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PubMed2000

Contribution of microbial amino acids to amino acid homeostasis of the host.

Among the reasons suggested for the discrepancy between N balance and tracer-derived indispensable amino acid (IAA) requirement estimates is the possibility that the metabolic requirement is met not only by the diet but also by IAA synthesized de novo by the gastrointestinal microflora, which are then absorbed. It is therefore crucial to better understand and quantify the microbial biosynthesis of amino acids in the human gastrointestinal tract and its potential role in providing IAA to meet human amino acid requirement. Here, the available evidence on the contribution of microbial amino acids to the host's amino acid homeostasis, applying the (15)N labeling paradigm, is summarized. Between 1 and 20% of circulating plasma lysine, urinary lysine and body protein lysine of the host, respectively, is derived from intestinal microbial sources and corresponds to a gross microbial lysine contribution of 11-68 mg. kg(-1). d(-1) in adult humans with an adequate protein intake when fecal or ileal microbial lysine enrichment is used as precursor. Factors affecting estimates of net microbial IAA contribution are discussed. It appears that the small intestine is responsible for a large part of microbial lysine uptake, although some absorption from the large intestine cannot be excluded. Nonoxidative lysine losses from the human gastrointestinal tract, which were found to be between 3.9 to 8.5 mg. kg(-1). d(-1), are necessary to estimate the net contribution of microbial IAA. It is reasonable to assume that microbial amino acid synthesis in the human gastrointestinal tract utilizes a mixture of various nitrogen sources, i.e., endogenous amino acids, urea and ammonia. Microbes in the small intestine may rely more on endogenous amino acids. Deprivation of nutrients, the intake of certain dietary nonstarch oligosaccharides, lipids, as well as protein intake level and source and level of consumption of certain amino acids can affect the composition and metabolic activity of the intestinal microflora and thus its fermentation products potentially available to the host. In conclusion, with the use of the (15)N labeling paradigm, a significant contribution of microbial lysine to the host lysine homeostasis is found. However, to assess the net contribution of microbial IAA and its importance in defining the adult IAA requirement, this is not the ultimately successful experimental strategy because the interpretation of results is complicated by the nitrogen recycling in the gut, the uncertainty of the precursor pool of absorption and the limited data on nonoxidative IAA losses from the human gastrointestinal tract.

Adult↗
PubMed1996

Effect of rumen protozoa on nitrogen utilization by ruminants.

Results obtained during the past decade indicate clearly that protozoa are actively involved in the degradation of dietary and microbial proteins in the rumen. Because of the great ability of protozoa to ingest the particulate matter suspended in the rumen, protozoa are more active in degrading insoluble than soluble proteins. This indicates that studies carried out using lysed and sonicated protozoa are not appropriate for quantifying the actual contribution of protozoa to protein degradation in the rumen. In vivo trials have confirmed that duodenal flow of both undegraded dietary protein plus bacterial protein generally is increased by defaunation. The decrease in ruminal ammonia concentration consistently observed after defaunation accounts for the lower urinary nitrogen (N) excretion found in defaunated animals, whereas the increase in fecal N excretion in the same animals probably results from a shift of plant cell wall digestion from the rumen to the large intestine. Total N excretion is not altered significantly by defaunation. A summary of literature data indicates there are contradictory effects of defaunation on ruminant performance. This implies that animal response to defaunation may depend on the specific nutrient-limiting performance on the one hand and on the modifications of digestion and metabolism resulting from defaunation on the other. Different methods are proposed to either eliminate or decrease the numbers of ruminal protozoa or to alter their makeup. However, none of these approaches has been tested under practical feeding conditions.

Animals↗
PubMed1975

Utilization of nitrogen from soybean meal, casein, zein, and urea by mature sheep.

