Source Of Energy For Growth Research Articles

Enrichment of fusobacteria from the rumen that can utilize lysine as an energy source for growth

Ruminal lysine degradation is a wasteful process that deprives the animal of an essential amino acid. Mixed ruminal bacteria did not deaminate lysine (50 mM) at a rapid rate, but lysine degrading bacteria could be enriched if Trypticase (5 mg/mL) was also added. Lysine degrading isolates produced acetate, butyrate and ammonia, were non-motile, stained Gram-negative and could also utilize lactate, glucose, maltose or galactose as an energy source for growth. Lactate was converted to acetate and propionate, and 16S rDNA indicated that their closest relatives were Fusobacterium necrophorum. Growing cultures produced ammonia at rates as high as 2400 nmol/mg protein/mL/min. Washed cell suspensions took up 14C lysine (3 μM) at an initial rate of 6 nmol/mg protein/min, and glucose addition did not affect the transport. Cells washed aerobically had the same transport rate as those handled anaerobically, but only if the transport buffer contained sodium. The affinity constant for sodium was 8 mM, and sodium could not be replaced by lithium. Cells treated with the sodium/proton antiporter, monensin (5 μM), did not take up lysine, but a protonophore that inhibited growth (tetrachlorosalicylanilide, 10 μM) had no effect. An artificial membrane potential created by potassium diffusion did not increase the rate of lysine transport, and an Eadie-Hofstee plot indicated the transport rate was directly proportional to the lysine concentration. Decreasing the pH from 6.7 to 5.5 caused an 85% decrease in the rate of lysine transport. The addition of F. necrophorum JB2 (130 μg protein/mL) to mixed ruminal bacteria increased lysine degradation 10-fold, but only if the pH was 6.7 and monensin was not present. Further work will be needed to see if dietary lysine enriches fusobacteria in vivo.

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath).

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

Chitinimonas taiwanensis gen. nov., sp. nov., a Novel Chitinolytic Bacterium Isolated from a Freshwater Pond for Shrimp Culture

A bacterial strain, designated cfT was isolated from surface water of a freshwater pond for shrimp (Macrobrachium rosenbergii) culture at Ping-Tung (Southern Taiwan). Cells of this organism were Gram-negative, slightly curved rods which were motile by means of a single polar flagellum. Strain cfT utilized chitin as the exclusive carbon, nitrogen, and energy source for growth, both under aerobic and anaerobic conditions. Optimum conditions for growth were between 25 and 37 degrees C, 0 and 1% NaCl and pH 6 to 8. Strain cfT secreted two chitinolytic enzymes with approximate molecular weight 52 and 64 kDa, which hydrolyzed chitin to produce chitotriose as major product. Sequence comparison of an almost complete 16S rDNA gene showed less than 92% sequence similarity with known bacterial species. Phylogenetic analysis based on the neighbour-joining and other methods indicated that the organism formed a distinct lineage within the beta-subclass of Proteobacteria. The predominant cellular fatty acids of strain cfT were hexadecanoic acid (about 29%), octadecenoic acid (about 12%) and summed feature 3 (16:1 omega7c or 15 iso 2-OH or both [about 49%]). Its DNA base ratio was 62.8 mol% G+C. We propose to classify strain cfT (= CCRC 17210T = LMG 22011T) as Chitinimonas taiwanensis gen. nov., sp. nov.

Degradation of pyrene and characterization ofSaccharothrix sp. PYX-6 from the oligotrophic Tianchi Lake in Xinjiang Uygur Autonomous Region, China

Bacterial strain PYX-6 that utilizes anthracene, phenanthrene, or pyrene for carbon and energy sources for growth was isolated from a non-polluted lake (Tianchi Lake) in Xinjiang Uygur Autonomons Region of China. Its morphology, physiological and biochemical properties, cell wall pattern and G+C mol% content of DNA molecules were characterized. The 16S rRNA gene of strain PYX-6 was sequenced and analyzed for similarities to related bacterial species. Results indicated that strain PYX-6 is a member of the GenusSaccharothrix, and the strain was namedSaccharothrix sp. PYX-6. When pyrene was the sole carbon source in cultural medium, the strain PYX-6 assimilated pyrene for growth and 0.005% of yeast extract stimulated pyrene degradation and assimilation. The optimal pH of cultural medium and the optimal shaking frequency during cultivation were 6–8 and 200 r/m, respectively. It was found that the disappearance of pyrene in medium occurred before significant growth of strain PYX-6 took place. Phthalic acid, benzylacetic acid, and benzylpropenoic acid were detected as catabolic intermediates during pyrene degradation with mass spectroscopy and this result indicated thatSaccharothrix sp. PYX-6 adopted a pathway that is different from the pathway of the previously reported pyrene-degradingMycobacterium sp. PYR-1.

