Stimulation of Nitrobacter agilis by Biotin.

Krulwich, Terry A. (Goucher College, Baltimore, Md.), and Helen B. Funk. Stimulation of Nitrobacter agilis by biotin. J. Bacteriol. 90:729-733. 1965.-Addition of biotin to nitrite-mineral medium greatly stimulated the autotrophic growth of four strains of Nitrobacter agilis. Comparisons of cultures of the organisms grown in parallel at 30 C in nitrite medium and in the medium supplemented with 150 mmug of biotin per ml showed that the vitamin promoted: (i) 2- to 4-fold greater rates of utilization of nitrite, and (ii) 100- to 1,000-fold greater populations of cells per milliliter. Avidin specifically inhibited the biotin stimulation of nitrite utilization at an avidin-biotin ratio of 133:1. Incubation of the four strains of N. agilis at 37 C imposed a requirement for biotin that could be met by daily addition of 150 mmug of the vitamin per ml of medium. The stimulatory effects of the vitamin at 30 C suggest that in N. agilis the synthesis of biotin is rate-limiting for growth.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations.

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

Metabolic and ultrastructural response to glucose of two eurytrophic bacteria isolated from seawater at different enriching concentrations

Two marine bacteria, an Acinetobacter sp. (strain GO1) and a vibrio sp. (strain G1), were isolated by extinction dilution and maintained in natural seawater supplemented with nitrogen, phosphorus, and glucose at 0.01 and 10 mg of glucose carbon per liter above ambient monosaccharide concentrations, respectively. After 3 days in unsupplemented natural seawater, growth in batch culture with glucose supplements was determined by changes in cell numbers and glucose concentration. The exponential growth of the Acinetobacter strain with added glucose was indistinguishable from that in natural seawater alone, whereas that of the Vibrio strain was more rapid in the presence of glucose supplements, suggesting that the Acinetobacter strain preferred the natural organic matter in seawater as a carbon source. The ultrastructure for both isolates was unaffected by glucose supplements during exponential growth, but there were marked changes in stationary-phase cells. The Vibrio strain formed polyphosphate at 10 mg of glucose carbon per liter, whereas poly-beta-hydroxybutyrate formation occurred at 100 mg and became excessive at 1,000 mg, disrupting the cells. In contrast, the Acinetobacter strain elongated at 100 and 1,000 mg of glucose carbon per liter but failed to show poly-beta-hydroxybutyrate formation. The diversity of responses shown here would not have been detected with a single concentration of substrate, often used in the literature to characterize both pure and natural populations of marine bacteria.     

VITAMIN B12, THIAMINE,AND BIOTIN CONTENTS OF MARINE PHYTOPLANKTON1

An extraction procedure was developed for determining vitamin B₁₂, thiamine, and biotin contents of marine phytoplankton. Phytoplankters were collected either by centrifugation or by retention on a glass fiber filter, then heated at 100 C for I hr in 100 ml of vitamin-free seawater acidified to pH 3.5 with HCl. The extract, after debris removal, was filter-sterilized and analyzed, for vitamin B₁₂, thiamine, and biotin with standard vitamin assay procedures. The vitamin contents of haeodactylum tricornutum, Skeletonema costatum, Stephanopyxis turris, and occolithus liuxleyi were determined during growth in batch cultures. P. tricornutum (non-vitamin requirer) growing in aerated cultures contained 0.29–0.96 ng B₁₂, 5–15 ng thiamine, and 0.45–1.70 ng biotin/mg C. Under similar conditions S. costatum (B₁₂-requirer) contained about 0.06 ng B₁₂, 5–36 ng thiamine, and 0.16–2.10 ng biotin/mg C. The concentrations of vitamin were generally similar during some portion of the growth curve, eg, logarithmic growth. The vitamin B₁₂, content of S. costatum growing under nonaerated conditions decreased when medium B₁₂, was reduced. The biotin content did not change when medium B₁₂ was decreased. The thiamine content per unit weight of C. huxleyi (thiamine-requirer) growing with either 10 or 120 ng/liter thiamine decreased under both medium concentrations, indicating no net synthesis of the vitamin.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

Microbiological Assay for Organic Compounds in Seawater. II: Distribution of Adenine, Uracil, and Threonine

Biochemically deficient strains of Serratia marinorubra have been isolated with specific requirements for adenine, uracil, and threonine. Standard curves for dose to growth response have been obtained showing a linear sensitivity from 0.5 to 4.0 mg of adenine per liter of seawater, 0.1 to 2.0 mg of uracil per liter of seawater, and 0.5 to 10 mg of threonine per liter of seawater. These mutants have been used to test for the presence of their required metabolites in natural seawater samples from the Gulf of Mexico and adjacent bays. Of the three compounds under investigation, adenine was found in 10 samples, uracil in 2 samples, and threonine in none. The possible significance of these findings to the marine environment is discussed.     

Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.     

