球孢白僵菌降解昆虫体壁蛋白酶基因 CDEP-1的克隆与序列分析

构建了球孢白僵菌不同诱导时间的混合cDNA文库。根据丝氨酸类蛋白酶的保守区域设计引物,以构建cDNA文库同样的mRNA为模板,采用RT-PCR法得到长度为594bp的片段BbP。序列测定表明BbP是球孢白僵菌类枯草杆菌蛋白酶Prl的一部分,以BbP为探针,从上述cDNA文库筛选得到长度为1557bp的克隆CDEP-1。CDEP-1含有一个1134bp的开放阅读框（ORF）,编码377个氨基酸,分子量为38616、PI=8.302的蛋白酶前体。CEDP-1的核苷酸序列与蛋白酶K、金龟子绿僵菌Prl、球孢白僵菌Prl的同源性分别为：57.9%、54.7%、83.3%。根据cDNA序列扩增到CDEP-1的基因组序列,分析表明其中含有3个内含子。Southern杂交表明CDEP-1在球孢白僵菌是单拷贝。     

Purification and characterisation of a novel 34,000-Mr cell-associated proteinase from the dermatophyte Trichophyton rubrum

Abstract A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton rubrum by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000- M _r (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15,000- M _r (reduced SDS-PAGE) and 37,000- M _r (substrate SDS-PAGE). It had a pH optimum of 7.5 and a p I of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000- M _r proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides immitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinase to be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the proteinase remains speculative although it may play a role in the development and subsequent proliferation of the fungus in vivo.     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

Abstract The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti- M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weigh ( M _r) M. leprae protein (MLP) with a subunit M _r of 22000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis . It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Gene structure and expression of the gene from Beauveria bassiana encoding bassiasin I, an insect cuticle-degrading serine protease

Genomic and cDNA encoding Beauveria bassiana bassiasin I, a potential cuticle-degrading serine protease, were isolated and analysed. Bassiasin I gene is comprised of 1137 bp (379 aa) and 3 introns which are 69, 62 and 68 bp long. The comparison of a deduced amino acid sequence with Metarhizium anisopliae Pr1, B. bassiana Pr1, and proteinase K showed high homology. When the cDNA including the intact signal peptide was expressed in E. coli, a clear proteolytic-degraded zone on LB-skimmed milk plates was observed.     

A fungal lectin and its apparent receptors on a nematode surface

Abstract A lectin was isolated from the nematode-trapping fungus Arthrobotrys oligospora . This carbohydrate-binding protein was developmentally regulated and was found only on trap-bearing mycelia. The lectin receptor on the surface of the nematode Panagrellus redivivus has, furthermore, been investigated using homogenates from whole nematodes or nematode cuticle. The ability of macromolecules from nematodes, fractionated according to M _r and lectin affinities, to inhibit the capture of nematodes by A. oligospora was tested using an inhibition assay, based on a simple dialysis membrane technique. One major lectin-binding glycoprotein, with apparent M _r of 65 000, was isolated from the nematode cuticle.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Molecular masses of cAMP-binding proteins in yeasts

Abstract The cAMP-binding proteins of different yeasts were photoaffinity labeled using 8- N ₃-[³²P]cAMP, and the M _r values of the labeled proteins estimated by SDS-polyacrylamide gel electrophoresis. The M _r values of the cAMP-binding proteins may be grouped into two size classes: (A) M _r of about 50 000 represented by Saccharomyces cerevisiae and S. uvarum , and (B) M _r of about 60 000 represented by Kluyveromyces fragilis, K. lactis, K. marxianus, S. globosus and S. rouxii .     

松褐天牛球孢白僵菌高毒力航天诱变菌株的筛选

【目的】松褐天牛Monochamus alternatus是我国松材线虫病Bursaphelenchus xylophilus的主要媒介昆虫。为了更好地开发利用松褐天牛病原微生物球孢白僵菌Beauveria bassiana, 本研究通过航天搭载诱变及室内筛选, 获得球孢白僵菌高毒力诱变菌株。【方法】将经神舟八号飞船航天搭载诱变后的孢子稀释液涂布在PDA平板上培养,获得单菌落菌株,进而筛选获得高毒力诱变菌株。观察所获9个航天诱变菌株的菌落形态、菌落生长速度、产孢量、孢子萌发率及抗高温胁迫能力等生物学特性, 在此基础上筛选出生物学性状优良的菌株B159, B252和B305, 并进一步对松褐天牛4龄幼虫进行生物测定。再通过撒菌粉和注射菌液方法, 检验B252和B305对松木段内松褐天牛幼虫的杀虫效果。【结果】球孢白僵菌航天诱变菌株的生物学特性与野生型菌株cfcc81357存在分化。9个航天诱变菌株的菌落形态发生了不同程度的改变,6个菌落生长速率出现负向变异,仅诱变菌株B159, B252和B305能产生分生孢子。航天诱变菌株B252和B305在浓度为1.0×107 cfu/mL时对松褐天牛4龄幼虫的校正死亡率均为100%, 半致死中时(LT₅₀)分别为8.08和8.56 d, 明显优于野生型菌株, 显示出对松褐天牛的极强毒力。使用撒菌粉和孢子液体注射方法, 诱变菌株B252和B305对松木段内松褐天牛幼虫死亡率比野生型菌株高。【结论】诱变菌株B252和B305可能是优良菌株, 对生物防治松褐天牛方面有潜在的实用价值。     

