Decrease of phosphate concentration in the medium by Brevibacterium casei cells

Brevibacteria able to decrease phosphate concentration in the medium are of interest for the study of the role of bacteria in the phosphorus cycle and for development of biotechnology of phosphate removal from waste. Brevibacterium casei, Brevibacterium linens, and Brevibacterium epidermidis grown in media with initial phosphorus concentrations of 1-11 mM were shown to decrease its concentration by 90%. The composition of the incubation medium required for B. casei to carry out this process was established. This process occurs in the absence of glucose but requires the presence of Mg2+, NH4+, and alpha-ketoglutarate. The latter two components may be replaced by amino acids metabolized to NH4+ and alpha-ketoglutarate: histidine, arginine, glutamine, proline, or glutamic acid. No formation of insoluble phosphate salts was observed when the media were incubated under the same conditions with heat-inactivated cells or without cells at pH 7-8.5.     

Direct Transition of Outgrowing Bacterial Spores to New Sporangia Without Intermediate Cell Division.

Vinter, Vladimir (Syracuse University, Syracuse, N.Y.), and Ralph A. Slepecky. Direct transition of outgrowing bacterial spores to new sporangia without intermediate cell division. J. Bacteriol. 90:803-807. 1965.-A direct transition was observed of the primary cell developed after germination of Bacillus cereus spores into new sporangia without intermediate division stages. Two simple methods were used for replacement of outgrowing spores into diluted medium or saline. Elongated primary cells prevented from division by limitation of nutrients in the suspending medium were able to form new forespores in 8 hr and sporangia in 12 hr. These new sporangia were still marked by attached envelopes of the original spore. Under the same conditions, cells replaced during the first divisions quickly lysed. Spores formed in the elongated primary cell during "microcycle sporogenesis" possessed normal heat resistance and refractility and were later released from sporangia.     

Inhibition of the development of the spore septum and membranes in Bacillus cereus by beta-phenethyl alcohol

Remsen, C. C. (Syracuse University, Syracuse, N.Y.), D. G. Lundgren, and R. A. Slepecky. Inhibition of the development of the spore septum and membranes in Bacillus cereus by beta-phenethyl alcohol. J. Bacteriol. 91:324-331. 1966.-The effect of phenethyl alcohol (PEA) upon the initial stages of sporulation in Bacillus cereus was studied with an electron microscope. PEA (0.35%) completely inhibited the development of the spore septum and forespore membranes. Some of the treated cells did form the axial filament of chromatin material regarded as the first stage in sporulation, but this was delayed by 4 to 5 hr compared with untreated cells. The definite effect upon these membrane systems lends support to the belief that the primary site of PEA inhibition may be upon the bacterial membrane. The fine-structure details observed during the initial stages of sporulation in untreated cells were in agreement with the structure published for other Bacillus species.     

Purification of human placental alkaline phosphatase. Salt effects in affinity chromatography

Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH(4))(2)SO(4). Retention on these columns was greatest at the highest concentrations of (NH(4))(2)SO(4). By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH(4))(2)SO(4)-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine-Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.     

Role of sodium in determining alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes

In contrast to the absolute Na(+) requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na(+). However, Na(+) (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na(+)-free medium possessed all the enzymes of the citric acid cycle including alpha-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na(+), which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na(+)-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Na(+) to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of alpha-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of alpha-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle.     

Influence of Nutritional Conditions on Production of l-Glutamine by Flavobacterium rigense

The nutritional conditions for the production of l-glutamine by Flavobacterium rigense strain 703 were investigated. The optimum concentration of ammonia for achieving the highest yield of l-glutamine (25 mg/ml of broth) was relatively broad, from 0.9 to 1.6%, whereas fumaric acid had a narrow optimum range, near 5.5%. High concentration of inorganic ions such as chloride or sulfate ion clearly inhibited cell growth. Therefore, ammonium salts other than (NH(4))(2)-fumarate were unsuitable for the highest production. The optimum concentration of (NH(4))(2)-fumarate was 7%. To reduce the concentration of fumaric acid in the medium, many substances were evaluated as substitutes. The fumaric acid concentration required for highest l-glutamine yield could not be replaced by any one of the compounds tested. However, part of fumaric acid could be replaced with succinic acid and cupric ion; 4% (NH(4))(2)-fumarate plus 2.5% succinic acid or 5% (NH(4))(2)-fumarate plus 1 mM cupric ion produced results similar to 7% (NH(4))(2)-fumarate in the fermentation medium.     