Construction of a Biotin-Overproducing Strain of Serratia marcescens

We have isolated mutants resistant to acidomycin, a biotin analog, from Serratia marcescens Sr41. Strain SB304, resistant to 0.5 mg of acidomycin (frequently called actithiazic acid) per ml, produced 5 mg of d-biotin per liter of a medium containing sucrose and urea. Strain SB412, which was isolated from SB304 on a minimal agar plate containing 2 mg of acidomycin per ml and 0.1 mg of 5-(2-thienyl)-valeric acid per ml, produced 20 mg of d-biotin per ml. The two enzymes related to biotin synthesis were found to be released from biotin-mediated feedback repression in these mutants. Transductional analysis revealed that SB412 had acquired at least two mutations, one in the biotin operon locus and the other in an unknown locus distant from the biotin operon locus.     

Formation of Valine in the Presence of Sodium Acrylate Monomer by Glutamic Acid Producing Bacteria

Glutamic acid producing bacteria accumulated a large amount of valine in the presence of the excess biotin, when sodium acrylate monomer (Na-AM) was added at the earlier phase of culture. Brevibacterium roseum ATCC 13825, particularly, accumulated the large amount of valine among bacteria tested and the conditions of valine accumulation by this strain were investigated.

The most effective addition time of Na-AM was at the earlier phase of logarithmic phase. The optimal concentration of Na-AM for the accumulation of valine was 1.0 per cent (v/v). Most effective nitrogen sources were the combination of 1.0 per cent urea and 0.2 per cent ammonium sulphate. The additions of Mn²⁺ and Fe²⁺ increased valine accumulation. By the excess concentration of biotin for growth, 20 μg/liter or more, did not affected valine accumulation, while the presence of the suboptimal condition of biotin for growth was not good for the formation of valine even in the presence of Na-AM. The accumulation of valine reached 9.0 mg/ml from 75.0 mg/ml of glucose in the presence of 50 μg/liter of biotin and 1.0 per cent (v/v) of Na-AM.

This strain possessed considerable activity of valine formation regardless of the addition of Na-AM and promoted the accumulation of valine by the addition of Na-AM.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater.

Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently.     

Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater.

Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently.     

Cultural Estimation of Yeasts on Seaweeds

Five per cent suspensions of freshly harvested seaweeds were used as an inoculum to develop a selective medium for epiphytic yeasts. Conditions for satisfactory yeast growth and visualization as red colonies on membrane filters were obtained by supplementing a basal glucose-Trypticase-yeast extract-agar at pH 7.0 with 100 mg each of chloramphenicol and 2,3,5-triphenyl tetrazolium chloride per liter. Maximal counts were obtained by triturating the algae in prechilled (4 C) seawater with a blender for 2 to 5 min. Inhibitory phenolic materials released from phaeophytes during this process were removed with a modified Cholodny filtration. A preliminary survey indicated that yeasts were epiphytic on all nine species of seaweeds and that maximal populations occurred on the chlorophytes and rhodophytes especially during the periods of warmer water.     

Effects of deep seawater on the growth of several species of marine micro-algae

Deep seawater (DSW; seawater under the euphotic layer), obtained from Deep Seawater Laboratory in Muroto, Japan, was applied to the culture ofDunaliella tertiolecta, D. salina, Nannochloropsis oculata, N. salina, Porphyridium cruentum, Tetraselmis tetrahele, andChaetoceros ceratosporum. DSW supported the exponential growth of every species. The growth yields were at 14.7 (±2.3 SD) mg dry weight per liter, and could be heightened by the addition of nitrate to DSW.Author for correspondence     

Increased Sensitivity of the Microbiological Assay for Biotin by Lactobacillus plantarum

Addition of Tween 80 to biotin assay medium containing acid-hydrolyzed casein as the amino acid source caused marked growth of Lactobacillus plantarum ATCC 8014 in the absence of added biotin. This growth-promoting activity could be eliminated by treating the "vitamin-free" Casamino Acids (Difco) with activated charcoal (Darco G-60) at pH 3.5 for 30 to 60 min. Incorporation of Tween 80 and charcoal-purified Casamino Acids (PCA) into the assay medium (0.8 g and 27 g, respectively, per liter of single strength medium) in place of unpurified Casamino Acids resulted in a medium in which L. plantarum responded to 30 to 50 times less biotin over an extended linear response range (1.3 logs versus 1.0 log) than was required for similar growth in the standard medium. Endogenous growth in the modified medium was absent if the inoculum used was of low density, if it was prepared from biotin-deficient cells, and if the reagents used were free from contaminating traces of biotin. Assays of biological materials for biotin content using the standard medium and the Tween 80-PCA-modified medium resulted in nearly identical values for all samples tested.     

