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Nowadays, the use of agricultural by‐products, as the cheap substrate for the production of value‐added products, is of high interest for the researchers and practitioners. Cellulase is a relatively expensive and a very important industrial enzyme where in this study was produced form rice by‐products under solid‐state fermentation. A new mutant of Trichoderma reesei was used for cellulase production. The effective variables were initially screened by “Plackett and Burman design.” Afterward, the main variables including moisture content, P source, incubation temperature, and incubation time were optimized by “one factor at a time design.” Finally, the resulting variables including 74% for moisture content, 2 g/L for K2 HPO 4, 30°C of incubation temperature, and 4 days of incubation time were reported as the ultimate optimal condition for cellulase production.

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production.

The use of polygalacturonase (PG) preparations in winemaking promotes the release of phenolic compounds. A PG from a new source, Aspergillus sojae mutant, was semi-purified and tested for grape mash maceration. Crude extract (CE), a commercial pectinase, and two high PG activity semi-purified preparations, FI and FII , were applied for maceration at PG activity of 3.5 U g−1 of grape for 46 h. Enzyme-assisted maceration significantly (P < 0.05) increased the total phenolic content from 255.8 to 916.3 ± 5.2, 5732.9 ± 9.9, 563.4 ± 6.7 and 620.6 ± 18.4 mg L−1 for CE, commercial pectinase, FI and FII, respectively. The content of individual phenolics such as gallic, protocatechuic, chlorogenic and p-coumaric acids was improved. Principal component and hierarchical clustering analyses suggested that CE has a better performance upon the release of phenols. Semi-purified preparations acted similar to commercial pectinase. These findings open an opportunity for the potential use of PG from the mutant strain as an alternative macerating enzyme., EU-FP7-Marie Curie Action (PIRSES-GA 2010-269211); Biotechnology and Synthetic Biology Focus Group (0020DII001 0820000100); National Council on Science and Technology of Mexico (CONACyT) (204152)

Summary Bacteria associated with free-living amoebae have attracted considerable attention because of their role in human disease and as models for studying endosymbiosis. However, the identification and analysis of such novel associations are hindered by the limitations of methods for isolation and axenization of amoebae. Here, we replaced the heat- inactivated Escherichia coli, which is typically used as food source during axenization, with a live E. coli tolC knockout mutant strain hypersensitive to antibi- otics. Together with the addition of otherwise suble- thal amounts of ampicillin, this approach tripled the success rate and reduced the time required for axenization by at least 3 days. Using this method for two environmental samples, 10 Acanthamoeba strains were isolated, seven of which contained bac- terial symbionts. In three cases, amoebae harbouring two phylogenetically distinct symbionts were recov- ered, supporting a more widespread occurrence of multi-partner symbiotic associations among free- living amoebae.

973 Programs [2010CB833802, 2012CB721005]; National Science Fund for Distinguished Young Scholars [30325044]

Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved mithramycin analogues mithramycin SK and mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to mithramycin SK. This work shows the ability to transform the previously useless mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.

Three rare carbamate-containing alkaloids, penicimutamides A–C (1–3), were isolated from a fungal mutant from the diethyl sulfate (DES) mutagenesis of marine-derived Penicillium purpurogenum G59. Their structures, including their absolute configurations, were determined by spectroscopic methods, especially the X-ray crystallography and CD analyses. HPLC-UV and HPLC-MS analyses evidenced that 1–3 were only produced in the mutant strain via biosynthetic pathways that were silent in the parental strain and activated by DES mutagenesis.

IS-Tlk/Kyo, a rat mutant strain derived from IS/Kyo strain, exhibits a kinked and/or short tail, in addition to a congenital anomaly of the lumbar vertebrae that is a hallmark of IS/Kyo rats. Homozygotes (Tlk/Tlk) of Tlk dominant gene are known to die during embryonic development. The present report deals with the morphological features of heterozygous IS-Tlk/Kyo rat fetuses in comparison with those of IS/Kyo rat fetuses. One of the morphological features was a high incidence of tail vertebral anomalies in IS-Tlk rats (81.6% versus 0% in IS/Kyo rats). Significantly low values in number of live fetuses and ossified 5th sternebra and sacral and caudal vertebrae were observed in IS-Tlk/Kyo rats compared with those in IS/Kyo rats as well as a low incidence of fetuses with ventral septal defects in IS-Tlk/Kyo (0% versus 54.4% in IS rats). These results suggest that the Tlk gene may be involved in the formation of the vertebral centra and the ventral septum when it expresses on the genetic background of the IS rat.

