Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis-nitrocellulose blotting

An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria.     

不同施肥模式对设施菜田土壤酶活性的影响

利用天津日光温室蔬菜不同施肥模式定位试验,研究了6种施肥模式对设施菜田土壤酶活性的影响.结果表明: 番茄生育期间不同施肥模式土壤α-葡萄苷酶、β-木糖苷酶、β-葡萄苷酶、β-纤维二糖苷酶、几丁质酶和磷酸酶的活性总体上均呈先增后降的趋势,土壤脲酶活性呈先增高后趋于平缓的趋势.与全部施用化肥氮相比,5种有机无机肥料配施模式土壤酶活性均有所提升,且随猪粪施用量的增加,尤其是配施秸秆条件下,土壤酶活性显著增加.番茄各生育期土壤酶活性与土壤微生物生物量碳、氮和可溶性有机碳、氮之间总体上呈显著或极显著正相关关系.同等养分投入量下,有机无机肥配施,特别是配施一定的秸秆可有效提高设施菜田土壤酶活性,维持较高的菜田土壤肥力,有利于设施蔬菜的可持续生产.     

Characterization of the branching patterns of glycogen branching enzyme truncated on the N-terminus

Truncation of 112 amino acids at the N-terminus (Nd(1-112)) changes the chain transfer pattern of the Escherichia coli glycogen branching enzyme (GBE) [Arch. Biochem. Biophys. 397 (2002) 279]. We investigated further the role of the N-terminus by engineering other truncated GBEs and analyzing the branching pattern by high-performance anion-exchange chromatography. The wild type GBE transfers mainly chains with a degree of polymerization (d.p.) of 8-14, the Nd(1-112) enzyme transfers a greater proportion of chains with higher d.p. 15-20, whereas the 63- and 83-amino acid deleted enzymes had an intermediate pattern of transferred chains (d.p. 10-20). These data showed that a progressive shortening of the N-terminus leads to a gradual increase in the length of the transferred chains, suggesting that the N-terminus provides a support for the glucan substrate during the processes of cleavage and transfer of the alpha-(1-4) glucan chains.     

Simultaneous carriage of Candida albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis

Abstract Genetic diversity of 160 Candida albicans isolates from the oral cavity of 16 HIV-infected adults prior to antifungal treatment was assessed using multilocus enzyme electrophoresis (10 C. albicans colonies were randomly chosen from each specimen culture). 20 electrophoretic types were distinguished from the analysis of 21 enzyme loci (10 were polymorphic). Five patients (31%) were found to be colonized by 2 or 3 genetically distinct strains. Nevertheless, in these five cases, one strain predominated (from 7 to 9 of the 10 colonies). Some HIV + patients with oral candidiasis appear to be simultaneously infected with several genetically different C. albicans strains before antifungal treatment.     

Comparative analysis of two-component signal transduction system in two streptomycete genomes

Species of the genus Streptomyces are major bacteria responsible for producing most natural antibiotics. Streptomyces coelicolor A3(2) and Streptomyces avermitilis were sequenced in 2002 and 2003, respectively. Two-component signal transduction systems (TCSs), consisting of a histidine sensor kinase (SK) and a cognate response regulator (RR), form the most common mechanism of transmembrane signal transduction in prokaryotes. TCSs in S. coelicolor A3(2) have been analyzed in detail. Here, we identify and classify the SK and RR of S. avermitilis and compare the TCSs with those of S. coelicolor A3(2) by computational approaches. Phylogenetic analysis of the cognate SK-RR pairs of the two species indicated that the cognate SK-RR pairs fall into four classes according to the distribution of their orthologs in other organisms. In addition to the cognate SK-RR pairs, some potential partners of non-cognate SK-RR were found, including those of unpaired SK and orphan RR and the cross-talk between different components in either strain. Our study provides new clues for further exploration of the molecular regulation mechanism of streptomycetes with industrial importance.     

