Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose.

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

Mitochondrial DNAs and plasmids as taxonomic characteristics in Trichoderma viride.

Mitochondrial DNA (mtDNA) was purified from 12 isolates of the Trichoderma viride aggregate and found to be, on the average, 32.7 kb in size. Plasmids were present in the mtDNA preparations from 8 of 12 strains of T. viride examined. Plasmids in four of the strains produced ladderlike banding patterns on gels, and these plasmids were studied in detail. The ladderlike patterns were produced by single molecules that were supercoiled to various degrees. Plasmids from two of the strains do not have homology with the mtDNA but do have a limited amount of homology with each other. No phenotype could be associated with the presence of a plasmid. Restriction endonuclease digestion of the mtDNAs produced patterns in which the presence or absence of certain fragments correlated with the classification of the strains into T. viride group I or II. Phenetic cluster analysis and parsimony analysis of the fragment patterns produced groups that corresponded to T. viride groups I and II. The fragment patterns were very diverse, with nearly all strains having a unique pattern. However, two strains of T. viride group I from widely different geographical locations did have identical restriction patterns for all the enzymes used in this study. This result indicates that it may not be possible to use mtDNA restriction patterns alone to identify Trichoderma strains.     

两种不同来源的绿色木霉降解木质纤维素研究

利用纤维素酶高产菌绿色木霉Trichoderma viride降解木质纤维素是实现废料资源化的重要手段。本研究选取来自不同生境的两株T. viride,分别以玉米秸秆和甘草药渣为基质,测定两者滤纸纤维素酶（filter paper cellulase,FPase）活性和还原糖产量。从时间、温度、水分、pH 4个方面比较两株T. viride的环境适应性和不同基质的差异性。结果表明,以玉米秸秆为基质,T. viride XJ最适初始料液比为1:4-1:5.5,T. viride AG最适初始料液比为1:5-1:5.5。初始料液比1:5.5时,T. viride AG产FPase活性显著高于T. viride XJ。两株T. viride最适发酵温度均为28℃,各温度处理下不同菌株间无显著差异。两株T. viride均表现为还原糖消耗。以甘草药渣为基质,T. viride XJ最适初始料液比为1:2-1:2.5,T. viride AG最适初始料液比为1:3-1:3.5。料液比高于1:3,T. viride AG产FPase活性显著高于T. viride XJ。T. viride AG最适发酵温度为28℃,T. viride XJ最适发酵温度为23-28℃。温度低于28℃,T. viride XJ产FPase活性显著高于T. viride AG。两株T. viride均表现为还原糖积累。两株T. viride最适初始pH均为6-7,最适发酵时间均为3d。最优发酵条件下FPase活性：T. viride AG>T. viride XJ。对T. viride产FPase诱导能力：甘草药渣>玉米秸秆。变差分解表明两株T. viride产FPase活性差异主要源于菌株对生境的生态适应。比较分析菌种来源、基质类型、环境条件对T. viride发酵效果的影响,将有助于该菌大规模应用性研究。     

Yeast mutants conferring resistance to toxic effects of cloned human insulin-like growth factor I

The production of extracellular human insulin-like growth factor I (IGF-I) in yeast is deleterious to the growth of the host organism. Mutants resistant to the toxic effects of IGF-I production were isolated. A subset of these mutants produced levels of IGF-I greater than the parent strain and were due to chromosomal recessive mutations at a single locus, hpx1. The overproduction of IGF-I was independent of the original promoter and vector expression system. The mutant strains also displayed enhanced extracellular production of other heterologous proteins.     

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol

Abstract Eleven strains of Trichoderma viride , 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica , using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum , a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.     

Comparison of chloroplast DNAs by specific fragmentation with EcoRI endonuclease

Summary Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide sugar heptose are deficient in proteinb. All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents. They excrete the periplasmic enzyme ribonuclease I. Mutants deficient in proteinsc and/ord have the same sensitivity towards these compounds as the parent strain. Cells of single, double and triple mutants are all rod-shaped. Electrophoretic analysis of cell evelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins. Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate.     

Mutants of Ralstonia (Pseudomonas) solanacearum sensitive to antimicrobial peptides are altered in their lipopolysaccharide structure and are avirulent in tobacco.