Amino acid transport by Sphingomonas sp. strain Ant 17 isolated from oil-contaminated Antarctic soil

The Antarctic bacterial isolate Sphingomonas sp. strain Ant 17 utilized a wide range of L-isomer amino acids as the sole carbon and energy source for growth. The pH and temperature optima for growth on amino acids were pH 7.0 and 15°C, respectively. Growth on serine and tryptophan was inhibited by uncouplers and inhibitors of oxidative phosphorylation, but not by monensin, a Na+/H+ antiporter, suggesting that sodium gradients were not specifically required for growth on these amino acids. Serine transport was via a high-affinity (apparent Km of 8 μM) permease specific for both the L- and D-isomer. Tryptophan transport exhibited biphasic kinetics with both high-affinity (apparent Km of 2.5 μM) and low-affinity (non-saturable) uptake systems detected. The high-affinity system was specific for L-tryptophan, L-tyrosine, and L-phenylalanine whereas the low-affinity permease was specific for L-tryptophan and L-phenylalanine, but not L-tyrosine. Neither orthovanadate nor sodium arsenate, inhibitors of ATP-dependent permeases, had any significant inhibitory effect on the rate of serine and tryptophan transport. The protonophore carbonyl cyanide m-chlorophenylhydrazone completely abolished serine and tryptophan transport; maximum rates of solute uptake were observed at acidic pH values (pH 4.0–5.0) for both amino acids. These results suggest that an electrochemical potential of protons is the driving force for serine and tryptophan transport by Ant 17. These high-affinity proton-driven permeases function over environmental extremes (e.g. broad temperature and pH range) that are likely to prevail in the natural habitat of this bacterium.

Amino acid deamination by ruminal Megasphaera elsdenii strains.

When ruminal fluid from a cow fed timothy hay was serially diluted (10-fold increments into anaerobic broth containing 15 mg ml(-1) Trypticase), the low dilutions (< or =10(-6)) had optical densities greater than 2.0 and ammonia concentrations greater than 100 m M. The optical densities and ammonia concentrations of the 10(-8) and 10(-9) dilutions were very low, but large cocci were observed in the 10(-8) dilution. The large cocci were isolated and identified by 16S rDNA sequencing as Megasphaera elsdenii. The freshly isolated strain (JL1) grew well on Trypticase, but less than 4% of the amino acid nitrogen in Trypticase was converted to ammonia. Optical density and ammonia production were twice as great if Casamino acids were provided, and similar results were obtained with seven other strains (B159, AW106, YT91, LC1, T81, J1, and YZ70). Specific activities of deamination (based on Casamino acids) of the eight strains ranged from 100 (strain JL1) to 325 (strain B159) nmol mg protein(-1) min(-1). None of the strains could utilize branched-chain amino acids as an energy source for growth, but specific activities of branched-chain amino acid deamination ranged from 15 to 65 nmol mg protein(-1) min(-1). All eight of the M. elsdenii strains grew well in the presence of 5 micro M monensin, and only two of the strains were strongly inhibited by 20 micro M monensin. On the basis of these results, it appears that M. elsdenii is deficient in peptidase activity and can utilize only a few amino acids. Some M. elsdenii strains produced ammonia and branched-chain volatile fatty acids nearly as fast as obligate amino acid-fermenting ruminal bacteria, but the extent of this production was at least fourfold lower. Because all of the strains could tolerate 5 micro M monensin, it is unlikely that this feed additive would significantly inhibit M. elsdenii in vivo.

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum.

