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1.
A multi-media biological aerated filter (MBAF) with clinoptilolite media was used to treat synthetic wastewater. Coal ash bioceramsite with supplemental metallic iron was added to the clinoptilolite media of MBAFs in a brick-wall embedded design. Performance parameters, such as hydraulic, organic, N and P loading capacity and microbial community composition were studied for different quantity of supplemental metallic iron contained in three MBAFs. The MBAFs with more metallic iron were found to have superior hydraulic and organic loading, and higher N and P capacities. COD, NH3-N and TP removal dropped by 7-10%, 6-7% and 4-5%, respectively, with when hydraulic loading was raised from 2.8 to 7.5 m3 m−2 d−1. NH3-N removal also decreased 8-9% when ammonia loading was elevated from 0.078 to 0.156 kg NH3-N m−3 d−1. Real-time PCR revealed a relatively stable bacterial community composed primarily of eubacteria that formed after an initial 120 d operational period. Doubling the amount of metallic iron in the bioceramsite media resulted in a twofold increase of eubacteria in the MBAF, but a decrease in the ratio of anaerobic ammonia-oxidizing bacteria to total bacteria.  相似文献   

2.
The effects of hydrodynamics on size, shape and distribution of benthic organisms are still not completely understood. Benthic organisms that inhabit wave-swept environments usually have small sizes and ethological adaptations to reduce drag and increase resistance force. Water speeds produced by waves in intertidal habitats can be more than 10 times higher than those in subtidal environments. However, comparatively small water speeds can produce high drag forces (Fd) on large subtidal organisms.Pinna nobilis is a subtidal epibenthic large bivalve-mollusc endemic to the Mediterranean Sea, a common inhabitant of Posidonia oceanica meadows. It lives partially buried in the seabed and shows a characteristic population structure. Small individuals are usually located at shallow sites whereas large individuals are only observed in deeper levels or sheltered locations. Also, some populations show a common orientation of the shell. These features are widespread throughout the Mediterranean, but their causes are unknown.The present work is a study on the relationship between population structure of P. nobilis and the habitat hydrodynamics. The main factor considered was Drag force due to the water flow produced by waves. Drag forces (Fd) supported by two populations located at different depths in the same P. oceanica meadow were estimated according to both the size and orientation of shells. Also, Fd acting on the individuals during the greatest storm recorded in the zone in the previous 9 years, were calculated. Drag coefficients (Cd), necessaries to estimate Fd, were estimated in the towing tank of the “Ecuela Técnica Superior de Ingenieros Navales (ETSIN)” of the Polytechnic University of Madrid.The results show significant differences in Fd acting in both populations. Despite the important increment of water speed with wave shoaling, individuals of the shallow population (SP), located at 6 m depth, withstand less Fd than those of the deep population (DP), at 13 m depth. The main reasons of this Fd reduction in SP are both the small size of the individuals and their common orientation, having the dorso-ventral side of the shell towards the main water flow. This fact, together with previous data showing higher mortality, less density of individuals, and less maximum asymptotic length, evidence that selective pressures regulate these population parameters, producing a trade-off between hydrodynamics, shell size and orientation, for each shore type and water depth.Combining the data of Fd supported by each population for different wave types, approximate values of the optimal mean Fd and the maximum dislodgement force withstood by P. nobilis were estimated (< 9 Newton (N) and ≈ 45 N respectively).  相似文献   

3.
The mass spectra of the trimethylsilyl ester trimethylsilyl ether derivatives of prostaglandins F, F and F are reported and discussed. Accurate masses from the high resolution spectra of these compounds are also presented. These spectra are interpreted with the aid of those of the corresponding d9-trimethylsilyl derivatives and selectively labeled trimethylsilyl ester-d9-trimethylsilyl ether derivatives. It was found that metastabledefocusing was helpful in elucidation of the mechanisms of formation of some ions.  相似文献   