Ralstonia solanacearum K60 was mutagenized with the transposon Tn5, and two mutants, M2 and M88, were isolated. Both mutants were selected based on their increased sensitivity to thionins, and they had the Tn5 insertion in the same gene, 34 bp apart. Sequence analysis of the interrupted gene showed clear homology with the rfaF gene from Escherichia coli and Salmonella typhimurium (66% similarity), which encodes a heptosyltransferase involved in the synthesis of the lipopolysaccharide (LPS) core. Mutants M2 and M88 had an altered LPS electrophoretic pattern, consistent with synthesis of incomplete LPS cores. For these reasons, the R. solanacearum gene was designated rfaF. The mutants were also sensitive to purified lipid transfer proteins (LTPs) and to an LTP-enriched, cell wall extract from tobacco leaves. Mutants M2 and M88 died rapidly in planta and failed to produce necrosis when infiltrated in tobacco leaves or to cause wilting when injected in tobacco stems. Complemented strains M2* and M88* were respectively obtained from mutants M2 and M88 by transformation with a DNA fragment harboring gene rfaF. They had a different degree of wild-type reconstituted phenotype. Both strains retained the rough phenotype of the mutants, and their LPS electrophoretic patterns were intermediate between those of the wild type and those of the mutants.     

Mutants ofStreptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites

Mutants ofStreptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, γ-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described inStreptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, ζ-rhodomycinone and glycosides of ε-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases.     

Production of fungal biomass protein using microfungi from winery wastewater treatment

This study was carried out to investigate the production of fungal biomass protein (FBP) in treatment of winery wastewater using microfungi. Three fungal strains, Trichoderma viride WEBL0702, Aspergillus niger WEBL0901 and Aspergillus oryzae WEBL0401, were selected in terms of microbial capability for FBP production and COD reduction. T. viride appeared to be the best strain for FBP production due to high productivity and less nitrogen requirement. More than 5 g/L of fungal biomass was produced in shake fermentation using T. viride without nitrogen addition, and by A. oryzae and A. niger with addition of 0.5-1.0 g/L (NH4)2SO4. The FBP production process corresponded to 84-90% COD reduction of winery wastewater. Fungal biomass contained approximately 36% protein produced by two Aspergillus strains, while biomass produced by T. viride consisted of 19.8% protein. Kinetic study indicated that maximum fungal cell growth could be achieved in 24h for T. viride and 48 h for A. oryzae and A. niger. Current results indicated that it could be feasible to develop a biotechnological treatment process integrated with FBP production from the winery waste streams.     

Screening of some wild fungal isolates for cellulolytic activities

Six wild fungal strains, Trichoderma viride, T. harzianum, Gliocladium virens, Aspergillus terreus, A. niger and Tiarosporella phaseolina , isolated from decomposed jute stacks and diseased jute stem, were tested for their cellulolytic and hemicellulolytic activities and compared with T. reesei MCG 77. Filter paper cellulase production by all these wild strains were lower than those produced by T. reesei while some strains ( T. viride, T. harzianum and G. virens ) possessed carboxymethyl cellulase, β-glucosidase, xylanase and β-xylosidase activities comparable to T. reesei. A. terreus and A. niger produced 3·2 and 1·2 times respectively, greater β-glucosidase activity compared to T. reesei when grown on microcrystalline cellulose.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

Role of outer membrane components in the nonspecific binding ofEscherichia coli to cellulose

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.     

重组pEGFP—C2-p100-TSN点突变质粒构建及表达

目的将6个不同的p100-TSN．Mutants基因片段分别定向连入PEGFP—C2质粒中,使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达,从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础。方法利用EcoR I和Xho I双酶切方法从6个pcDNA3．1（＋）-p100-TSN．Mutants重组质粒中分别获得p100-TSN．Mutants的cDNA片段,将其连人pEGFP—C2质粒载体中,再将成功构建的6个pEGFP—C2-p100-TSN．Mutants质粒分别转染COS7细胞中,荧光显微镜下观察绿色荧光蛋白表达。结果①将重组质粒进行双酶切鉴定可见p100-TSN．Mutants的cDNA片段;②转染重组质粒后可观察到绿色荧光蛋白的表达。结论①6个pEGFP—C2-p100-TSN．Mutants重组质粒构建成功;②p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达。     

Isolation and characterization of Zymomonas mobilis mutants resistant to octadecyltrimethylammonium chloride,a detergent acting on hopanoid-producing bacteria