National Science Fund for Distinguished Young Scholars [30325044]; Chinese Ministry of Education [306010]

Inducamide A (Ia), B (Ib) and C (II) are shown to not exhibit significant antibacterial activity, but (II) reveals modest cytotoxicity.

Members of the Mycobacterium tuberculosis group synthesize a family of long-chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non-oxygenated α-mycolic acid and the oxygenated keto- and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.

Two new avemectin analogues 1 and 2 belonging to 'b' series of components were isolated from a ∆aveCDE mutant Streptomyces avermectinius strain. Their structures were elucidated by detailed analysis of NMR and MS spectroscopic data. Compounds 1 and 2 displayed moderate cytotoxicities against B16, MG-63, and Saos-2 cell lines in an in vitro assay.

Eight new metabolites, including new sesquiterpenoids (I), a new backbone sesquiterpene rearranged from guaiane, two disecoguaianes (II), two new dihydroisocoumarins (IV), and one new tetralone (III) are isolated together with ten known compounds.

The polyenoyltetramic acid physarorubinic acid (2) has been isolated from microplasmodia of the slime mold Physarum polycephalum. The structure was elucidated by means of 1D- and 2D-NMR, IR, and UV spectroscopy and mass spectrometry. The stereochemistry was proven by synthesis of the tetramic acid unit and application of circular dichroism. It was shown that plasmodia of a white mutant strain also contain yellow pigments.

The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.

New cis-diol metabolites of both aromatic and non-aromatic heterocyclic rings have been isolated from growing cultures of a mutant strain of the soil bacterium Pseudomonas putida(UV4) and stereochemically assigned; a novel heterocyclic cis-diol of benzothiophene, 2,3-dihydroxy-2,3-dihydrobenzothiophene is found to exist exclusively in the cis-configuration in water but equilibrates readily with the trans-isomer.

Three new metabolites were isolated from a pathogenic bacterium, Nocardia brasiliensis IFM 0075 strain, a producer of a new anthracycline antibiotic (SO-075R1) and its mutant strain (IFM 0075-13-1). The structural studies showed that they are reduced anthracyline related compounds. Some biosynthetic routes of these matabolites were discussed.

Lactobacillus helveticus strain TN-4, a spontaneous mutant strain of Lactobacillus helveticus TY1-2, produced an exocellular polysaccharide from reconstituted skim milk. On the basis of the results of methylation analysis, enzymatic digestion, mild Smith degradation, mild acid hydrolysis, acetolysis, and 1D and 2D 1 H-NMR spectroscopy, it was concluded that the polysaccharide has a d -galactofuranose containing hexasaccharide repeating unit with the following structure

The polyenoyltetramic acid physarorubinic acid (2) has been isolated from microplasmodia of the slime mold Physarum polycephalum. The structure was elucidated by means of 1D- and 2D-NMR, IR, and UV spectroscopy and mass spectrometry. The stereochemistry was proven by synthesis of the tetramic acid unit and application of circular dichroism. It was shown that plasmodia of a white mutant strain also contain yellow pigments.

National Science Fund for Distinguished Young Scholars [30325044]; Chinese Ministry of Education [306010]

Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate.

The isolation and structure elucidation of the new Spirostaphylotrichins N (2), O (3), P (4), and T (7) from a mutant strain of S. coccosporum are described. The biogenetic relationship of the known spirostaphylotrichins is discussed.

After UV-irradiation an alkaloid-blocked mutant of Claviceps paspali was isolated from an ergometrine and lysergic acid amides (1:1) producing strain. The formation of ergometrine is blocked in this particular mutant strain which accumulates only lysergic acid amides. A significant morphological differentation of colonies of mutant as well as of mycelium in submerged cultures could be observed and total alkaloid yields are rather influenced.

