Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.     

Esterases in mycobacteria

It was found that a submerged culture ofMycobacterium phlei degrades simple esters (ethylacetate and ethylbutyrate) as well as synthetic lipids (triacetine and tributyrine). The effect of pH on the rate of degradation of tributyrine was investigated and the maximum activity of esterases found within a wide range of pH. The activity of esterases was followed during growth of a submerged culture ofMycobacterium phlei. Esterases were not released into the cultivation medium during growth or even during the early stationary phase. Only a low steady activity of esterases could be demonstrated in a filtrate of the cultivation liquid. The total activity of esterases reached its maximum after a 6–11 day incubation. The specific activity of esterases reached a maximum on the 6th day of incubation; its value decreased to about one half and did not change substantially on prolonged incubation. Changes in the specific activity of esterases were found to be time-related with changes of pH and a decrease of the specific activity was associated with a release of macromolecular compounds into the incubation medium. Esterases as well as other macromolecular compounds were isolated from the filtrate of the cultivation medium ofMycobacterium phlei. The isolated preparation contained 60–72% total activity of esterases present in the filtrate of the cultivation liquid.     

Nutrition of three species of microsporum

Summary The growth ofMicrosporum Cookei, M. distortum, andM. nanum was compared on solid media containing 23 different carbon sources and 25 different nitrogen sources.M. nanum grew well only on media containing ribose, xylose, levulose, or erythritol as the carbon source.M. distortum andM. Cookei were found to utilize a far greater variety of carbon sources. Both grew well on dextrose, levulose, galactose, mannose, ribose, arabinose, rhamnose, sucrose, cellobiose, lactose, trehalose, melibiose, melezitose, raffinose, dextrin, and mannitol; in additionM. Cookei grew well on xylose, maltose, and erythritol. Aspartic acid, arginine, and citrulline were favorable sources of nitrogen for all three species. M. nanum also grew well on alanine, ornithine, histidine, and proline;M. distortum on glycine, serine, asparagine, glutamic acid, leucine, and cysteine; andM. Cookei on asparagine, tyrosine, ornithine, histidine, leucine, isoleucine, and proline.WithM. Cookei andM. distortum grown in liquid media in shake culture, mannitol was the best carbon source and tyrosine the best of the nitrogen sources tested. Nutritional differences exist among these three species,M. Audouini, M. canis, andM. gypseum.From a thesis, under the direction of Dr.Frederick T. Wolf, submitted in partial fulfillment of the requirements for the degree Master of Arts, August 1961.     

Physicochemical Characterization of Tween 80-Hydrolyzing Esterases Produced by Rapidly Growing Mycobacteria

Tween 80-hydrolyzing esterases produced by various species of rapidly growing mycobacteria were partially purified from sonicated cell lysates by diethylaminoethyl (DEAE) cellulose and subsequent Sephadex G-150 column chromatographies. The amount of the esterase produced per gram of bacterial cells varied markedly with each species. Mycobacterium smegmatis, M. chelonei, and M. phlei were high producers and M. chitae and M. diernhoferi were low producers of Tween-hydrolyzing esterase. The resistance of each mycobacterial strain to oleic acid correlated well with their esterase-producing ability. All the esterases studied were adsorbed on DEAE cellulose in 50 mM Tris-HCl buffer (pH 7.5), indicating that they are acidic proteins. Esterases of M. smegmatis, M. chitae, M. fortuitum, and M. phlei were eluted from DEAE at high concentrations (0.11–0.18 m) of ammonium sulfate, while those of M. parafortuitum and M. diernhoferi were eluted at lower concentrations (0.05–0.08 m). With Sephadex G-150 gel filtration, all esterases were shown to have similar molecular weights (36,000 to 58,000). On the basis of heat-stability and trypsin- or chymotrypsin-sensitivity, these esterases were divided into three groups: one was heat-stable and protease-sensitive (M. smegmatis and M. fotuitum), another was heat-labile and protease-resistant (M. chelonei), and the other was the intermediate of the above two groups (M. diernhoferi).     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.     

Cell cycle inMycobacterium phlei

The initial replication region of the chromosome on the replication map ofM. phlei constructed by means of sequential mutagenesis in synchronous populations was accurately determined. By following the time shift of the replication moment of the genesbac andmet in the control culture and in the culture with the initial inhibition of DNA synthesis by nalidixic acid the start of replication of the chromosome was determined at 15 min before replication of the geneile. On the basis of the results obtained a scheme of the cell cycle inM. phlei was proposed. Intervals C and D depend on the generation time, become prolonged independently of each other and assume the whole cycle. The ratio C/(C + D) equals to 0.56 and the interval D has a value of 0.76 of the interval C. The mutual ratio of the intervalsC: D is 1.3: 1.0. The obtained results make it possible to form the assumption about mutual ratios between the chromosome replication and cell division in bacteria exhibiting slow growth rates.     