4.
The concept of solid retention time (SRT) was applied in the trickling-filter process. A rational model of the trickling-filter process employing activated-sludge-process operational parameters was presented. The design equation was developed as follows; 1/SRT = [(S0 ? Sn)/X ]·(F/VY ? kd, where SRT is the sludge retention time, S0 is the influent substrate concentration; Sn is the effluent substrate concentration; X is the total cell mass retained per unit filter volume; V is the total volume of the filter; F is the influent flow rate; Y is the cell yield, and kd is the cell decay rate. A laboratory-scale trickling-filter pilot plant treating synthetic sucrose waste-water was studied to verify the present design equation. The solid retention time was evaluated from the total slime mass (active and inactive) retained and the sludge wasted daily. It was found that the present design equation could be applied for designing the trickling-filter process by the application of SRT employed in the activated sludge process. Also, the SRT could be related to the hydraulic loading and influent substrate concentration for a given filter medium. The variation of SRT by the hydraulic loading at constant organic loading was observed and could be expressed by the mechanistic model. When SRT was maintained more than 12 days, it provided the highest five-day biological oxygen demand (BOD5) removal, minimum sludge production, and lowest sludge volume index (SVI) value. The present model does include both microbial growth kinetic concepts, which can be more practical and meaningful for the design of a trickling filter.  相似文献   

5.
Summary Physical characteristics, namely floc density function, floc size distribution, and relative floc strength, of a number of flocculent yeast types were measured. A straight-line relationship was found to exist between log values of size and density for the yeasts examined. Each yeast type had coefficients from this relationship which could be used to interpret settling behaviour. Indices of relative floc strength were also obtained and together with the floc density function allowed fuller interpretation of yeast settling than with simpler theories.Symbols a constant (g·cm-3) - B 2/B 1 floc binding strength of floc2 relative to floc1 - d f floc diameter (cm) - d i image diameter on print (cm) - d max maximum floc diameter (cm) - f d Ploc effective density (g·cm-3) - g gravitational constant (981 cm·s-1) - K p constant (-) - R l rate of enlargement on film - R 2 rate of enlargement on print - S s density of suspending liquid phase (g·cm-3) - S f density of solid (floc) phase (g·cm-3) - U t terminal settling velocity (cm·s-1) - u liquid viscosity (g·cm-3·-1)  相似文献   

6.
污水地下渗滤系统脱氮效果及动力学过程   总被引:2,自引:0,他引:2  
李海波  李英华  孙铁珩  王鑫 《生态学报》2011,31(24):7351-7356
建立了模拟污水地下渗滤过程的中试系统,重点考察了水力负荷对系统脱氮效率的影响情况,建立了描述地下渗滤系统微生物脱氮过程的动力学模型.结果表明:地下渗滤系统脱氮效果好,抗水力负荷冲击能力强,处理最佳水力负荷0.125m3· m-2· d-1,出水中氮浓度低于《城市污水再生利用——景观环境用水水质》标准( GB/T 18921-2002).地下渗滤系统硝化过程符合一级动力学模型NE=Noe-0.4812t,温度是影响硝化速率的主要因素,两者的关系是KT=0.2218×1.035(T-20);出水硝态氮浓度与水力停留时间之间呈负指数关系,可描述为C=16.3475e-0.2548t,碳源是引起反硝化速率变化的主要因子.在基质层垂直深度65 cm处二次补加生活污水,反硝化速率常数由0.0355提高到0.0488.强调地下渗滤系统的污水净化功能而忽视其生态服务功能,是系统运行中普遍存在的认识误区,过高的水力负荷不利于硝化-反硝化反应的顺利进行.地下渗滤系统运行应采取适宜的水力负荷方式,促进硝化-反硝化作用.  相似文献   

7.
Rotation of the γ subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α3β3γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH0 and ΔG0 values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α3β(Y341W)3γ subcomplex had similar Kd and ΔG0 values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.  相似文献   