Isoniazid Uptake in Relation to Growth Inhibition of Mycobacterium tuberculosis

(14)C-isoniazid (INH) was used to study the relationship between drug uptake or binding by Mycobacterium tuberculosis and growth inhibition of the organism, which is dependent upon the concentration of drug and the duration of exposure. When strain H37R(a), grown in modified Sauton's liquid medium, was treated with 0.1 mug of INH per ml for 2 to 6 hr, followed by 10 mug of nicotinic hydrazide (NH) per ml to block further INH uptake, growth was retarded but not completely inhibited upon continued incubation. NH itself did not retard growth. However, cells treated in a similar manner with INH alone grew normally when diluted 1:100 in fresh, drug-free media. Uptake data showed that bacilli exposed to 0.1 mug of INH per ml accumulated 5.5, 9.7, and 12 mmug/mg of dry cells at 2, 4, and 6 hr, respectively. Other experiments suggested that once isoniazid is bound, it is not rapidly lost when NH is added or when the cells are diluted in fresh media. In the presence of 1.0 mug of INH per ml, tubercle bacilli took up 10 to 37 mmug/mg of dry cells in 20 to 90 min. These cells were not markedly inhibited when diluted 1:40 in fresh NH-containing media and incubated for 6 days. Growth inhibition of tubercle bacilli by INH depends on the uptake of sufficient drug, but the evidence obtained in this study suggests that the absolute concentration of bound INH is not as important in the action of the drug as is the maintenance of a critical cellular concentration for a requisite period of time.     

Enumeration, isolation, and characterization of n(2)-fixing bacteria from seawater

Marine pelagic N(2)-fixing bacteria have not, in general, been identified or quantified, since low or negligible rates of N(2) fixation have been recorded for seawater when blue-green algae (cyanobacteria) are absent. In the study reported here, marine N(2)-fixing bacteria were found in all samples of seawater collected and were analyzed by using a most-probable-number (MPN) method. Two different media were used which allowed growth of microaerophiles, as well as that of aerobes and facultative anaerobes. MPN values obtained for N(2)-fixing bacteria ranged from 0.4 to 1 x 10 per liter for water collected off the coast of Puerto Rico and from 2 to 5.5 x 10 per liter for Chesapeake Bay water. Over 100 strains of N(2)-fixing bacteria were isolated from the MPN tubes and classified, yielding four major groups of NaCl-requiring bacteria based on biochemical characteristics. Results of differential filtration studies indicate that N(2)-fixing bacteria may be associated with phytoplankton. In addition, when N(2)-fixing bacteria were inoculated into unfiltered seawater and incubated in situ, nitrogenase activity could be detected within 1 h. However, no nitrogenase activity was detected in uninoculated seawater or when bacteria were incubated in 0.2-mum-filtered (phytoplankton-free) seawater. The ability of these isolates to fix N(2) at ambient conditions in seawater and the large variety of N(2)-fixing bacteria isolated and identified lead to the conclusion that N(2) fixation in the ocean may occur to a greater degree than previously believed.     

Effect of iron on the biodegradation of petroleum in seawater.

The biodegradation of South Louisiana (SL) crude oil and the effects of nitrogen, phosphorus, and iron supplements on this process were compared in a polluted (10,900 oil degraders per liter) and in a relatively clean (750 oil degraders per liter) littoral seawater sample taken along the New Jersey coast. Without supplements, the biodegradation of SL crude oil was negligible in both seawater samples. Addition of nitrogen and phosphorus allowed very rapid biodegradation (72% in 3 days) in polluted seawater. Total iron in this seawater sample was high (5.2 muM), and the addition of iron did not increase the biodegradation rate further. In the less polluted and less iron-rich (1.2 muM) seawater sample, biodegradation of SL crude oil was considerably slower (21% in 3 days) and the addition of chelated iron had a stimulating effect. Ferric octoate was shown to have a similar stimulating effect on SL crude oil biodegradation as chelated iron. Ferric octoate, in combination with paraffinized urea and octylphosphate, is suitable for treatment of floating oil slicks. We conclude that spills of SL crude and similar oils can be cleaned up rapidly and efficiently by stimulated biodegradation, provided the water temperatures are favorable.     

Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga neapolitana

Growth medium components and cultivation conditions for the extremely thermophilic bacterium Thermotoga neapolitana were optimized. A defined marine salts medium was formulated. Trace amounts of iron stimulated growth of T. neapolitana, while zinc inhibited growth at concentrations exceeding 11.1 μM. Other trace metals had no effect on its growth. Of the vitamins tested, only biotin was required for optimal growth. A defined mineral medium containing 5 g of carbohydrates per liter as the carbon source and 0.5 g of cysteine per liter as the sulfur source and reductant supported growth. Growth was stimulated by inclusion of vitamin-free Casamino Acids. Elemental sulfur, cystine, and dimethyl disulfide in the growth medium enhanced growth. Elemental sulfur and cystine relieved growth inhibition by hydrogen. T. neapolitana formed colonies in 2 days on plates of complex medium solidified with gellan gum and in 4 days on defined medium. The efficiency of plating was determined when growing cultures were sampled both aerobically and anaerobically and plated under aerobic and anaerobic conditions. Mean plating efficiencies were improved by sampling the growing cultures under strictly anaerobic conditions. Little or no improvement was obtained by inoculating plates inside an anaerobic chamber. Plating efficiencies of approximately 80% were obtained. Polycarbonate jars with aluminum lids withstood repeated incubation at 77°C without significant deterioration of the anaerobic seal and provided the most consistent results.