Protoplast formation,L-colony growth,and regeneration ofClostridium beijerinckii NRRL B-592 and B-593 andClostridium acetobutylicum ATCC 10132

Summary Protocols for protoplast formation, L-colony cultivation, and regeneration ofClostridium beijerinckii NRRL B-592, B-593 andC. acetobutylicum ATCC 10132 were developed. Two osmotically reinforced media were formulated. Protoplasts of B-592, B-593, and ATCC 10132 grew as cell wall-deficient forms (L-colonies) when plated on the first medium (BLM) and continued to do so through at least 3 passages on this medium. The second (BRM) permitted the L-colonies to regenerate cell walls after transfer to this medium. TransferredC. beijerinckii B-592 L-colonies reverted to bacillary colonies at a frequency of 25%. Likewise, L-colonies of B-593 andC. acetobutylicum ATCC 10132 could be regenerated at frequencies of 7.0 and 8.6%, respectively. Thus, these procedures are suitable for genetic engineering of these industrial microorganisms using protoplast manipulation techniques.     

Adsorption of a hydrophobic bacterium onto hematite: Implications in the froth flotation of the mineral

Summary The present study reports on the relationship between adsorption ofMycobacterium phlei onto hematite and flotation of the mineral. From light and scanning electron microscopy, contact angle and electrophoretic mobility observations it was found thatM. phlei is more negatively charged than hematite, that it readily accumulates onto the mineral and that it functions as a flotation collector for the mineral with optimum flotation taking place at about pH 2.5.     

Morphogenesis in callus tissue cultures of someMatricaria andAchillea Species

In the present paper we deal with the possibility of morphogenesis induction in callus tissue cultures of some representatives ofMatricaria andAchillea species. Shoot regeneration from calli ofMatricaria chamomilla andM. inodora has been induced by 0.1 mg l^-1 kinetin or by combination of 0.5 mg l^-1 kinetin and 0.5 mg l^-1 NAA added to Murashige-Skoog culture medium. Rhizogenesis took place without any other addition of auxin. In callus tissue cultures ofAchillea ptarmica cultivated on Murashige-Skoog medium with 1 mg l^-1 2,4-D after a year long cultivation the whole plant has been regenerated without any change of nutrient requirements. In callus tissue ofA. nobilis under the same conditions only roots wore regenerated.     

The formation of phenol in the degradation ofp-hydroxybenzoic acid byKlebsiella aerogenes (Aerobacter aerogenes)

After growth ofK. aerogenes in chemically defined media consisting of mineral salts andp-hydroxybenzoate with or without glucose, phenol was found in the culture fluid at concentrations inhibiting further growth. Bacteria adapted to mineral salts medium containingp-hydroxybenzoate as sole source of carbon and energy produced small but isolable quantities of 3,4-dihydroxybenzoic acid and catechol and oxidized these substances as rapidly asp-hydroxybenzoate. Bacteria adapted to mineral salts medium containing glucose as sole carbon and energy source did not oxidizep-hydroxybenzoate, 3,4-dihydroxybenzoate or catechol. Bacteria adapted to glucose medium or top-hydroxybenzoate medium did not oxidize or utilize phenol as sole carbon and energy source. A metabolic pathway forp-hydroxybenzoate degradation is proposed and the formation of phenol is attributed to a side reaction.     

The comparison of a new mutant ofMycobacterium phlei with some strains of rapidly growing mycobacteria

A red, scotochromogenic mutant of the non-acid-fast strain ofMycobacterium phlei was obtained. Mutual relationship between this mutant and rapidly growing mycobacteria was studied using complex morphological, cultivation, biochemical and serological analyses as well as determination of the base composition in DNA. Taxonomical aspects of individual analyses are discussed.     

Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain Mycobacterium phlei WU-0103

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50°C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45°C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography–mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45°C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.     