8.
F1-ATPase is an ATP-driven motor in which γε rotates in the α3β3-cylinder. It is attenuated by MgADP inhibition and by the ε subunit in an inhibitory form. The non-inhibitory form of ε subunit of thermophilic Bacillus PS3 F1-ATPase is stabilized by ATP-binding with micromolar Kd at 25 °C. Here, we show that at [ATP] > 2 μM, ε does not affect rotation of PS3 F1-ATPase but, at 200 nM ATP, ε prolongs the pause of rotation caused by MgADP inhibition while the frequency of the pause is unchanged. It appears that ε undergoes reversible transition to the inhibitory form at [ATP] below Kd.  相似文献   

9.
During the course of our study to develop analytical methodology for quantitating the investigative antitumor agent 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one (DAF; NSC 686288) in plasma, a significant concentration of a metabolite was observed in a post-dosed rat. The results of electron-ionization (EI) mass spectrometric analysis of the metabolite suggested that N-acetylation had occurred, but, interestingly, that only one of the compound’s two primary amino groups had been transformed. Comparing the mass spectra and gas chromatographic retention times of a mono-acetylated sample of DAF and that of the metabolite showed both to be the same. A retro-Diels–Alder (RDA) fragmentation of the B ring of DAF results in formation of two abundant product ions, each retaining one of the amino groups. The EI mass spectrum of mono-N-acetamido-d3 DAF shows loss of ketene-d2, leading to formation of an –NHD group. The ensuing RDA fragmentation easily identifies which of the two product ions contains the deuterium, thereby allowing us to assign the site of N-acetylation as the amino group on ring C (the 4′ position) of DAF.  相似文献   

10.
Background and AimsLeaf biomechanical resistance protects leaves from biotic and abiotic damage. Previous studies have revealed that enhancing leaf biomechanical resistance is costly for plant species and leads to an increase in leaf drought tolerance. We thus predicted that there is a functional correlation between leaf hydraulic safety and biomechanical characteristics.MethodsWe measured leaf morphological and anatomical traits, pressure–volume parameters, maximum leaf hydraulic conductance (Kleaf-max), leaf water potential at 50 % loss of hydraulic conductance (P50leaf), leaf hydraulic safety margin (SMleaf), and leaf force to tear (Ft) and punch (Fp) of 30 co-occurring woody species in a sub-tropical evergreen broadleaved forest. Linear regression analysis was performed to examine the relationships between biomechanical resistance and other leaf hydraulic traits.Key ResultsWe found that higher Ft and Fp values were significantly associated with a lower (more negative) P50leaf and a larger SMleaf, thereby confirming the correlation between leaf biomechanical resistance and hydraulic safety. However, leaf biomechanical resistance showed no correlation with Kleaf-max, although it was significantly and negatively correlated with leaf outside-xylem hydraulic conductance. In addition, we also found that there was a significant correlation between biomechanical resistance and the modulus of elasticity by excluding an outlier.ConclusionsThe findings of this study reveal leaf biomechanical–hydraulic safety correlation in sub-tropical woody species.  相似文献   

11.
The kinetics of renaturation of the β2-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F1 and F2 are studied and compared. Steps corresponding to the refolding of F1, to the association of the folded F1 and F2 fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β2 starting from the denatured F1 and F2 fragments.The step corresponding to the refolding of the F1 domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β2-protein. It is concluded that the refolded F1 fragment corresponds to a folding intermediate on the pathway of renaturation of the β2-subunit.  相似文献   

12.
The modern horse (Equus caballus) is the product of over 50 million yrs of evolution. The athletic abilities of the horse have been enhanced during the past 6000 yrs under domestication. Therefore, the horse serves as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. The structure and function of skeletal muscle show remarkable plasticity to the physical and metabolic challenges following exercise. Here, we reveal an evolutionary layer of responsiveness to exercise-stress in the skeletal muscle of the racing horse. We analysed differentially expressed genes and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. By estimating genome-wide dN/dS ratios using six mammalian genomes, and FST and iHS using re-sequencing data derived from 20 horses, we were able to peel back the evolutionary layers of adaptations to exercise-stress in the horse. We found that the oldest and thickest layer (dN/dS) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (FST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.  相似文献   