Indocarbazostatins C (3) and D (4), new inhibitors of NGF-induced neurite outgrowth were isolated from culture broth of a mutant strain, Streptomyces sp. MUV-6-83. The structural elucidation of 3 and 4 revealed that these inhibitors were methyl ester analogs of the corresponding ethyl ester compounds, indocarbazostatin (1) and indocarbazostatin B (2), respectively.

Aspergillus niger van Teighem, a soil isolate, was mutagenized by u.v. light. A mutant strain with 3 times increased productivity was generated on exposure of the parent strain to u.v. for 15 min. This mutant on further exposure to u.v. yielded second generation mutants with only marginal increase in inulinase productivity with respect to the parent mutant, but the best yield of 377 U ml-1 was considered promising for industrial use.

The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N-methyl, N′-nitro N-nitrosoguanidine (NTG). One of the mutant strains designated as Sat-20 failed to grow in a medium containing sulphate (MgSO4 · 7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3 · 5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.

From the Engineering Biology Department of Sofia University' microbiological collection a Trichoderma species mutant strain was found to be most promissing on preparation of cellulase enzymic products. Based on solid-phase and submerged cultures laboratory and industrial scale procedures for obtaining cellulase products were developed. Different enzyme application possibilities were investigated and the results were presented.

Salmonella typhimurium 1195 mutant bacteria were allowed to produce clusters of lipopolysaccharide with new, wild-type specificity over the export sites. Ferritin-labeling experiments showed that the lipopolysaccharide diffuses away from the export sites at 37 °C and is found dispersed on the cell surface, whereas at 0 °C it remains immobile. The density of lipopolysaccharide molecules on the bacterial surface, measured as number of lipopolysaccharide molecules per μm2, or as percentage of the bacterial surface covered by lipopolysaccharide was determined in two wild-type S. typhimurium strains and one mutant strain, which was grown under conditions either permissive or nonpermissive for the formation of wild-type lipopolysaccharide. There are 0.7—1.0 × 105 lipopolysaccharide molecules per μm2 surface area. About 15—25% of the bacterial surface was found to be covered by lipopolysaccharide. The mean intermolecular distance at hypothetical uniform distribution of lipopolysaccharide was calculated to be in the range of 3—4 nm. It was of no consequence to the lipopolysaccharide surface density whether the mutant strain produced wildtype or mutant lipopolysaccharide. An estimate of the lateral diffusion constant D (D∼ 3 × 10−13 cm2/s) of lipopolysaccharide in the outer membrane was made from earlier published data [2]. Possible reasons for the low order of magnitude of D, based on the observed surface density, are discussed.

SUMMARY Envelope fragments of E. coli K12 have been produced by freeze-fracturing ‘by hand’ and negatively stained after thawing. The outer leaflet of the plasma membrane disintegrated upon thawing whereas the outer leaflet of the outer membrane did not. Negative staining revealed the following structural features on the outer membrane fragments: (i) ‘grooves’ 4–6 nm wide, (ii) spherical particles 6–8 nm in diameter, (iii) ‘black dots’ 3–8 nm in diameter. Treatment of cells with EDTA before freeze-fracturing caused dilatation of grooves into holes eventually leading to fragmentation of the outer membrane. A mutant strain deficient in two outer membrane proteins fractured exclusively through the outer membrane. The outer leaflets so obtained disintegrated upon thawing similarly as observed for the outer leaflet of the plasma membrane.

Chimeric hydra were constructed from ectoderm of one strain and endoderm of a morphologically different strain. Body size and budding rate were assayed in three pairs of ectoderm/endoderm chimeras, which were chosen on the basis of their differences in size and budding rates. Reciprocal chimeras of one parental pair were the same size as their endodermal parent, while chimeras of another pair were closer to the ectodermal parents in size. Chimeras of the third pair were similar to one another in size, and intermediate in size between the parental strains. The budding rates of the three reciprocal pairs were similar to their ectodermal parents. This suggests that ectoderm exerts more influence than endoderm on budding rate, while both ectoderm and endoderm are capable of influencing the size of a hydra. The morphology of these three pairs of reciprocal chimeras and of a fourth pair of chimeras constructed from normal H. attenuata and a mutant strain which produces multiple basal discs were compared to the parental morphologies. Both ectoderm and endoderm contribute to the morphology of the chimeric strains.

