Proline excretion by Escherichia coli K12

Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion.     

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 μg ml-1. Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition. This revised version was published online in November 2006 with corrections to the Cover Date.     

粘杆菌素高产菌株的选育

用亚硝基胍对多粘类芽胞杆菌 (Paenibacilluspolymyxa)AS1.541进行诱变 ,采用其自身次生代谢产物粘杆菌素进行筛选时正突变率为 32.3% ,获得一株粘杆菌素发酵单位比出发菌株提高 106%的菌株。对发酵单位最高的一株菌再次诱变 ,用甲硫氨酸结构类似物乙硫氨酸筛选时正突变率为 46.9%并得到一株产量提高 57%的菌株。     

AN ISOLATION UNIT FOR SUPPLYING RADIOISOTOPES TO SPECIFIC SEGMENTS OF AN INTACT ROOT

A device useful for supplying radioactive ions to specific segments of intact roots is described. A small volume of aerated, radioactive culture solution may be circulated around an isolated segment of a root, while the remainder of that root and the other roots of the plant are exposed to non-radioactive culture solution. Any root which does not have lateral roots extending beyond the epidermis can be inserted into the device without handling the root itself. No sealants are required to achieve a leak-proof seal above and below the root segment. The use of flexible rubber membranes to isolate root segments causes no detectable damage to root cells where the membranes contact the root epidermis.     

Extracellular Polysaccharide from the Black Yeast NRRL Y-6272: Improved Methods for Preparing a High-Viscosity, Pigment-Free Product

When the extracellular polysaccharide from the black yeast NRRL Y-6272, composed of two parts N-acetyl-D-glucosamine and one part N-acetyl-D-glucosaminuronic acid, is isolated at maximum culture viscosity, adhering black pigment gives the polysaccharide preparations a gray-to-black appearance. Precipitation of the polysaccharide from cell-free culture supernatants with either ethanol of hexadecyltrimethylammonium bromide failed to remove the pigment. Various other methods were therefore tried for obtaining a high-viscosity polysaccharide product free of pigment. By systematically varying ingredients of defined and semidefined media, an improved medium was found that not only gave polysaccharide preparations of increased viscosity, but also increased yield. A key ingredient in this medium is L-asparagine. Also, adding autoclaved bovine serum albumin or egg albumin to this medium at the time of inoculation allowed a pigment-free polysaccharide to be isolated by standard procedures. None of several other proteins of synthetic polyamides tested were as effective as bovine serum albumin or egg albumin. In an alternate approach, pink mutants obtained by irradiation of the parent black strain with ultraviolet light, apparently produce the same extracellular polysaccharide free of any pigment but in lower yields or inferior in quality.     

Production and Characterization of a New Fungal Metabolite,Cotylenol

A new metabolite, cotylenol, has been isolated from the culture filtrate of a fungal strain, 501–7 w. Production and characterization of cotylenol are described. The chemical relationship between cotylenol and cotylenin A has been elucidated.     

一株高产黑色素细菌的分离及鉴定*

从武汉东湖中分离出一株高产胞外黑色素的细菌(BFHM2002)。BFHM2002具有产黑色素速度快,产量高且不需要酪氨酸诱导等优点;而且经酪氨酸诱导可显提高BF-HM2002胞外黑色素的产量,初步鉴定BFHM2002属坚强芽孢杆菌(Bacillus firmus)。鉴于BFHM2002的产黑色素特性,将成为芽孢杆菌属的一个新的菌种资源。     

Screening of lysine-producing strains of brevibacterium with a rotating liquid culture system using a large number of micro-holes for individual cell cultivation

Summary With the technique of rotating liquid culture system in 7000 bottomless micro-holes, mutant strains having urease activity higher than that of the original strain were screened out in a medium with urea as a sole nitrogen source. A strain having a lysine yield of 12.5% and urease activity 20% higher than those of the original strain was isolated. The technique is suitable for screening strains where visual methods are available to estimate the amount of metabolite.     

A Simple Method For Plating and Cloning Ciliates and Other Protozoa

SYNOPSIS. A simple method is described for plating and cloning ciliates and other protozoa, based on a principle differing from that traditionally used for plating and cloning bacteria and other microorganisms. This procedure, referred to as the silicone-oil-plating-procedure (SOPP), involves vortexing small volumes of culture medium containing protozoa with larger volumes of a non-toxic silicone oil and plating the resulting unstable emulsion in small plastic petri plates. Discrete microdroplets of culture medium form containing protozoa entrapped and immobilized between the hydrophobic surfaces of the plastic petri dish and the oil. Protozoa, isolated by this method grow, divide, and multiply to form clones. the procedure may be used for plating and cloning protozoa in bacterized and axenic culture. Variations of the basic method may be applied to isolating protozoa from the wild, washing protozoa to remove microorganisms, screening for potential mutants, and for replica plating.     