13.
Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in d-alanyl-d-lactate. A key enzyme in this process is a d-alanyl-d-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide d-alanyl-d-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for d-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.The origin of antibiotic resistance determinants is of significant interest for several reasons, including the prediction of the emergence and spread of resistance patterns, the design of new antimicrobial agents, and the identification of potential reservoirs for resistance elements. Antibiotic resistance can occur either through spontaneous mutation in the target or by the acquisition of external genetic elements such as plasmids or transposons which carry resistance genes (7). The origins of these acquired genes are varied, but it has long been recognized that potential reservoirs are antibiotic-producing organisms which naturally harbor antibiotic resistance genes to protect themselves from the actions of toxic compounds (6).High-level resistance to glycopeptide antibiotics such as vancomycin and teicoplanin in vancomycin-resistant enterococci (VRE) is conferred by the presence of three genes, vanH, vanA (or vanB), and vanX, which, along with auxiliary genes necessary for inducible gene expression, are found on transposons integrated into plasmids or the bacterial genome (1, 20). These three genes are essential to resistance and serve to change the C-terminal peptide portion of the peptidoglycan layer from d-alanyl-d-alanine (d-Ala-d-Ala) to d-alanyl-d-lactate (d-Ala-d-Lac). This change results in the loss of a critical hydrogen bond between vancomycin and the d-Ala-d-Ala terminus and in a 1,000-fold decrease in binding affinity between the antibiotic and the peptidoglycan layer, which is the basis for the bactericidal action of this class of compounds (5). The vanH gene encodes a d-lactate dehydrogenase which provides the requisite d-Lac (3, 5), while the vanX gene encodes a highly specific dd-peptidase which cleaves only d-Ala-d-Ala produced endogenously while leaving d-Ala-d-Lac intact (19, 21). The final gene, vanA or vanB, encodes an ATP-dependent d-Ala-d-Lac ligase (4, 8, 10). This enzyme has sequence homology with the chromosomal d-Ala-d-Ala ligases, which are essential for peptidoglycan synthesis but which generally lack the ability to synthesize d-Ala-d-Lac (9).We have recently cloned vanH, vanA, and vanX homologues from two glycopeptide antibiotic-synthesizing organisms: Amycolatopsis orientalis C329.2, which produces vancomycin, and Streptomyces toyocaensis NRRL 15009, which produces A47934 (14). In addition, the vanH-vanA-vanX gene cluster was identified in several other glycopeptide producers. We have also demonstrated that the VanA homologue from S. toyocaensis NRRL 15009 can synthesize d-Ala-d-Lac in vitro and in the glycopeptide-sensitive host Streptomyces lividans (15, 16). We now report the expression of the A. orientalis C329.2 VanA homologue DdlN in Escherichia coli, its purification, and its enzymatic characterization. These data reinforce the striking similarity between vancomycin resistance elements in VRE and glycopeptide-producing organisms and support the possibility of a common origin for these enzymes.

Expression, purification, and specificity of DdlN.

DdlN was overexpressed in E. coli under the control of the bacteriophage T7 promoter. The construct gave good yields of highly purified enzyme following a four-step purification procedure (Table (Table1;1; Fig. Fig.1).1). Like other dd-ligases, DdlN behaved like a dimer in solution (not shown).