Colicin B consists of a single polypeptide chain

Abstract Colicin B was isolated in pure form from Escherichia coli Cl139 and was shown to consist of a single polypeptide chain with an apparent M _r of 70000. Therefore, it does not differ from other colicins where the toxic activity resides in one polypeptide.     

Potential of Beauveria bassiana (Hyphomycetes: Moniliales) for controlling the white pine weevil, Pissodes strobi (Col., Curculionidae)

Abstract:  Studies were conducted under laboratory conditions to document the potential of Beauveria bassiana conidia applications for controlling the white pine weevil, Pissodes strobi (Peck). A screening test including six B. bassiana isolates allowed us to demonstrate that CFL (Centre de Foresterie des Laurentides) and IP-CPB (Île Perrault-charançon du pin blanc) were the most virulent isolates among the ones tested, with percentages of mortality after 3 weeks of 73% and 85.5%, respectively. These two B. basiana isolates were applied either onto soil or branch sections to compare the effectiveness of these potential control strategies. Greater than 75% mortality was observed within 3 weeks for both modes of application using suspensions of B. bassiana at a concentration of 1.0 × 10⁸ (soil application) and 1.0 × 10⁹ conidia/ml (branch application). The results demonstrate for the first time that B. bassiana is an effective entomopathogen against P. strobi .     

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal

Abstract We have isolated two alkaline phosphatases (H-AP and L-AP, for high and low molecular mass, respectively) from Pseudomonas aeruginosa PA01. These two enzymes were found to differ in mobility on sodium dodecyl sulphate polyacrylamide gels (H-AP, M _r = 51 000 and L-AP, M _r = 39 500), amino-terminal amino acid sequence and did not cross-react. Both enzymes were active as phosphomonoesterases while only L-AP demonstrated any phosphodiesterase activity. Both enzymes were purified from P. aeruginosa grown in phosphate limiting conditions using the same protocol and were identified in both periplasmic and extracellular locations. A low level of H-AP was produced constitutively whereas L-AP was produced only after induction by reduced phosphate concentration in the growth medium. An L-AP-like enzyme has been previously described, however, this is the first report of a second P. aeruginosa alkaline phosphatase.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

Methanol dehydrogenase from Hyphomicrobium MS 223

Abstract The monomethyl sulfate-degrading bacterium, Hyphomicrobium MS 223 , contains a NAD(P)-independent methanol dehydrogenase (EC 1.1.99.8) which was isolated and characterized. The enzyme was activated by ammonium ions, had an M _r of 118000 and was composed of two subunits of identical M _r. It showed a broad substrate specificity for primary alcohols and was able to oxidize secondary alcohols and several aliphatic aldehydes. The new competitive inhibitor acetaldehyde oxime inhibited aldehyde oxidation more strongly than alcohol oxidation.     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .     

Intergeneric protoplast fusion in lactic acid bacteria

Abstract Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of M _r 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The M _r 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the M _r 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the M _r 130 000 and 28 000 proteins are structurally different.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Activation of Clostridium botulinum type B and E derivative toxins with lysine-specific proteases

Abstract Clostridium botulinum type B and E derivative toxins were activated with lysyl endopeptidase or endoproteinase Lys-C, which splits only the bond involving the carboxyl group of a lysine residue. Type B toxin was more efficiently activated with lysyl endopeptidase; type E toxin was more efficiently activated with trypsin. Type B toxin was split by the lysine-specific protease into 2 fragments of molecular sizes indistinguishable from those induced with trypsin. Type E toxin was split by the same protease into 3 fragments, 2 of which had M _r identical to those obtained with trypsin, the other having an M _r less than that of the heavy chain but greater than that of the light chain. These results attest that both activation and nicking of type B and E derivative toxins are ascribable to cleavage, not of an arginyl, but of a lysyl bond.