Agrochemical resistant mutants of nitrogen fixing cyanobacteriumTolypothrix tenuis as nitrogen fertilizer for rice

Summary Agrochemical resistant mutants of nitrogen fixing cyanobacteriumTolypothrix tenuis were isolated after MNNG mutagenesis. The mutants exhibited higher nitrogenase activity and released more quantities of extracellular nitrogenous, substances such as ammonia, indole acetic acid like substances and amino acids when compared to the parent. They also increased the available nitrogen status of the soil in rice culture. Significant increase in the growth and yield upon inoculation of these mutants into rice culture was observed in comparison with chemical nitrogen fertilizer urea, as well as the parent strain treament.     

Conception and characterization of a continuous plug flow bioreactor

This report describes the design and use of a new type of Continuous Plug Flow Reactor (CPFR). The reactor comprises a continuous glass tube (diameter of 19 mm) arranged into a spiral (0.4 m external diameter) consisting of five loops, which rotate around an axle. The CPFR has advantages over other reactors in that continuous fermentation is possible without dilution of the culture and biomass or metabolite yield is increased. In addition temperature and water activity may be adjusted to a different optimum level for each physiological stage of the cultivated microorganism.     

Method for the isolation of Escherichia coli mutants with enhanced recombination between chromosomal duplications.

A method is described for the isolation of Escherichia coli mutants that show increased recombination between a pair of chromosomal duplications. These "hyper-rec" mutants display a variety of secondary phenotypes. I have isolated a large number of hyper-rec mutants and found them useful in screening for mutants that accumulate labeled DNA fragments after short pulses with [3H]thymidine. The mutants so recovered include ones that are defective in deoxyribonucleic acid ligase, deoxyribonucleic acid polymerase I and its associated 5' yields 3' exonuclease, and a group of mutants, dnaS, that accumulate abnormally short Okazaki fragments. Evidence is presented that suggests that the lac-att80 segment of the chromosome cannot be inverted.     

Elucidating cytokinin response mechanisms using mutants

Cytokinins (CKs) have powerful effects on many elements of plant development, but little is known about the cellular and molecular mechanisms of CK action. This review describes recent progress in identification and characterization of mutants of flowering plants that may permit elucidation of CK response mechanisms. Several Nicotiana plumbaginifolia mutants that are resistant to high levels of exogenous CK have been isolated. Characterization of these mutants has led to information about relationships between CKs and other hormones, and CKs and nutrient metabolism. Two Arabidopsis thaliana mutants that are specifically resistant to CKs in a root elongation assay, cyr1 and stp1, have been described, and may represent lesions in the CK signal transduction pathway. A mutant that produces elevated levels of CKs, amp1, has provided surprising information about the role of CKs in cotyledon formation. A set of tagged mutations that result in CK independent growth in culture has been identified, and the affected gene, CKI1, cloned. The possibility that this gene encodes a CK receptor is discussed. Continued molecular/genetic analysis of CK responses is predicted to result in rapid progress in the next few years in understanding how CKs act.     

Methods for Mutation and Selection of the Ergot Fungus

A new method is described in which the Salkowski reaction is used for the rapid selection of alkaloid-producing mutants of the ergot fungus. This method was used to investigate the influence of a second mutation with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) on various mutants selected by a preliminary NTG mutation of Claviceps sp. strain SD 58. Three groups of mutants were used: high alkaloid producers, low alkaloid producers, and auxotrophs. Results indicated that a second mutation of all three types of mutants could improve alkaloid yield and vegetative vigor. In addition, a second mutation increased the frequency of auxotroph production. The difficulty of producing stable mutants from ergot strains that have multinucleated cells and that do not readily produce conidia in culture, such as an ergotamine-producing strain, was overcome by first forming protoplasts of the fungus and then subjecting them to the mutagen. Stable auxotrophs were obtained in this manner.     

Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.     

Accumulation of Nucleic Acid-related Substances by Microorganisms

The induction of adenosine-producing mutants from an inosine-producing mutant previously derived from a Bacillus strain was attempted, and it was found out that the xanthine-requiring mutants lacking of adenase produce a large amount of adenosine.

The outline of the processes for the derivation of these mutants was described. Main product of these mutants was adenosine, and the culture broth contained a little amount of adenine as a by-product.

The culture conditions optimal for the production of adenosine were investigated, and the yield of adenosine in the culture broth was more than 16 mg/ml.     

General method for the isolation of conditional lethal mutants in any required region of the virus genome: its application to the 'semi-essential' region of phage Mu

A general method is presented that enables viral mutants to be isolated in any required region of their genome. An application of the method is reported relating to the isolation of conditional lethal mutants in the "semi-essential' region of phage Mu. Some properties are described of one mutant found in this way, Mucts62SEEam8 Apl, which is typical of a class of mutants that were isolated.     

Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.