TABLE 1

Purification of DdlN from E. coli BL21 (DE3)/pETDdlN
SampleProtein (mg)Activity (nmol/min)Sp act (nmol/ min/mg)Recovery (%)Purification (fold)
Lysate1248436.82100
Ammonium sulfate (20–50% saturation)67.678011.5921.7
Sephacryl S20011.682571.49811
Q Sepharose2.87422658839
Phenyl Superose0.429974835110
Open in a separate windowOpen in a separate windowFIG. 1Purification of DdlN from E. coli BL21 (DE3)/pETDdlN. Proteins were separated on an SDS–11% polyacrylamide gel and stained with Coomassie blue. Lane 1, molecular mass markers (masses are noted at the left in kilodaltons); lane 2, whole-cell lysate; lane 3, ammonium sulfate fraction (20 to 50% saturation); lane 4, Sephacryl S200; lane 5, Q Sepharose; lane 6, phenyl Superose.The amino acid substrate specificity of DdlN was assessed by incubation of 14C-d-Ala with all 20 common amino acids in the d configuration. Purified DdlN catalyzed the synthesis of d-Ala-d-Ala in addition to that of several other mixed dipeptides, including d-Ala-d-Met and d-Ala-d-Phe (Fig. (Fig.2).2). Thus, DdlN exhibits a substrate specificity which is similar to that of VanA (4), with the capacity to synthesize not only d-Ala-d-Ala but also mixed dipeptides with bulky side chains in the C-terminal position.Open in a separate windowFIG. 2Substrate specificity of DdlN. Autoradiogram from thin-layer chromatography analysis of DdlN substrate specificity. All reaction mixtures contained 2.5 mM d-Ala and 1 mM ATP, and the radiolabel was 14C-d-Ala, except where noted. Lane 1, d-Ala; lane 2, d-Lac with 14C-d-Lac label; lane 3, d,l-methionine; lane 4, dl-phenylalanine; lane 5, d-Hbut; lane 6, d-hydroxyvalerate. Letters indicate the following: A, d-Ala-d-Lac; B, d-Lac; C, d-Ala-d-Met; D, d-Ala-d-Phe; E, d-Ala-d-Hbut; F, d-Ala-d-hydroxyvalerate.Importantly, DdlN is a depsipeptide synthase with the ability to synthesize d-Ala-d-Lac, d-Ala-d-hydroxybutyrate (Hbut), and d-Ala-d-hydroxyvalerate (Fig. (Fig.2).2). However, unlike VanA (5), d-hydroxycaproate and d-phenyllactate are not substrates (not shown). Thus, DdlN is a broad-spectrum d-Ala-d-X ligase with depsipeptide synthase activity.

Characterization of d-Ala-d-X ligase activity.

Following the initial assessment of the specificity of the enzyme, several substrates were selected for quantitative analysis by evaluation of their steady-state kinetic parameters (Table (Table2).2). DdlN has two amino acid (or hydroxy acid) Km values. Steady-state kinetic plots indicated that, like other dd-ligases, the N-terminal Km (Km1) was significantly lower (higher specificity) than the C-terminal Km (Km2). Since the former value is expected to be independent of the C-terminal substrate, only Km2 values were determined and are reported here.

TABLE 2

Characterization of steady-state parameters of DdlN and VanA
LigaseSubstrateKm2 (mM)kcat (min−1)kcat/Km2 (M−1 s−1)
DdlNd-Ala21 ± 2229 ± 71.8 × 102
d-Lac0.4 ± 0.0555 ± 12.3 × 103
d-Hbut2.5 ± 0.332 ± 22.1 × 102
ATPa1.2 ± 0.271 ± 50.98 × 102
DdlMbd-Ala166 ± 27
d-Lac1.08 ± 0.10
VanAcd-Ala382951.3 × 102
d-Lac7.1942.2 × 102
d-Hbut0.601083.0 × 103
Open in a separate windowa Determined in the presence of 10 mM d-Lac. b Data from reference 16c Data from reference 5. DdlN showed good d-Ala-d-Ala ligase activity but with a very high and physiologically questionable Km2 (21 mM). On the other hand, d-Ala-d-Lac synthesis was excellent, with a 4-fold decrease in kcat, compared to d-Ala-d-Ala synthesis, which was offset by a 52-fold drop in Km that resulted in a >12-fold increase in specificity (kcat/Km2). d-Hbut was also a good substrate, with a kcat/Km2 comparable to that of d-Ala.Steady-state kinetic parameters for d-Ala-d-X formation showed trends similar to those found with both VanA and DdlN. For example, the kcat values between VanA and DdlN were virtually the same for most substrates. There were significant differences, however. For instance, while the Km2 values for d-Ala were very high for all three enzymes, DdlN does have greater affinity for d-Ala, with a 1.8- and 7.9-fold lower Km2 than those of VanA and DdlM, respectively. Additionally, the Km2 for d-Lac was 17.8- and 2.7-fold lower than those for VanA and DdlM. Thus, DdlN has a more restrictive specificity for the C-terminal residue than VanA, which is compensated for by a higher affinity for the critical substrate d-Lac.

pH dependence of peptide versus that of depsipeptide synthesis activity.