In many laboratories cellulolytic enzyme components from culture filtrates of different species of the fungus Trichoderma have been isolated and characterized [l-lo]. These components have shown great diversity as regards both enzymic and physicochemical properties. More than a dozen ‘different’ enzymes have been isolated. The great diversity among these enzymes is somewhat puzzling in view of the fact that they must perform their functions outside the organism in a rather hostile environment that would not be expected to favour a complex multienzyme system sensitive to minor changes in the conditions. The aim of the present work is therefore to investigate the>elationships among components present in culture filtrates of the enhanced mutant strain QM 9414 of Tnkhoderma reesei using a new approach based on immunochemical techniques.

The optimal parameters for the cultivation in 10-l fermenters of a mutant strain Humicola lutea 120-5 were established:temperature 30°C, inoculum size 6%, inoculum age 24 h, aeration rate 0,6 vol/vol · min, medium agitation 620 rpm and cultivation time 72 h. A maximal proteolytic activity of 2000 µg tyrosine liberated from 2%casein ml−1 culture filtrate min−1 at pH 3.0 and 40°C was obtained under the fixed conditions. α-Amylase biosynthesis during the cultivation of H. lutea 120-5 was observed but it was insignificant to the 72nd h. It is demonstrated that starch can be used as alternative to glucose carbon source. It is proved that the mutant strain H.lutea 120-5 produced two acid proteinases.

The composition of the lipopolysaccharides from E. coli K12 P678 and from a mutant strain PM61 was investigated. No difference was found between the lipopolysaccharides of both strains. These bacteria belong to the CR34 serologic type in the group of E. coli K12.

The effect of larval age range on survival of twoTribolium castaneum strains in mixed cultures was studied in an experimental situation in which other selective forces were reduced to a minimum. Increasing the difference in larval age setween the strains by one day resulted in a reduction of survival of the faster-developing mutant strain, because more of its earlier pupae were cannibalized by the larvae. General aspects of the effects of pupal cannibalism by larvae on the outcome of intra-specific competition (sensu lato) inTribolium are discussed.

THE STUDY OF sequentially blocked mutant strains of various microorganisms has led to the further elucidation of many biosynthetic pathways (Snell, 1954; Cheldelin and King, 1954). However, no definite clues to the biosynthesis of riboflavin have been provided by the study of a temperaturesensitive, riboflavin-requiring mutant strain (51602t, rib-i) of Neurospora crassa previously reported by Mitchell and Houlahan (1946). The availability of a second riboflavin-deficient strain (Tatum et al., 1950) suggested that its thorough investigation might lead to further information on the biosvnthesis of this important vitamin. A number of related compounds have been tested on this strain (Y30539, rib-2), and exhaustive tests have been made to detect the accumulation of intermediates in riboflavin biosynthesis. Although these attempts have been unsuccessful, the examination of the new strain has provided interesting genetic and other biological information. This paper summarizes the results of these investigations.

Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.

A caffeine-resistant mutant of Aspergillus parasiticus NRRL 2999 was isolated and subsequently designated strain BCR1. The mutant strain grew in the presence of > 8 mg/mL caffeine, while growth of the parent strain was delayed by 1 mg/mL and inhibited by 2 mg/mL. Strain BCR1 produced abundant amounts of aflatoxin only when cultured in media containing caffeine. Residual caffeine analyses indicated that caffeine-resistance in BCR1 was not due to the metabolic elimination of caffeine.

A mutant formed after UV irradiation of a potent aflatoxin producing strain ofAspergillus parasiticus elaborated 80% less aflatoxin than did the parent strain and produced an orange-red pigment. This new metabolite which represents 1% of the mycelial mass has been identified as 2-hexanoyl-1,3,6,8-tetrahydroxyanthraquinone (norsolorinic acid), mol wt 370, mp 256–257 C, and molecular formula C20H18O7.

Production of aflatoxins and norsolorinic acid by a mutant strain ofAspergillus parasiticus follows a similar course. Both substances are completely or partially inhibited when the mutant is grown in a chemically defined medium while illuminated continuously, at 37 C, in medium lacking zinc, and in the presence of para-aminobenzoic acid. Higher yields of both compounds are obtained when the mold is grown in an enriched medium.