A sporulation medium for strict anaerobes

The production of spores, and spore position and shape, are taxonomic criteria in a number of genera of bacteria. The heat resistance of spores is also of interest taxonomically and with respect to the survival of bacteria, particularly pathogens, in a number of habitats. The demonstration of spore formation by growth of bacteria in various standard media and under different conditions (e.g. at sub-optimum temperature) is often not easy. Thus, a number of media designed to encourage spore formation and to produce spores with maximum heat resistance have been introduced, for instance for Clostridium perfringens (Ellner 1956; Angelotti et al. 1962; Kim et al. 1967; Duncan & Strong 1968) and Sporolactobacillus inulinus (Kitahara & Lai 1967).     

A vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes

A highly efficient method is described for producing at room temperature anoxic solutions of 50 ml or less in test tubes or serum vials by combining negative pressure with strong vortexing so that the liquid-surface, gas exchange area is increased by orders of magnitude. Liquid media suitable for the cultivation of methanogens may be rendered anoxic after three short vacuum-vortex steps.     

A novel three-compartmented electrochemical bioreactor for enrichment of strict anaerobes based on metabolite production

In this study, a novel three-compartmented electrochemical bioreactor (3-CEB) was designed in an effort to overcome the disadvantages of the two-compartmented electrochemical bioreactor (2-CEB) separated with a cation-selective membrane for enrichment of strict anaerobes. The 3-CEB was comprised of an anode, outlet, and a cathode compartment. The outlet compartment was positioned between the anode and cathode compartment, and it was separated with the anode side by a rubber plate and with the cathode side by a porous glass membrane. A platinum wire bridging the anode and outlet compartment operated as a redox passage, however, through which no material could permeate. Butyrate fermentation bacteria were enriched on the basis of the metabolite production. Butyrate generated by strict anaerobes was significantly more abundant in the 3-CEB than in the 2-CEB. Acetic acid and lactic acid generated by facultative anaerobes was relatively higher in the 2-CEB than in the 3-CEB. Meanwhile, butyrate was not generated in the bioreactor utilized for the control test, to which the electrochemical potential was not charged. In a continuous culture using the 3-CEB, the majority of the glucose was fermented to butyrate, and the acetate additionally supplied to the bacterial culture was metabolically reduced to butyrate. More lactate than butyrate was generated from glucose in the 2-CEB.     

Seasonal changes in the frequency of isolating strict anaerobes in purulent-inflammatory processes

Seasonal changes in the isolation rate of obligate anaerobes from the pathological material of patients with purulent inflammatory diseases were studied. For this purpose 707 samples of pathological material were analysed in the course of 1982-1986. Anaerobes were detected in 160 samples, which constituted 22.6% of all samples under study and 33.5% of the samples showing microbial growth. A statistically significant increase in the isolation rate of anaerobes from pathological material at the period of March-April was established. It was considered expedient to regard this newly found effect as an additional risk factor in the appearance of anaerobic infections and to take it into account in planning and carrying out prophylactic and diagnostico-therapeutic measures.     

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His₆ encoding recombinant ANEP with a His₆-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.     

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His(6) encoding recombinant ANEP with a His(6)-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.     

Development of an apparatus for cultivation of anaerobic microorganisms

Summary A novel apparatus with an L-shaped test tube was developed for anaerobic cell cultivation. Anaerobic condition was achieved without the rigorous gassing with CO₂ during the various stages of medium preparation. dispensation and cell inoculation. The growth of both moderate and strict anaerobes in this apparatus were similar to those obtained with the glove box method.     

Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.     

Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970

Several varieties of peptone supported growth of A. culbertsoni to different extents reaching a maximum cell density of 1-2 X 10(6)/ml. Proteose peptone and tryptone also yielded good growth when combined with thiamine and vitamin B12. A combination of proteose peptone with glucose, yeast extract and salts promoted excellent growth of A. culbertsoni with cell density reaching 1-2 X 10(7) cells/ml; tryptone and one of the indigenous peptones also yielded comparable growth when substituted for proteose peptone in this medium. Casamino acids also supported good growth of amoebae and requirement of yeast extract could be met by a combination of thiamine, vitamin B12 and biotin. Bacto peptone did not support good growth of this amoeba but supplementation of peptone with casamino acids or amino acid mixture improved the growth supporting capacity of the medium. Development of several media with or without glucose will aid in cultivation of A. culbertsoni, studies on its metabolism as well as screening of potential drugs.