The partitioning of the syntheses of d-Ala-d-Ala and d-Ala-d-Hbut in VanA and other depsipeptide-competent dd-ligases has been shown to be pH dependent (17). Determination of the pH dependence of DdlN in synthesizing peptide versus depsipeptide (Fig. (Fig.3)3) directly paralleled the results obtained with VanA in similar experiments. At lower pHs (<7), d-Ala-d-Hbut synthesis predominates and is exclusive at a pH of <6 (Fig. (Fig.3).3). At pH 7.5, levels of synthesis of d-Ala-d-Hbut and d-Ala-d-Ala are relatively equal, while at a pH greater than 8, the capacity to synthesize peptide overtakes the capacity to synthesize depsipeptide, although the latter is never abolished. Open in a separate windowFIG. 3pH dependence of partitioning of the syntheses of peptide and depsipeptide by DdlN. (A) Autoradiogram of a thin-layer chromatography separation of the products of reaction mixtures containing 14C-D-Ala, unlabeled D-Ala, and d-Hbut. (B) Quantification of reaction products following phosphorimage analysis. Filled circles, D-Ala-d-Hbut; open circles, D-Ala-D-Ala.The partitioning of the formation of peptide versus depsipeptide as a function of pH by DdlM is comparable to that by VanA and depsipeptide-competent mutants of DdlB (17), which show essentially exclusively depsipeptide formation at lower pHs and increasing peptide formation as the pH increases. This implies a potential role for the protonated ammonium group of d-Ala2 in second-substrate recognition and suggests a mechanism for the discrimination between d-Ala and d-Lac at physiologic pH. The structural basis for this distinction remains obscure for DdlB and VanA or DdlN.

Concluding remarks.