Organic acid production by Basidiomycetes. 3. Cultural conditions for L-malic acid production

Cultural conditions were examined for the purpose of increasing yields of l-malic acid by the Basidiomycetes Schizophyllum commune and Merulius tremellosus, which have the ability to produce this acid as a main product in CaCO(3)-containing medium in shaken culture. The most favorable nitrogen sources selected were 0.3% (NH(4))(2)SO(4) and 0.18% NH(4)Cl. Effective combinations of inorganic salts in the medium were 0.1% KH(2)PO(4), 0.05% MgSO(4).7H(2)O, and 0.05% KCl, and suitable concentrations of glucose were 5 to 10%. Several nonionic surface-active agents promoted the filamentous mycelial growth of these strains and increased acid production. In particular, Tween 80 in 0.3% concentration markedly stimulated malic acid production by S. commune, and yields greater than 50% based on available glucose, were obtained after 10 to 14 days. Acid production by M. tremellosus was stimulated most with 0.5% Carbowax 4000 (polyethylene glycol), and the resultant yields were more than 40%.     

Clostridium perfringens: I. Sporulation in a Biphasic Glucose-Ion-Exchange Resin Medium

A biphasic culture medium suitable for cultivation and sporulation of Clostridium perfringens, C. botulinum, and C. sporogenes was devised. The medium designed for use in a disposable, compartmented, plastic film container contained peptones, yeast extract, minerals, an anion exchange resin, and glucose in 4% agar as the solid phase and (NH(4))(2)SO(4) and 0.1% agar as the liquid phase. With the biphasic system, it was not necessary to use active cultures as inocula. Growth was at least equal to that obtained in conventional media, and spore production of 9 out of 12 strains of C. perfringens equalled or usually exceeded that of conventional media.     

Keto acid metabolism in Desulfovibrio.

Four strains of Desulfovibrio each excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth. One of the strains (Hildenborough) excreted alpha-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the alpha-ketoglutarate. Arsenite (I mM) prevented the accumulation of alpha-ketoglutarate; I mM-malonate did not affect the accumulation of keto acids. Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. Alpha-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated. This and previous work indicate that a tricarboxylic acid pathway from citrate to alpha-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.     

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.     

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Microbial decomposition of wood in streams: distribution of microflora and factors affecting [C]lignocellulose mineralization

The distribution and lignocellulolytic activity of the microbial community was determined on a large log of Douglas fir (Pseudotsuga menziesii) in a Pacific Northwest stream. Scanning electron microscopy, plate counts, and degradation of [C]lignocelluloses prepared from Douglas fir and incubated with samples of wood taken from the surface and within the log revealed that most of the microbial colonization and lignocellulose-degrading activity occurred on the surface. Labeled lignocellulose and surface wood samples were incubated in vitro with nutrient supplements to determine potential limiting factors of [C]lignocellulose degradation. Incubations carried out in a nitrogenless mineral salts and trace elements solution were no more favorable to degradation than those carried out in distilled water alone. Incubations supplemented with either (NH(4))(2)SO(4) or organic nitrogen sources showed large increases in the rates of mineralization over incubations with mineral salts and trace elements alone, with the greatest effect being observed from an addition of (NH(4))(2)SO(4). Subsequent incubations with (NH(4))(2)SO(4), KNO(3), and NH(4)NO(3) revealed that KNO(3) was the most favorable for lignin degradation, whereas all three supplements were equally favorable for cellulose degradation. Supplementation with glucose repressed both lignin and cellulose mineralization. The results reported in this study indicate that nitrogen limitation of wood decomposition may exist in streams of the Pacific Northwest. The radiotracer technique was shown to be a sensitive and useful tool for assessing relative patterns of lignocellulose decay and microbial activity in wood, along with the importance of thoroughly characterizing the experimental system before its general acceptance.     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Effect of Ethyl Picolinate on Germination, Growth and Sporulation of Bacillus spp.