Growth of the hopanoid-producing bacterium Zymomonas mobilis was inhibited at low concentrations of the cationic detergent octadecyltrimethylammoniumchloride (OTAC). A relationship between sensitivity of Zymomonas mobilis to OTAC, presence of hopanoids and ethanol tolerance was postulated. Mutants resistant to OTAC were isolated from strains ZM1 and ZM4. They did not present any alteration of the hopanoid content and their squalene cyclases showed the same sensitity to OTAC as the parent enzymes. Resistance to OTAC paralleled pleiotropic effects including, enhanced accessibility of the membrane-bound alkaline phosphatase, important release of proteins from cells by Tris/HCl treatment, increased resistance to antibiotics and increased sensitivity to ethanol. In addition, OTAC^R mutants were also characterized by the synthesis or the overproduction of an outer membrane protein (F53) not detected on 2D-PAGE maps of parent strains and by a normal heat shock response. The role of hopanoids, heat shock proteins, protein F53 and membrane organization in ethanol tolerance is discussed.Abbreviations OTAC octadecyltrimethylammoniumchloride - SLS sodium lauryl sarcosinate     

Genes affecting the major outer membrane proteins of Escherichia coli K-12: Mutations at nmpA and nmpB

Summary Strains of Escherichia coli K-12 carrying mutations at either of two distinct loci (nmpA and nmpB) produce a new outer membrane pore protein which is not present in wild type cells. Mutations at either of these loci result in sensitivity to phage TC45, which can use this new protein as its receptor, and the new protein (the NmpAB protein) appears to be identical in both NmpA and NmpB mutants. In order to determine whether both of these loci contain structural genes for the NmpAB protein, strains carrying secondary mutations at either of these loci which produced altered proteins were sought by selecting for resistance to phage TC45. Mutants which produced proteins with altered electrophoretic mobility and altered peptide maps were isolated from strains carrying both nmpA and nmpB mutations, and these secondary mutations mapped at the same sites as the original mutations leading to production of the NmpAB protein. This suggests that both loci contain structural genes. Strains mutant at nmpB which can no longer produce the protein can mutate at the nmpA locus to produce the new protein, indicating that both genes can exist in the same cell. Since the altered proteins of mutant strains could be distinguished from one another, we attempted to construct strains in which both nmpA and nmpB were expressed. In all cases only the protein produced by the nmpB mutation was produced, indicating some form of cooperative regulation of the two genes.     

Utilization of γ-Aminobutyric Acid as the Sole Carbon and Nitrogen Source by Escherichia coli K-12 Mutants

Wild-type strains of Escherichia coli K-12 cannot grow in media with gamma-aminobutyrate (GABA) as the sole source of carbon or nitrogen. Mutants were isolated which could utilize GABA as the sole source of nitrogen. These mutants were found to have six- to ninefold higher activities of gamma-aminobutyrate-alpha-ketoglutarate transaminase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) than those of the wild-type parent strains. Secondary mutants derived from these GABA-nitrogen-utilizing strains were able to grow on GABA as the sole source of carbon and nitrogen. They also grew faster on a variety of other carbon and nitrogen sources, and their growth was more strongly inhibited by different metabolic inhibitors than was that of the parent strains. The nature of the two mutations and the possible genes involved are discussed. A scheme of the pathway for GABA breakdown in E. coli K-12 is presented.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

EFFECT OF CERTAIN CHLORONITROBENZENES ON GERMINATION, GROWTH AND SPORULATION OF SOME FUNGI

When grown in an atmosphere saturated with tetrachloronitrobenzene (TCNB) vapour, Botrytis cinerea showed retarded germination and colony growth and its sporulation was completely suppressed. Retardation of growth was greater on dilute or acidified media and at lower temperatures.
Similar effects were shown by Bhizoctonia solani, Phytophthora parasitica, Mucor hiemalis and Trichoderma viride but none of these was as sensitive as Botrytis cinerea , especially as regards linear growth. Of two strains of Fusarium caeruleum , one was much more sensitive than the other, both in linear growth and sporulation. Pythium deBaryanum was unaffected in its growth rate.
TCNB, as a growth repressant, is fungistatic without being fungicidal.
With fungi which are sensitive, tetrachloronitrobenzene was more effective than the penta-derivative, except for Trichoderma viride where the converse applied.     

Plasmid R46-mediated protection against bleomycin is poLA+-dependent

Strains of Escherichia coli deficient in post-replication recombination repair were more sensitive to bleomycin than wild-type, repair-proficient strains. Mutants lacking excision repair functions were no more sensitive to bleomycin than the wild-type strains, indicating that this pathway is not involved in the repair of bleomycin-damaged DNA. Plasmid R46 not only protected repair-proficient strains but also those with recB, recC, uvrA or lig genotypes, suggesting that R46 protection against bleomycin is independent of these host repair functions. However, R46 protection was abolished in recA or polA strains, indicating that these gene functions are necessary for plasmid-mediated protection. It is suggested that protection may be due to a recA+-dependent interaction of a plasmid-encoded product with host DNA polymerase I, resulting in an increase in the DNA repair capacity of cells.