Fusobacterium mortiferum utilizes sucrose [glucose-fructose in alpha(1-->2) linkage] and its five isomeric alpha-D-glucosyl-D-fructoses as energy sources for growth. Sucrose-grown cells are induced for both sucrose-6-phosphate hydrolase (S6PH) and fructokinase (FK), but the two enzymes are not expressed above constitutive levels during growth on the isomeric compounds. Extracts of cells grown previously on the sucrose isomers trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5) and palatinose alpha(1-->6) contained high levels of an NAD+ plus metal-dependent phospho-alpha-glucosidase (MalH). The latter enzyme was not induced during growth on sucrose. MalH catalysed the hydrolysis of the 6'-phosphorylated derivatives of the five isomers to yield glucose 6-phosphate and fructose, but sucrose 6-phosphate itself was not a substrate. Unexpectedly, MalH hydrolysed both alpha- and beta-linked stereomers of the chromogenic analogue p-nitrophenyl glucoside 6-phosphate. The gene malH is adjacent to malB and malR, which encode an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system and a putative regulatory protein, respectively. The authors suggest that for F. mortiferum, the products of malB and malH catalyse the phosphorylative translocation and intracellular hydrolysis of the five isomers of sucrose and of related alpha-linked glucosides. Genes homologous to malB and malH are present in both Klebsiella pneumoniae and the enterohaemorrhagic strain Escherichia coli O157:H7. Both these organisms grew well on sucrose, but only K. pneumoniae exhibited growth on the isomeric compounds.

Transformation of thiodiglycol by resting cells of Alcaligenes xylosoxydans PGH10.

A new strain of Alcaligenes xylosoxydans able to aerobically cometabolize thiodiglycol, the primary hydrolysis product of sulfur mustard, was isolated and tested in a laboratory scale stirred tank reactor. The strain, named PGH10, cannot use TDG as sole carbon and energy source for growth, but resting cells previously grown on either rich broth or defined mineral media efficiently metabolize this compound through [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid as intermediates. Degradation of TDG by PGH10 is shown to take place at late exponential and stationary phase but is not triggered by carbon exhaustion. Cultures pregrown to saturation for 48 h in the absence of TDG can be stored and used for degradation of TDG, reducing significantly the time required to achieve the reduction of the compound concentration to undetectable levels. Degradation can take place in buffered media with no carbon source added, although best results were obtained in mineral media supplemented with citrate or fructose. Oxidation to [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid was proposed to be catalyzed by a butanol-dehydrogenase activity. Inhibition of TDG transformation in the presence of several alcohols is also shown.

Novel alkylsulfatases required for biodegradation of the branched primary alkyl sulfate surfactant 2-butyloctyl sulfate.

Recent reports show that contrary to common perception, branched alkyl sulfate surfactants are readily biodegradable in standard biodegradability tests. We report here the isolation of bacteria capable of biodegrading 2-butyloctyl sulfate and the identification of novel enzymes that initiate the process. Enrichment culturing from activated sewage sludge yielded several strains capable of growth on 2-butyloctyl sulfate. Of these, two were selected for further study and identified as members of the genus Pseudomonas. Strain AE-A was able to utilize either sodium dodecyl sulfate (SDS) or 2-butyloctyl sulfate as a carbon and energy source for growth, but strain AE-D utilized only the latter. Depending on growth conditions, strain AE-A produced up to three alkylsulfatases, as shown by polyacrylamide gel electrophoresis zymography. Growth on either SDS or 2-butyloctyl sulfate or in nutrient broth produced an apparently constitutive, nonspecific primary alkylsulfatase, AP1, weakly active on SDS and on 2-butyloctyl sulfate. Growth on 2-butyloctyl sulfate produced a second enzyme, AP2, active on 2-butyloctyl sulfate but not on SDS, and growth on SDS produced a third enzyme, AP3, active on SDS but not on 2-butyloctyl sulfate. In contrast, strain AE-D, when grown on 2-butyloctyl sulfate (no growth on SDS), produced a single enzyme, DP1, active on 2-butyloctyl sulfate but not on SDS. DP1 was not produced in broth cultures. DP1 was induced when residual 2-butyloctyl sulfate was present in the growth medium, but the enzyme disappeared when the substrate was exhausted. Gas chromatographic analysis of products of incubating 2-butyloctyl sulfate with DP1 in gels revealed the formation of 2-butyloctanol, showing the enzyme to be a true sulfatase. In contrast, Pseudomonas sp. strain C12B, well known for its ability to degrade linear SDS, was unable to grow on 2-butyloctyl sulfate, and its alkylsulfatases responsible for initiating the degradation of SDS by releasing the parent alcohol exhibited no hydrolytic activity on 2-butyloctyl sulfate. DP1 and the analogous AP2 are thus new alkylsulfatase enzymes with novel specificity toward 2-butyloctyl sulfate.

Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture.

The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.

Studies on effects of dietary fatty acids as related to their position on triglycerides.