Ethyl picolinate inhibited outgrowth and sporulation of Bacillus cereus T and laboratory isolates of B. megaterium AH2 and AV1 and B. brevis AG4. The outgrowth-inhibition was relieved by supplementing the medium before inoculation with any of the following: aspartate, asparagine, leucine, lysine, phenylalanine, tyrosine and vitamin-free casamino acids. The uptake of radioactive precursors in the synthesis of proteins, ribonucleic acid and deoxyribonucleic acid was prevented when ethyl picolinate was added before inoculation. Cultures incubated for a short period before the addition of ethyl picolinate assimilated radioactive precursors, outgrew and divided after a lag. None of the cultures containing ethyl picolinate sporulated. Growth was not inhibited when a portion of vegetatively growing culture was transferred to medium containing ethyl picolinate.     

Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Effects of Ammonium and Non-Ammonium Salt Additions on Methane Oxidation by Methylosinus trichosporium OB3b and Maine Forest Soils

Additions of ammonium and non-ammonium salts inhibit atmospheric methane consumption by soil at salt concentrations that do not significantly affect the soil water potential. The response of soils to non-ammonium salts has previously raised questions about the mechanism of ammonium inhibition. Results presented here show that inhibition of methane consumption by non-ammonium salts can be explained in part by ion-exchange reactions: cations desorb ammonium, with the level of desorption varying as a function of both the cation and anion added; differential desorption results in differential inhibition levels. Differences in the extent of inhibition among ammonium salts can also be explained in part by the effects of anions on ammonium exchange. In contrast, only minimal effects of cations and anions are observed in liquid cultures of Methylosinus trichosporium OB3b. The comparable level of inhibition by equinormal concentrations of NH(4)Cl and (NH(4))(2)SO(4) and the insensitivity of salt inhibition to increasing methane concentrations (from 10 to 100 ppm) are of particular interest, since both of these patterns are in contrast to results for soils. The greater inhibition of methane consumption for NH(4)Cl than (NH(4))(2)SO(4) in soils can be attributed to increased ammonium adsorption by sulfate; increasing inhibition by non-ammonium salts with increasing methane concentrations can be attributed to desorbed ammonium and a physiological mechanism proposed previously for pure cultures.     

Improved growth medium for Campylobacter species.

Campylobacter species were grown in a base containing proteose peptone no. 3, yeast extract, K2HPO4, (NH4)2SO4, NA2SO3, soluble starch, and agar. Concentrations and sources of organic nitrogen and growth factors were critical, and the optimal pH range was 7.0 to 7.5. Cultures tolerated 0.7% NaCl in addition to the salt present in the organic constituents and were sensitive to surface-active agents at concentrations recommended for enrichment of other gram-negative bacteria. Cultures were maintained on the proposed medium for 1 year with transfer every 2 weeks.     

Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium

Ellar, D. J. (Syracuse University, Syracuse, N.Y.), and D. G. Lundgren. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J. Bacteriol. 92:1748-1764. 1966.-A study was made of the fine structure of sporulating cells of Bacillus cereus grown in a chemically defined medium. The developmental stages of sporulation occurred in a fairly synchronous manner and were complete by 14 hr. This time period was shortened when spore wall peptide components were added to the medium, but the addition had no effect upon fine structure except to thicken the cell wall. Sporulation could be separated into six morphological stages which generally agreed with those published for other sporulating bacteria. The initiation of the spore (forespore) septum takes the form of an inward folding of the cytoplasmic membrane toward the pole of the cell. The inward folding forms a characteristic Y-shaped membrane structure enclosing an area within which vesicles are found. These vesicles comprise the perisporal mesosome of the cell. The membranes on opposite sides of the cell progress toward the cell center where they fuse to form the double unit membrane of the spore septum. As the proliferation of the spore septum continues, the vesicular areas move towards the pole. The end result is a double forespore membrane which completely encloses a part of the vegetative cell's chromatin. Sporal mesosomes, as well as membrane vesicles, are involved in the proliferation of the forespore. Vesicles are generally bounded by a single unit membrane, whereas in the sporal mesosomes several unit membranes are arranged concentrically. The latter become associated with the segregation of a portion of the nuclear material into the forespore region of the cell.