The utilization of nitrogen contained in soybean meal, casein, zein, and urea was studied in 12 mature wethers. Net microbial synthesis rates during the 6-hour period after feeding corresponded to ruminal ammonia concentrations, suggesting that the ruminal ammonia level in these experiments was a limiting factor in microbiol protein synthesis. Maximum conversion of dietary nitrogen to microbial nitrogen in the rumen during a 24-hour period was estimated to be 62.5, 85.3, 40.9, and 90.1%, respectively, for soybean meal, casein, zein, and urea. Estimated production rates of acetic, propionic, butyric, isovaleric, and valeric acids during the first 6 hours after feeding suggested that zein supported the lowest microbial activity in the rumen, and that urea supplementation resulted in isovaleric and valeric acids production rates equivalent to or greater than rates when the other nitrogen supplements were present in the diet. Daily endogenous urinary and metabolic fecal nitrogen determined by regression analysis utilizing soybean meal as the only nitrogen supplement were 72 mg/kg body weight0.75 and 340 mg/100 g of dry matter intake. Biological values of 84.8, 78.3, 84.3, and 76.4 were obtained for soybean meal, casein, zein, and urea, respectively.

Animal Feed↗
PubMed2002

Characterizing man-made and natural modifications of microbial diversity and activity in coastal ecosystems.

The impacts of growing coastal pollution and habitat alteration accompanying human encroachment are of great concern at the microbial level, where much of the ocean's primary production and biogeochemical cycling takes place. Coastal ecosystems are also under the influence of natural perturbations such as major storwns and flooding. Distinguishing the impacts of natural and human stressors is essential for understanding environmentally-induced change in microbial diversity and function. The objective of this paper is to discuss the applications and merits of recently developed molecular, ecophysiological and analytical indicators and their utility in examining anthropogenic and climatic impacts on the structure and function of coastal microbial communities. The nitrogen-limited Neuse River Estuary and Pamlico Sound, North Carolina are used as examples of ecosystems experiencing both anthropogenic (i.e., accelerating eutrophication) and climatic stress (increasing frequencies of tropical storms and hurricanes). Additional examples are derived from a coastal monitoring site (LEO) on the Atlantic coast of New Jersey and Galveston Bay, on the Gulf of Mexico. In order to assess structure, function, and trophic state of these and other coastal ecosystems, molecular (DNA and RNA-based) characterizations of the microbial taxa involved in carbon, nitrogen and other nutrient transformations can be combined with diagnostic pigment-based indicators of primary producer groups. Application of these methods can reveal process-level microbial community responses to environmental variability over a range of scales. Experimental approaches combined with strategic monitoring utilizing these methods will facilitate: (a) understanding organismal and community responses to environmental change, and (b) synthesizing these responses in the context of ecosystem models that integrate physical, chemical and biotic variability with environmental controls.

Animals↗
PubMedDate not supplied

[Microbial destruction of cyanide and thiocyanate].

The role played by a bacterial community composed of Pseudomonas putida, strain 21, Pseudomonas stutzeri, strain 18, and Pseudomonas sp., strain 5, and by physical and chemical factors in the degradation of CN- and SCN- was studied. It was shown that the degradation of CN- is determined both by the action of bacteria and by abiotic physical and chemical factors (pH, O2, temperature, the medium agitation rate, etc.). The contribution of chemical degradation was found to increase drastically at pH below 9.0; when air was blown through the medium (irrespective of the pH value); under active agitation of the medium; and when the medium surface interfacing air was increased. Even at elevated pH values (9.0-9.2), suboptimal for bacterial growth, the microbial degradation could account for at most 20-25 mg/l of CN-, regardless of its initial concentration. When CN- and SCN- were concurrently present in the medium, the former compound was the first to be degraded by microorganisms. The rate of bacterial degradation of SCN- under continuous cultivation in a chain of reactors was found to depend on its concentration, the medium flow rate, agitation rate, and the pattern of carbon source supply and could exceed 1 g/(1 day). CN- and SCN- are utilized by bacteria solely as nitrogen sources. The mechanism of CN- and SCN- degradation by the microbial community is discussed.

Biodegradation, Environmental↗
PubMed1998

Different mathematical approaches to estimating microbial protein supply in ruminants.