Resistance to vancomycin and other glycopeptides is mediated through the synthesis of a peptidoglycan which does not terminate with the canonical d-Ala-d-Ala dipeptide. Thus, enterococci which exhibit the VanC phenotype, which consists of low-level, noninducible resistance to vancomycin only, have peptidoglycan terminating in d-Ala-d-Ser (19). On the other hand, bacteria which are constitutively resistant to high concentrations of glycopeptides, such as lactic acid bacteria and VRE exhibiting the VanA or VanB phenotype (high-level inducible resistance to vancomycin), incorporate the depsipeptide d-Ala-d-Lac into their cell walls (2, 12, 13). The enzymes responsible for the intracellular synthesis of d-Ala-d-Lac not surprisingly have significant amino acid sequence similarity with d-Ala-d-Ala ligases, which are responsible for d-Ala-d-Ala synthesis in all bacteria with a cell wall (9).The d-Ala-d-Lac synthases can be subdivided into two groups based on sequence homology: those found in the constitutively resistant lactic acid bacteria and those found in glycopeptide-producing organisms and VanA or VanB VRE (9, 14). The former have more similarity with exclusive d-Ala-d-Ala ligases. Indeed, single point mutations in d-Ala-d-Ala ligases which yield sequences more similar to those of lactic acid bacterium d-Ala-d-Lac ligases are sufficient to induce significant depsipeptide synthase activity in these enzymes (17). Similarly, mutational studies of the d-Ala-d-Lac ligase from Leuconostoc mesenteroides have demonstrated that the converse also holds (18). On the other hand, the molecular basis for depsipeptide synthesis by the VanA or VanB ligases is unknown, in large part due to the lack of protein structural information on which to base mutational studies, unlike the situation with d-Ala-d-Ala ligases, where the E. coli DdlB structure serves as a template for mechanistic research (11).Significantly, a major difference in the VanA or VanB ligases and other dd-ligases lies in the amino acid sequence of the ω-loop region, which closes off the active site of DdlB (11) and has been shown to contribute amino acid residues with the capacity to control the syntheses of d-Ala-d-Ala and d-Ala-d-Lac, notably, Tyr216 (17, 18). Until recently, the VanA and VanB ligases were exceptional in amino acid structure and had no known homologues. The sequencing of resistance genes from glycopeptide-producing bacteria has uncovered enzymes with >60% homology to VanA or VanB and which are virtually superimposable in the critical ω-loop region (14, 15). One of these, DdlM from S. toyocaensis NRRL 15009, has been shown to have d-Ala-d-Lac ligase ability (15, 16), although no rigorous analysis of this activity has been performed. The results presented here demonstrate that DdlN from the vancomycin producer A. orientalis C329.2 not only is a d-Ala-d-Lac ligase but also has significant functional homology with VanA. It is not known at present if, like S. toyocaensis NRRL 15009 (16), A. orientalis C329.2 also possess a d-Ala-d-Ala-exclusive ligase, though the presence of a vanX gene (14) suggests that it may.These studies demonstrate that DdlN cloned from a vancomycin-producing bacterium is a d-Ala-d-Lac ligase which has not only amino acid sequence homology with the dd-ligases from VRE but also functional homology. Thus, VanA, VanB, DdlN, and DdlM have likely evolved from similar origins. The fact that a vanH-vanA-vanX gene cluster can be found in other glycopeptide producers as well (14) suggests that the genes now found in VRE may have originated in glycopeptide-producing bacteria. Our finding that overexpressed, purified, DdlN shows many enzymatic characteristics similar (though not identical) to those of VanA suggests that the genes from glycopeptide-producing bacteria can be important in elucidating biochemical and protein structural aspects of the VRE proteins.  相似文献   

14.
This study was conducted to identify the factors affecting the performance of membrane bioreactor (MBR) for piggery wastewater treatment. The change of organic and nitrogen concentrations in piggery wastewater was studied to investigate the treatment efficiency. The increase of COD, BOD and NH3–N from 1150 to 2050 mg/L, 683 to 1198 mg/L and 154 to 248 mg/L has led to the decrease of treatment efficiency. Removal efficiencies of COD, BOD and NH3–N have decreased from 96.0% to 92.0%, 97.0% to 92.7% and 93.2% to 69.5%, respectively. The effects of biomass characteristics on membrane fouling were determined based on Pearson’s correlation coefficient (rp). It was found that MLSS had a negative correlation with permeate flux (rp = −0.745, at significant level of 0.05) while sludge floc size a positive correlation (rp = 0.731, at significant level of 0.05). MLSS and sludge floc size were found to be the dominant factors that controlled the membrane filterability while sludge viscosity, EPS, SMP and SV30 have taken as the sub-factors affecting membrane fouling.  相似文献   

15.
The spatial variability of the photochemical efficiency of phytoplankton photosystem II was analyzed in two contrasting regions of the Black Sea: the western part of the deepwater region and a region influenced by the Danube River discharge. The fluorescence values for open (F0) and closed (Fm) photosystem II reaction centers in the investigated areas varied by an order of magnitude and correlated closely. The potential photochemical efficiency of phytoplankton photosystem II (Fm ? F0)/Fm varied from 0.16 to 0.70. Three types of the vertical distribution of this index were found. In the first type, the values increased from the surface to the top of the thermocline and then remained stable down to the bottom of the euphotic zone; in the second type, they increased from the surface to the bottom of the euphotic zone; in shallow areas, they were stable within the euphotic zone. With an increase in light intensity, the phytoplankton photochemical efficiency decreased. The light inhibition of the photosystem II efficiency was more intense in the deeper than in the upper layers of the euphotic zone.  相似文献   

