather than drift. There was no significant correlation between population differentiation at quantitative traits and molecular markers. Significant spatial genetic structure consistent with models of isolation by distance was detected within all studied populations. The site-of-origin environmental factors explain about 9.07% of total phenotypic genetic variation among populations. The eco-geographical factors that influence more the variation of measured traits among populations are the soil texture and altitude. Nevertheless, there were no consistent pattern of associations between gene diversity (He) and environmental factors.     

On the integration of subthreshold inputs from Perforant Path and Schaffer Collaterals in hippocampal CA1 pyramidal neurons

Using a realistic model of a CA1 hippocampal pyramidal neuron, we make experimentally testable predictions on the roles of the non-specific cation current, I _h , and the A-type Potassium current, I _A , in modulating the temporal window for the integration of the two main excitatory afferent pathways of a CA1 neuron, the Schaffer Collaterals and the Perforant Path. The model shows that the experimentally observed increase in the dendritic density of I _h and I _A could have a major role in constraining the temporal integration window for these inputs, in such a way that a somatic action potential (AP) is elicited only when they are activated with a relative latency consistent with the anatomical arrangement of the hippocampal circuitry.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Genomics of the<Emphasis Type="Italic"> ccoNOQP</Emphasis>-encoded<Emphasis Type="Italic"> cbb</Emphasis><Subscript>3</Subscript> oxidase complex in bacteria

Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb ₃) complex with a high affinity for O₂, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group (Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O₂. The most widespread strategy involves direct regulation by the O₂-responsive protein Fnr. The second strategy involves an O₂-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O₂-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixK genes. Another, not yet identified, strategy is likely to operate in the -Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the -subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered.     

Gas Exchange Responses to CO2 Concentration Instantaneously Elevated in Flag Leaves of Winter Wheat Cultivars Released in Different Years

Three winter wheat (Triticum aestivum L.) cultivars, representatives of those widely cultivated in Beijing over the past six decades, were grown in the same environmental conditions. Net photosynthetic rate (P _N) per unit leaf area and instantaneous water use efficiency (WUE) of flag leaves increased with elevated CO₂ concentration. With an increase in CO₂ concentration from 360 to 720 µmol mol^–1, P _N and WUE of Jingdong 8 (released in 1990s and having the highest yield) increased by 173 and 81 %, while those of Nongda 139 (released in 1970s) increased by 88 and 66 %, and Yanda 1817 (released in 1945, with lowest yield) by 76 and 65 %. Jingdong 8 had the highest P _N and WUE values under high CO₂ concentration, but Yanda 1817 showed the lowest P _N. Stomatal conductance (g _s) of Nongda 139 and Yanda 1817 declined with increasing CO₂ concentration, but g _s of Jingdong 8 firstly went down and then up as the CO₂ concentration further increased. Intercellular CO₂ concentration (C _i) of Jingdong 8 and Nongda 139 increased when CO₂ concentration elevated, while that of Yanda 139 increased at the first stage and then declined. Jingdong 8 had the lowest C _i of the three wheat cultivars, and Yanda 1817 had the highest C _i value under lower CO₂ concentrations. However, Jingdong 8 had the highest P _N and lowest C _i at the highest CO₂ concentration which indicates that its photosynthetic potential may be high.     

黄花棘豆脱水素OoY_2K_4的鉴定及表达分析

该研究以黄花棘豆cDNA为模板,采用同源克隆法,从黄花棘豆转录组数据库中克隆获得1个响应逆境胁迫的胚胎发育晚期丰富蛋白基因,命名为OoY_2K_4;OoY_2K_4基因ORF为786bp,编码261个氨基酸,含有2个保守的Y片段和4个K片段,为典型的Y_2K_4类脱水蛋白亚家族成员;OoY_2K_4蛋白不具有跨膜结构域,不存在信号肽,亲水性极强,含有1个糖基化位点和17个磷酸化位点;亚细胞定位显示,OoY_2K_4蛋白定位于细胞质中。多序列比对发现,OoY_2K_4蛋白与其他物种第二组LEA蛋白(脱水素)序列高度保守;进化树分析显示,该序列与三叶草、蒺藜苜蓿和紫花苜蓿相似度最高,亲缘关系最近。采用qRT-PCR对OoY_2K_4基因在干旱、高盐、低温以及脱落酸、乙烯、赤霉素处理下的表达分析显示,干旱和高盐胁迫可显著诱导OoY_2K_4基因表达,而低温胁迫下基本无变化;激素处理均可诱导OoY_2K_4基因高效表达,其中脱落酸诱导下OoY_2K_4基因表达最显著。研究推测,OoY_2K_4基因可能通过依赖ABA的信号途径参与黄花棘豆对干旱和高盐逆境胁迫的应答反应。     