This article reviews published literature on how the stereospecific structure of dietary triglycerides may affect lipid metabolism in humans. Animal studies have shown enhanced absorption of fatty acids in the sn-2 position of dietary triglycerides. Increasing the level of the saturated fatty acid palmitic acid in the sn-2 position (e.g., by interesterification of the fat to randomize the positions of the fatty acids along the glycerol backbone) has been shown in rabbits to increase the atherogenic potential of the fat without impacting levels of blood lipids and lipoproteins. In contrast, enhancing the level of stearic acid in the sn-2 position has not been found to affect either atherogenic potential or levels of blood lipids and lipoproteins in rabbits. Fatty acids other than palmitic and stearic have not been studied systematically with respect to possible positional effects. A limited number of human studies have shown no significant effects of interesterified fats on blood lipid parameters. However, it is unknown whether modifying the stereospecific structure of dietary triglycerides would affect atherogenicity or other long-term health conditions in humans. It is possible that incorporation of palmitic acid into the sn-2 position of milk fat is beneficial to the human infant (as a source of energy for growth and development) but not to human adults. Additional research is needed to determine whether processes like interesterification, which can be used to alter physical parameters of dietary fats (e.g., melting characteristics), may result in favorable or unfavorable long-term effects in humans.

Phosphorylation and metabolism of sucrose and its five linkage-isomeric α- d-glucosyl- d-fructoses by Klebsiella pneumoniae

Not only sucrose but the five isomeric α- d-glucosyl- d-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at O-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6′-O-phosphorylated α- d-glucosyl-fructoses are hydrolyzed by an inducible (∼49–50 Kda) phospho-α-glucosidase from Family 4 of the glycosylhydrolase superfamily.

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum.

Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Escherichia coli.

Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the beta-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40, respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.

Effect of growth conditions on thiocyanate degradation and emission of carbonyl sulfide by Thiobacillus thioparus THI115

Thiobacillus thioparus THI115 utilizes thiocyanate as its sole source of energy for growth. Carbonyl sulfide (COS) was found as an intermediate in the headspace of a sealed flask during the degradation of thiocyanate, and ammonia and sulfate were produced as the final products which slightly decreased the medium pH. In a pH-controlled TC medium, the highest degradation activity for thiocyanate was obtained at pH 6.2 and the activity was inhibited at pH 6.0. When whole gases in both liquid medium and headspace were removed from the culture vessel by forced aeration, neither growth of T. thioparus THI115 nor the degradation of thiocyanate could be observed. On the other hand, thiocyanate was degraded after a lag period of about 150 h when only the gases in the headspace were removed. These results suggest that the entire COS produced was easy to diffuse outside the cells and that the evolved COS was re-incorporated to be metabolized by cells. Despite the fact that COS is toxic to organisms, thiocyanate degradation by T. thioparus THI115 was tolerated when a total of 500 μmol of COS, which is about 17 times higher than the lethal dose of 50 for 90 min in rats, was added to the culture. T. thioparus THI115 degraded up to 60 mM thiocyanate in 130 h when supplied with sufficient oxygen and keeping the medium pH over 6.2.

Proteolysis and utilization of albumin by enrichment cultures of subgingival microbiota.

Subgingival dental plaque consists mainly of microorganisms that derive their energy from amino acid fermentation. Their nutrient requirements are met by the subgingival proteolytic system, which includes proteases from microorganism and inflammatory cells, and substrate proteins from sulcus exudate, including albumin. To determine the selective effect of individual proteins on microbiota, we used albumin as the main substrate for growth. Eight subgingval plaque samples from untreated periodontal pockets of patients with adult periodontitis were inoculated in peptone yeast medium with bovine albumin (9 g/l). After three subculture steps, cell yields of the enrichment cultures at the medium with 0, 1.25, 2.5, 5, 10, and 20 g/l albumin were determined. Proteolytic activity (U/absorbance at 550 nm) of the enrichment cultures and different isolates derived from the cultures was estimated by the degradation of resorufin-labeled casein. It was observed that the yield of the mixed culture was albumin limited, and the proteolytic activities of the cultures in albumin broth were higher than in control (peptone broth). Among the isolates from the enrichment cultures, Peptostreptococcus micros, Prevotella melaninogenica, Prevotella buccae and Prevotella bivia demonstrated proteolysis. The frequent occurrence of Streptococcus gordonii and Streptococcus anginosus in the albumin cultures is explained by their ability to utilize arginine as an energy source for growth. Albumin in the medium was partly degraded by pure cultures but completely consumed in enrichment cultures, indicating synergy of bacterial proteinases. It is concluded that the subgingival microbiota possesses proteolytic activity and may use albumin as a substrate for their growth. Enrichment cultures on albumin may serve as a relatively simple in vitro model to evaluate the effects of proteinase inhibitors.