Use of internal standards in electrophoretic surveys of enzyme polymorphism

General criteria are described for selecting internal standards for electrophoretic gels. Gel sieving behavior is used to characterize the size and charge of enzymes of interest and of putative standard proteins. For a variety of enzymes of the butterfly Colias meadii, hemoglobin and ferritin are shown to be effective standards.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

Abstract Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78^T, S. innocens strain B256^T, Brachyspira aalborgi strain 513A^T, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes ( genetic distances between groups = 0.6–0.9). Chicken spirochaete strain C1 and B. aalborgi 513A^T represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3–99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/ Serpulina species.     

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus

The isolated cell wall of Streptomyces griseus 52–1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.     

海洋来源链霉菌MY0504产纤溶酶的发酵条件优化

【目的】以发酵液纤溶酶活力为指标,优化海洋来源的链霉菌菌株MY0504的发酵条件。【方法】在菌株生长曲线及单因素试验基础上,采用Plackett-Burman设计筛选影响纤溶酶活性的主要因素,进一步用最陡爬坡试验及Box-Behnken中心组合设计法优化发酵条件。【结果】纤溶酶活性最高的发酵条件为:葡萄糖21.68 g/L,酵母粉25.31 g/L,NaCl5.0 g/L,K_2HPO_4·3H_2O3.0 g/L,MgSO_4·7H_2O 0.5 g/L,FeSO_4·7H_2O 0.02 g/L,装液量50 mL(250 mL摇瓶),接种量10%(体积比),初始pH 7.5,温度24°C,转速200 r/min,培养时间4.5 d。发酵液纤溶酶活性可达2 190.6 U/mL。【结论】确定了MY0504菌株产纤溶酶的最优发酵条件,为该酶的进一步分离纯化及性质研究奠定基础。     

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.     

一株生防圆突起链霉菌的分类鉴定

菌株SCY311是从河南省凤凰山土壤样品中分离到的对多种植物病原真菌具有拮抗活性的一株放线菌。为了明确其分类地位, 在形态特征、培养特征、生理生化特征、细胞壁组分测定等传统分类学方法的基础上, 测定和分析了菌株的16S rRNA基因序列。结果表明, 菌株SCY311在高氏一号培养基上生长良好, 基内菌丝呈褐色; 气生菌丝灰色至鼠灰色, 不产生可溶性色素, 无吸水现象; 孢子链卷曲, 末端形成闭合或开放螺旋; 孢子椭圆或圆柱状, 表面形成结节状突起; 生理生化特征和在国际链霉菌计划(ISP)培养基上的培养特     

A new electrophoretic approach to biochemical systematics of bees

SDS electrophoresis of soluble proteins from 49 species of bees from 8 families produce a conservative character set. Differences between species appear to be actual differences in polypeptides and their concentrations rather than spurious differences. The data set based on the absorption values of bands proves to be more useful than does a data set based on the molecular weights of bands present or one based on the presence and absence of bands. Numerical analysis of a matrix of distance coefficients based on absorption values of bands shows much overlap between families of bees. Also, some taxa of questionable affinities when compared morphologically can be shown to be biochemically quite similar to other members of their traditional morphological families. Other taxa, which appear very similar morphologically, are highly differentiated biochemically. Examples are presented to compare and contrast the data from the SDS-separated character set to predictions based on the theories of regulatory and constant rate protein evolution. The results of the study largely agreed with predictions based on constant rate protein evolution. Exceptions may be understood in the framework of regulatory evolution. Consequences of discordance between morphological and biochemical phenetics are discussed as they affect systematic classifications.     

生物炭施用方式对黑土和潮棕壤养分及氮磷转化相关酶活性的影响

为探明不同生物炭施用方式对农田土壤养分含量及元素转化的影响,通过设置在黑土和潮棕壤的两个为期5年的田间定位试验,研究每年低量施用(AL,22.5 t· hm-2·a-1)和间隔性高量施用(IH,112.5 t·hm-2·5 a-1)玉米秸秆生物炭对土壤碳、氮、磷含量,土壤生物学性质及氮磷转化相关酶活性的影响,以期为秸秆...     