Carbon dioxide and methane fluxes in boreal peatland microcosms with different vegetation cover—effects of ozone or ultraviolet-B exposure

O₃ concentrations in the troposphere are rising and those in the stratosphere decreasing, the latter resulting in higher fluxes of solar ultraviolet-B (UV-B) radiation to the earth's surface. We assessed whether the fluxes of CO₂ and CH₄ are altered by enhanced UV-B radiation or elevated tropospheric O₃ concentrations in boreal peatland microcosms (core depth 40 cm, diameter 10.5 cm) with different vegetation cover. At the end of the UV-B experiment which lasted for a growing season, net CO₂ exchange (NEE) and dark ecosystem respiration (R _TOT) were sevenfold higher, and CH₄ efflux 12-fold higher, in microcosms with intact vegetation dominated by Eriophorum vaginatum L. and Sphagnum spp., compared to microcosms from which we removed E. vaginatum. Vegetation treatment had minor effects on CH₄ production and consumption potentials in the peat, suggesting that the large difference in CH₄ efflux is mainly due to efficient CH₄ transport via the aerenchyma of E. vaginatum. Ambient UV-B supplemented with 30% and elevated O₃ concentrations (100 and 200 ppb, for 7 weeks) significantly increased R _TOT in both vegetation treatments. Elevated O₃ concentrations reduced NEE over time, while UV-B had no clear effects on the fluxes of CO₂ or CH₄ in the cloudy summer of the study. Field experiments are needed to assess the significance of increasing UV-B radiation and elevated tropospheric O₃ concentration on peatland gas exchange in the long-term.     

Genes determining leucine aminopeptidase and mildew resistance from the ornamental apple, ‘White Angel’

Mildew resistance in the ornamental apple White Angel was found to be determined by complementary genes. The gene R _w was found to be necessary for the expression of resistance controlled by the resistance gene Pl _w . The close linkage between the isoenzyme gene, Lap-2, for leucine aminopeptidase and P1 _w was confirmed. The efficiency of Lap-2 as a marker in screening for mildew resistance is limited, as it cannot account for susceptible plants with the r _w r _w P1 _w p1 _w genotype. It has, however, an important role to play in combining resistance genes from different sources. The genotypes of White Angel (R _w r _w , Pl _w pl _w , Lap-2an), Jester (R _w r _w , p1 _w p _w , Lap-2an) Katja (R _w r _w ,p1 _w p1 _w , Lap-2an) and Gloster 69 (r _w r _w , p1 _w p1 _w , Lap-2an) were determined. It also appeared that R _w might influence Lap-2 activity in young seedlings.     

A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

Expression of the mosquitocidal cryIVB gene under the control of different promoters in Bacillus sphaericus 2362 and acrystalliferous Bacillus thuringiensis subsp. israelensis c4Q2-72

The 3.7 kb XbaI fragment harbouring the cryIVB gene which encoded a 130 kDa mosquitocidal toxin protein from Bacillus thuringiensis subsp. israelensis (B.t.i.) was placed downstream to the cat-86 gene promoter (P _cat-86, spore stage specific expression) or bgaB gene promoter (P _bgaB , vegetative stage specific expression). The constructs were subcloned into pBC16 to obtain pBTC3 and pBTC6, respectively. Both plasmids and the other construct, pBTC1 were successfully transferred into B. thuringiensis subsp. israelensis c4Q2-72 and B. sphaericus 2362. Western blot analysis showed that P _bgaB in front of P _cryIVB could enable cells to produce a 130 kDa protein from the vegetative stage (4 h) whereas those with P _cat-86 could not. The positive detection of 130 kDa crystal protein during the vegetative stage (4 h) by Western blot analysis indicated the vegetative-stage-specific expression of P _bgaB , while the 130 kDa crystal protein produced from cryIVB gene under control of P _cat-86 was detected only at 48 h. The strong activity of P _bgaB , together with P _cryIVB within pBTC6 in both bacterial hosts was also shown by the toxicity assay against Aedes aegypti larvae (B.t.i. c4Q2-72, 5.6 ± 3.6 × 10² c.f.u./ml; B. sphaericus 2362, 5.4 ± 2.5 × 10² c.f.u./ml) which were 100-fold and 10-fold more toxic to such larvae when compared with pBTC3 (P _cat-86 together with P _cryIVB ) and pBTC1 (contained only its self promoter) in the same bacterial host strains, respectively. The plasmid pBTC6 is not stable in either Bacillus host.     