Sodium-dependent glutamate uptake by an alkaliphilic, thermophilic Bacillus strain, TA2.A1.

A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (DeltapNa) across the cell membrane. N, N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (DeltaPsi), but only when sodium was present. In the absence of sodium, the rate of DeltaPsi-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of DeltapNa alone (i.e., no DeltaPsi). Glutamate uptake was specifically inhibited by monensin, and the Km for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 microM, and the Vmax was 0.7 nmol. min-1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues.

Degradation of 3-phenylpropionic acid by Haloferax sp. D1227.

Haloferax sp. D1227, isolated from soil contaminated with highly saline oil brine, is the first halophilic archaeon to demonstrate the utilization of aromatic compounds (i.e., benzoic acid, cinnamic acid, and 3-phenylpropionic acid) as sole carbon and energy sources for growth. The degradation of 3-phenylpropionic acid in this strain was studied to examine the strategies utilized by Archaea to metabolize aromatic compounds. Based on our findings of (1) the extracellular accumulation of cinnamic acid, benzoic acid, 3-hydroxybenzoic acid, and gentisic acid in cultures of Haloferax D 1227 grown on 3-phenylpropionic acid, (2) the presence of an 3-phenylpropionylCoA dehydrogenase, (3) the ATP, CoA, and NAD-dependent conversion of cinnamic acid to benzoylCoA, and (4) the presence of gentisate 1,2-dioxygenase, we propose that Haloferax D1227 metabolizes 3-phenylpropionic acid by initial 2-carbon shortening of the side chain to benzoylCoA via a mechanism similar to fatty acid beta-oxidation, followed by aromatic degradation using a gentisate pathway. The upper aliphatic pathway from 3-phenylpropionic acid to benzoic acid is regulated separately from the lower gentisate pathway.

Insecticidal activity and biodegradation of the toxin from bacillus thuringiensis subsp. kurstaki bound to humic acids from soil

The equilibrium adsorption and binding of the active toxin from Bacillus thuringiensis subsp. kurstaki, toxic to lepidopteran larvae, to humic acids extracted from two forest and two cultivated soils, as well as the insecticidal activity and the biodegradation of the bound toxin, were studied. From 75 to 85% of the toxin added was rapidly adsorbed to the humic acids at equilibrium, and adsorption to a constant amount of humic acids increased with the concentration of the toxin until a plateau was reached. Differences in total acidity and in the content of phenolic groups of the humic acids appeared to be primarily responsible for differences in the amounts of toxin bound (45–80% of the adsorbed toxin) after extensive washing with distilled water. The content of carboxyl groups and the degree of polymerization (E4/E6) did not appear to influence significantly the differential binding. Bound humic acid–toxin complexes were toxic to larvae of the tobacco hornworm ( Manduca sexta). The lethal concentration necessary to kill 50% of the larvae (LC 50) of the bound toxin was comparable with that of the free toxin, indicating that the binding of the toxin to humic acids did not affect its insecticidal activity. The bound toxin did not support the growth of a mixed microbial culture from soil, although the free toxin was rapidly utilized as a carbon and energy source for growth, indicating that binding of the toxin to humic acids reduced its biodegradability. The result of these studies indicate that the toxins from B. thuringiensis introduced in transgenic plants and microbes could persist, accumulate, and remain insecticidal in soil as a result of binding to humic acids, as well as on clays, as previously described. This persistence could pose a hazard to non-target organisms and enhance the selection of toxin-resistant target species.

Biotoxicity of mercury to Chlorella vulgaris as influenced by amino acids

The toxicity of mercury ion, on Chlorella vulgaris, is largely influenced by amino acids. Five amino acids, namely alanine, asparagine, glutamic acid, cysteine and histidine, were added separately to the medium containing static dose of mercury. Survival (%) of the alga was reduced with the increasing concentrations of mercury. Of these five amino acids, cysteine was found to be the most effective while alanine and glutamic acid were the least effective on reducing the toxic effect of mercury on the alga measured in terms of growth, chlorophyll and protein content. The order of detoxification was Alanine < Glutamate < Asparargine < Histidine < Cysteine. Amino acids from ligands with Hg2+ making it less toxic to the alga and produce an additional source of energy for growth and development.