Many of the amino acids that are available for absorption in ruminants are derived from microbial protein that has been synthesized in the reticulorumen. This paper focuses on the prediction of the microbial protein supply and evaluates different approaches to represent mathematically the process of microbial protein synthesis. In current protein evaluation systems for ruminants, the microbial protein supply is predicted using empirical equations that relate microbial protein production to the amounts of ruminally available energy and nitrogen. In contrast, mechanistic models of rumen function endeavor to describe quantitatively the microbial protein production that is based on underlying identifiable processes. A brief description is presented of two culture techniques used to examine microbial ecosystems, namely, batch culture and chemostat culture. The mathematical equations describing these cultures are helpful in understanding key parameters of microbial production for inclusion in models, including specific growth rate, growth yield, and substrate affinity. The availability of carbohydrates is a primary determinant of microbial protein production in the rumen, and the adequacy of mathematical representations of this relationship in empirical and mechanistic models is assessed. The representation of substrate utilization for nongrowth processes and the relationship between microbial protein production and the availability of various nitrogen sources are discussed. A variable part of the synthesized microbial protein does not reach the duodenum but is degraded in the rumen, and its representation is examined. The prediction of microbial protein supply should be based on a sound representation of the underlying mechanisms, including the interactions among microbes and between microbial activity and substrate degradation.

Animals↗
PubMed2000

Influence of TNT transformation on microbial community structure in four different lake microcosms.

After World War II, large amounts of obsolete ammunition were dumped in various lakes in Sweden. Trinitrotoluene, TNT, was one of the main components of the dumped explosives. In this study, four different lake microcosms originating from lakes where relatively large amounts of ammunition were dumped were used to mimic the effect of TNT release on the natural microbial community. Increased microbial growth was found in lake microcosms amended with TNT. However, negligible mineralization of TNT was detected, suggesting that TNT was not utilized as a carbon source, but as a nitrogen source. Random amplified polymorphic DNA (RAPD) analysis indicated that the TNT induced no significant differences in microbial community composition and therefore, no major changes in natural selection, despite the increased microbial growth in the presence of the compound. More than 95% of the added TNT bound irreversibly to the sediments, possibly as a result of microbial transformation to reactive metabolites that subsequently bound covalently to components of the sediment. The results, taken together, suggest that no permanent change in the microbial ecology occurred as a result of the TNT amendment. This was probably due partly to the transient exposure of the microbial communities to the TNT before it became irreversibly bound to the sediment, and partly to the fact that TNT was not a primary growth substrate that strongly affects natural selection.

Bacteria↗
PubMed2000

NADPH oxidase, Nramp1 and nitric oxide synthase 2 in the host antimicrobial response.

Using highly conserved, complex enzyme systems, leukocytes utilize the toxic nature of free radical intermediates, derived from oxygen and nitrogen, to control microbial pathogens as part of the innate immune response. Upon activation, NADPH oxidase generates superoxide anion radicals, which in turn give rise to further reactive oxygen intermediates. Similarly, activated nitric oxide synthase 2 catalyses the production of nitric oxide radicals, which leads to the formation of reactive nitrogen intermediates. Nitrogen- and oxygen-centered reactive intermediates can interact to form further reactive species. In addition, presence of the cationic transporter, Nrampl, may exacerbate the effects of these toxic compounds on invading microbes. While each of these antimicrobial systems can operate independently, the combination of their activities is synergistic in the successful containment of almost all invading pathogens. These systems are activated and modulated by microbial products and a series of temporally expressed cytokines. They also feed directly into the initiation of the adaptive immune response, which culminates in lasting specific immunity. The effector molecules, generated in the early innate immune response, are not specific to the invading pathogen and may also cause damage to the host. It is the critical balance of these processes in the initial stages of infection that determines the outcome of infectious disease.

Animals↗
PubMed2000

Microbial Biomass and Community Structure in a Sequence of Soils with Increasing Fertility and Changing Land Use.

The microbial biomass and community structure of eight Chinese red soils with different fertility and land use history was investigated. Two community based microbiological measurements, namely, community level physiological profiling (CLPP) using Biolog sole C source utilization tests and phospholipid fatty acid (PLFA) profiles, were used to investigate the microbial ecology of these soils and to determine how land use alters microbial community structure. Microbial biomass-C and total PLFAs were closely correlated to organic carbon and total nitrogen, indicating that these soil microbial measures are potentially good indices of soil fertility in these highly weathered soils. Metabolic quotients and C source utilization were not correlated with organic carbon or microbial biomass. Multivariate analysis of sole carbon source utilization patterns and PLFAs demonstrated that land use history and plant cover type had a significant impact on microbial community structure. PLFAs showed these differences more than CLPP methods. Consequently, PLFA analysis was a better method for assessing broad-spectrum community differences and at the same time attempting to correlate changes with soil fertility. Soils from tea orchards were particularly distinctive in their CLPP. A modified CLPP method, using absorbance readings at 405 nm and different culture media at pH values of 4.7 and 7.0, showed that the discrimination obtained can be influenced by the culture conditions. This method was used to show that the distinctive microbial community structure in tea orchard soils was not, however, due to differences in pH alone.