The chloroplast cytochrome b 6 f complex can exist in monomeric and dimeric states

The cytochrome b ₆ f complex isolated from spinach chloroplast membranes can be resolved into two forms, a monomeric and a dimeric form, by centrifugation on sucrose gradients. The conversion of the dimeric form of the complex into the monomeric form could be prevented by cross-linking with the homobifunctional reagent, dithiobis(succinimidylpropionate) but not by cross-linking with disuccinimidyltartrate or glutaraldehyde. SDS-PAGE analyses of the monomeric and dimeric forms of the cytochrome complex showed the presence of specific cross-linked products in each respective form of the complex. For example, the monomeric form contained a cross-linked product of cytochrome f, cytochrome b ₆ f and subunit IV while the dimeric form contained a cross-linked dimer of cytochrome b ₆ f. The presence of the former in the isolated cytochrome b ₆ f complex prepared by the method of Hurt and Hauska (Eur J Biochem 117: 591–599, 1981) indicates the presence of the monomer in his preparation.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSP dithiobis(succinimidylpropionate) - DST disuccinimidyltartrate     

Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae)

Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q _ST = 0.47, and F _ST = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q _ST and F _ST . Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P₂0₅) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, H _e , were established in five-microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

The Influences of I h on Temporal Summation in Hippocampal CA1 Pyramidal Neurons: A Modeling Study

Recent experimental and theoretical studies have found that active dendritic ionic currents can compensate for the effects of electrotonic attenuation. In particular, temporal summation, the percentage increase in peak somatic voltage responses invoked by a synaptic input train, is independent of location of the synaptic input in hippocampal CA1 pyramidal neurons under normal conditions. This independence, known as normalization of temporal summation, is destroyed when the hyperpolarization-activated current, I _h, is blocked [Magee JC (1999a), Nature Neurosci. 2: 508–514]. Using a compartmental model derived from morphological recordings of hippocampal CA1 pyramidal neurons, we examined the hypothesis that I _h was primarily responsible for normalization of temporal summation. We concluded that this hypothesis was incomplete. With a model that included I _h, the persistent Na⁺ current (I _NaP), and the transient A-type K⁺ current (I _A), however, we observed normalization of temporal summation across a wide range of synaptic input frequencies, in keeping with experimental observations.     

Subunit Organization of the Stator Part of the F 0 Complex from Escherichia coli ATP Synthase

Membrane-bound ATP synthases (F₁F₀) catalyze the synthesis of ATP via a rotary catalyticmechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potentialis supposed to propel rotation of a subunit c ring of F₀ together with subunits and of F₁,hereby forming the rotor part of the enzyme, whereas the remainder of the F₁F₀ complexfunctions as a stator for compensation of the torque generated during rotation. This reviewfocuses on our recent work on the stator part of the F₀ complex, e.g., subunits a and b. Usingepitope insertion and antibody binding, subunit a was shown to comprise six transmembranehelixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circulardichroism (CD) spectroscopy, the secondary structure of subunit b incorporated intoproteoliposomes was determined to be 80% -helical together with 14% turn conformation, providingflexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplexwas shown to be active in proton translocation and functional F₁ binding revealing the nativeconformation of the polypeptide chain. Chemical crosslinking in everted membrane vesiclesled to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32Ccould be crosslinked to subunit a, indicating a close proximity of subunits a and b near themembrane. Further evidence for the proposed direct interaction between subunits a and b wasobtained by purification of a stable ab ₂ subcomplex via affinity chromatography using Histags fused to subunit a or b. This ab ₂ subcomplex was shown to be active in proton translocationand F₁ binding, when coreconstituted with subunit c. Consequences of crosslink formationand subunit interaction within the F₁F₀ complex are discussed.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.