Use of a plant-derived enzyme template for the production of the green-note volatile hexanal

Hexanal is a key organoleptic element of green-note that is found in both fragrances and flavors. We report a novel process for the production of hexanal using immobilized enzyme templates extracted from different plant sources in combination with hollow-fiber ultrafiltration for in situ separation. Enzyme templates, known to be responsible for the synthesis of hexanal from linoleic acid (18:2), were isolated from naturally enriched tissues including carnation petals, strawberry and tomato leaves. These templates were immobilized in an alginate matrix and used as a biocatalyst in a packed-bed bioreactor. Continuous product recovery was achieved using a hollow-fiber ultrafiltration unit. The effects of pH, reaction temperature, and substrate and enzyme concentrations were studied and their effects on hexanal generation identified and optimized. Utilizing optimized conditions, hexanal production 112-fold higher than endogenous steady-state levels in a corresponding amount of plant tissue could be achieved over a 30-minute period. Based on the reactor studies, product inhibition also appears to be an important factor for bioreactor-based hexanal production.     

Blasticidin S-producing Streptomyces morookaensis possesses an enzyme activity which hydrolyzes puromycin

Abstract Puromycin was inactivated without the presence of acetyl coenzyme A when incubated with extracts of blasticidin S-producing Streptomyces morookaensis . The two derivatives from puromycin, contained in the reaction mixture, were detected by thin-layer chromatography, purified by high performance liquid chromatography and analyzed for determination of the chemical structures by ¹H-nuclear magnetic resonance and positive-ion fast atom bombardment mass spectrometries. The analytical data revealed that puromycin was inactivated by the hydrolysis of amide linkage between the aminonucleoside and 0 -methyl-l-tyrosine moieties, suggesting that S. morookaensis possesses an enzyme activity which hydrolyzes puromycin.     

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen-fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.     

链霉菌Z94-2碱性脂肪酶产生条件及酶学性质

在１５２株脂肪酶产生菌中,链霉菌Ｚ９４－２产脂肪酶活力为５９６ｕ／ｍＬ,其最适培养基（ｇ／Ｌ）为：糊精１０、黄豆饼粉３０、尿素１０、Ｋ２ＨＰＯ４０．５、ＭｇＳＯ４０．５、ＮａＣｌ１和ＡＥＯ９０．５,产酶的最适条件为：初始ｐＨ９．５～１０．０,在２６℃培养４８ｈ。用ＰＶＡ橄榄油乳化系统测定该酶的最适ｐＨ９．８,最适温度３７℃,在ｐＨ８．６～１０．２于５℃存放２４ｈ,酶活力不变。０．１４ｍｏｌ／Ｌ的氯     

Glucose-6-phosphate dehydrogenase and malate dehydrogenase enzyme electrophoretic patterns amongst strains of Bacteroides fragilis

Fifty-two strains of Bacteroides fragilis were examined for their enzyme electrophoretic patterns of glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH). All strains tested possessed high levels of both enzymes but the G6PDH reduced NADP whereas MDH was NAD-dependent. Twenty-seven strains produced single bands of both G6PDH and MDH. In all cases G6PDH migrated faster than MDH. Strains clustered by a single linkage algorithm were recovered in eight clusters at the 77% similarity level. The remaining 25 strains produced multiple bands of one or both enzymes. These were recovered in six clusters at the 72% similarity level using the same algorithm. The results of this study revealed considerable heterogeneity of enzyme patterns within B. fragilis.     

Conversion of Kepone by Methanosarcina thermophila

Abstract Amino acid sequences of protease inhibitors ( Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Müller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Müller, but belongs to the same taxon as S. lividans 66.