16.
Gonadal steroids alter the apparent molecular size of intrapituitary Follicle-Stimulating Hormone (FSH) in rats and monkeys as well as increase the percentage of acidic FSH isohormones in sheep. Hence, we hypothesized that the molecular size of ovine (o) FSH would be increased by gonadal steroids. Extracts of pituitaries from rams and wethers, as well as, from wethers which had been implanted with dihydrotestosterone (DHT), 17β-estradiol (E2) or both steroids (n=4–6 per treatment group) were subjected to analytical gel permeation chromatography using Sephadex G-100 Superfine. FSH concentrations in chromatographic fractions were determined by radioimmunoassays. Although FSH in pituitaries of non-implanted wethers eluted slightly earlier (i.e. larger) than FSH in pituitaries from E2-implanted wethers as evaluated by distribution coefficients (Kds) during chromatography (P<0.05), gonadal steroids did not consistently increase Kds but tended to decrease them. When Kds were extrapolated to apparent molecular weights using a series of standard proteins (bovine serum albumin (bSA), ovalbumin (OA), carbonic anhydrase (CA) and cytochrome c (CC)) that were included in each chromatogram, the differences between treatment groups were not statistically significant (P>0.05). Thus, in contrast to rats and monkeys, neither castration nor steroid-replacement appears to alter the molecular size of FSH in the sheep pituitary as evaluated by analytical gel permeation chromatography.  相似文献   

17.
The paper is concerned with the existence and asymptotic character of the nonlinear boundary value problemdG/dt=F(t,G,F, ¦α?β¦) (1) ¦α?β¦dF/dt=g(t,G,F, ¦α?β¦)G(o,¦α?β¦)=k 1,G(∞,¦α?β¦)=k 2 (2) as ¦α?β¦→ o+ The discussion is related to the problem of particle-number fluctuations in the theory of cosmic radiation andG andF denote respectively the probability generating functions for the electron distribution in an electron-initiated and a photon-initiated shower. A solution of the system (1) satisfying the boundary conditions (2) is constructed so that specified limiting conditions are fulfilled.  相似文献   

18.
F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3β3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3β3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.  相似文献   

19.
In the present study, we used genomic data, generated with a medium density single nucleotide polymorphisms (SNP) array, to acquire more information on the population structure and evolutionary history of the synthetic Frizarta dairy sheep. First, two typical measures of linkage disequilibrium (LD) were estimated at various physical distances that were then used to make inferences on the effective population size at key past time points. Population structure was also assessed by both multidimensional scaling analysis and k-means clustering on the distance matrix obtained from the animals’ genomic relationships. The Wright’s fixation FST index was also employed to assess herds’ genetic homogeneity and to indirectly estimate past migration rates. The Wright’s fixation FIS index and genomic inbreeding coefficients based on the genomic relationship matrix as well as on runs of homozygosity were also estimated. The Frizarta breed displays relatively low LD levels with r2 and |Dʹ| equal to 0.18 and 0.50, respectively, at an average inter-marker distance of 31 kb. Linkage disequilibrium decayed rapidly by distance and persisted over just a few thousand base pairs. Rate of LD decay (β) varied widely among the 26 autosomes with larger values estimated for shorter chromosomes (e.g. β=0.057, for OAR6) and smaller values for longer ones (e.g. β=0.022, for OAR2). The inferred effective population size at the beginning of the breed’s formation was as high as 549, was then reduced to 463 in 1981 (end of the breed’s formation) and further declined to 187, one generation ago. Multidimensional scaling analysis and k-means clustering suggested a genetically homogenous population, FST estimates indicated relatively low genetic differentiation between herds, whereas a heat map of the animals’ genomic kinship relationships revealed a stratified population, at a herd level. Estimates of genomic inbreeding coefficients suggested that most recent parental relatedness may have been a major determinant of the current effective population size. A denser than the 50k SNP panel may be more beneficial when performing genome wide association studies in the breed.  相似文献   

20.
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