Utilization of oligosaccharides by three imperfect fungi

Summary The utilization of oligosaccharides (maltose, sucrose, lactose and raffinose) by three imperfect fungi viz.Alternaria tenuis Auct.,Colletotrichum capsici (Syd.)Butler &Bisby andColletotrichum gloeosporioides Penz. isolated from diseased leaves of tomato,Scindapsus pictus andPolyscias balfuriana respectively, was studied chromatographically. Except for lactose, all the sugars were utilizzed through a hydrolytic pathway and almost all proved to be a suitable source of carbon for the growth of fungi. Along with the utilization of sucrose, a simultaneous synthesis of an oligosaccharide was also observed in case of two species ofColletotrichum. C. gloeosporioides, however, also synthesized an oligosaccharide, maltotriose if it is grown in the medium containing maltose. During the utilization of raffinose, melibiose and fructose were detectted in the culture medium ofA. tenuis. Melibiose was further broken down into glucose and galactose by the two species ofColletotrichum.All the sugars were consumed from the media within 15 days except for lactose which persisted in the media even after 15 days.     

Properties of beta-lactamases produced by three species of mycobacteria

1. Mycobacterium smegmatis (N.C.T.C. 8158), M. fortuitum and M. phlei (MPI) produce a constitutive β-lactamase that has penicillinase and cephalosporinase activity. 2. The β-lactamases of these three species of acid-fast bacteria were mainly cell-bound, only small amounts of activity being liberated into the extracellular fluid. The total β-lactamase activity of these mycobacteria was much lower than that of certain Gram-positive organisms, but comparable with that reported for species of Gram-negative bacteria. 3. The β-lactamases of intact cells of the mycobacteria were not freely accessible to any of the substrates tested, but the apparent crypticity factor to benzylpenicillin was greater than that to cephaloridine and cephalosporin C. 4. Attempts to induce β-lactamase activity in M. smegmatis and M. phlei failed even with high concentrations of inducer. 5. The β-lactamases obtained from the three species of mycobacteria showed different substrate specificities, including different relative activities as cephalosporinases and penicillinases respectively. 6. Certain derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid were found to be resistant to hydrolysis by β-lactamases of M. smegmatis and M. fortuitum. 7. The β-lactamase of M. smegmatis was competitively inhibited by a number of β-lactamase-resistant derivatives of 6-aminopenicillanic acid, but not by similar derivatives of 7-aminocephalosporanic acid.     

Regulation of biosynthesis of monensins on chemically defined medium

Addition ofL-valine andDL-isoleucine to the cultivation medium ofStreptomyces cinnamonensis was found to affect the ratio of synthesized monensins A and B. In the presence ofL-valine monensin A is synthesized predominantly, whereas in the presence ofDL-isoleucine the production of monensin B increases.     

Larval settlement of the bivalvesMya arenaria andM. uzenensis on scallop collectors in Vostok Bay, Sea of Japan

This paper discusses data on the horizontal and vertical distribution of the spat ofMya arenaria andM. uzenensis on collectors for the cultivation of the scallopMizuhopecten yessoensis in Vostok Bay. The maximum settlement of the mollusks on scallop collectors occurred in the innermost part of the bay. The spat density ofM. arenaria on collectors was 2.6 times that ofM. uzenensis. M. arenaria larvae preferred settling on the upper portions of collectors, while the distribution ofM. uzenensis was random. The time of collector placement in the sea influenced spat numbers.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Growth inhibition of bacteria by tributylgermanium acetate and its reversal by blood constituents

Growth inhibition ofMycobacterium phlei by tributylgermanium acetate can be reversed by addition of large amounts of blood or blood serum to the nutrient medium; the blood erythrocyte fraction is inactive.On the other hand growth inhibition of the lactic acid bacteriaStreptococcus lactis andLeuconostoc mesenteroides can be counteracted by traces of blood or its crythrocyte fraction, by hemin and by catalase. Blood serum even in large amounts is ineffective.Whereas tributylgermanium acetate is highly active against most lactic acid bacteria studied, the antagonistic action of blood or hemin is found only with the species named. The possible mechanism of this antagonism is discussed.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Effect of Na+ and K+ on the growth of marine speciesVibrio pelagius andAlteromonas haloplanktis in chemostat cultures

The effect of decreasing NaCl gradients on the cell yields of the marine speciesVibrio pelagius andAlteromonas haloplanktis was studied in chemostat cultures using media containing different ionic compositions as well as glucose and succinate as the carbon and energy sources. While there were a number of dissimilarities both within and between the species that related to the particular conditions of cultivation, the major difference between them was the ability of K to spare their requirement for Na. In the case ofV. pelagius, K greatly decreased the levels of Na required; withA. haloplanktis, no significant reduction occurred. These findings indicate a substantial difference in the nature of the ionic requirements of these two species, a result consistent with the fact that these two species are from distinct evolutionary lineages.