Journal Article↗
PubMed1980

Aspects of urea metabolism in ruminants with reference to the goat.

In goats and other ruminants, urea functions as a source of nitrogen for protein biosynthesis in the digestive tract. Ammonia can be absorbed in the digestive system when formed in excessive quantitites and enhance formation of urea, or it can be derived from urea of blood plasma when its formation from feed sources is small. Entry rates of urea into plasma may vary from 4 to 80 mumol/min per kg.75 body weight depending on dietary conditions. Urea formation is related to nitrogen intake of which approximately 70% passes into the urea pool of plasma. Irreversible losses of urea of plasma into the digestive tract vary between 10 and 90% depending on the protein to energy ratios of the diet. Entry of urea from plasma into the rumen appears to be a passive process which is sensitive to short-term changes of urea concentrations in plasma. Permeability of ruminal epithelium to urea may be altered by fermentation products of rumen (ammonia, carbon dioxide, volatile fatty acids). The influx of nitrogen into the rumen is related to needs for nitrogen of microbial populations and is associated with changes of renal excretion and tubular reabsorption of urea. Combined gastrointestinal and renal responses exert a synergistic effect on improved utilization of urea of plasma when uptake of dietary nitrogen is limited in goats and other ruminants.

Animals↗
PubMed1984

The nutritive value of rumen micro-organisms in ruminants. 4. The limiting amino acids of microbial protein in growing sheep determined by a new approach.

Four experiments were carried out to identify and quantify the limiting amino acids (AA) in rumen microbial protein (RMP). A method was developed which involved first, an assessment of the efficiency of utilization of absorbed AA-nitrogen (U) of RMP, defined as the retention of AA-N from RMP absorbed from the small intestine, and second, addition of a mixture of AA similar to the absorbed AA profile in a quantity defined by the U of RMP and equal to (1-U)/U. Third, it involved removal of each AA in turn and measurement of the resultant N retention. Using this approach it was possible to calculate both the order and extent of AA limitations in RMP. Apart from methionine which was found to be the most limiting AA, only lysine, arginine and histidine reduced N retention when omitted, and accordingly only these AA were limiting in RMP. The method is discussed in detail and the amount of supplementary AA required to utilize RMP fully is calculated.

Amino Acids↗
PubMed1985

Nitrogen metabolism by the microbial flora of the rabbit caecum.

The dense microbial flora of the rabbit caecum consisted chiefly of bacteria (10(11)/g) with small numbers of yeast cells (10(6)/g). Using strictly anaerobic technique, 23% of the direct microscopic cell count was cultivated and 55% of the cultivatable bacteria utilized ammonia as the sole source of nitrogen. Ureolytic bacteria were isolated from the caecal lumen and mucosa and were identified as Bacteroides vulgatus, Clostridium clostridiiforme, Bacillus spp. and Staphylococcus spp. Ammonia assimilation by the bacterial flora of the caecum was by incorporation into alpha-oxoglutarate catalysed by NADPH-linked glutamate dehydrogenase.

Ammonia↗
PubMed1980

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.

Acetates↗
PubMed1977

Microbial metabolism of aromatic nitriles. Enzymology of C-N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216.

1. An organism utilizing benzonitrile as sole carbon and nitrogen source was isolated by the enrichment-culture technique and identified as a Nocardia sp. of the rhodochrous group. 2. Respiration studies indicate that nitrile degradation proceeds through benzoic acid and catechol. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme that catalyses the conversion of benzonitrile directly into benzoic acid without intermediate formation of benzamide. 4. This nitrilase enzyme was purified by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 in the presence and absence of substrate. The purity of the enzyme was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme shows a time-dependent substrate-activation process in which the substrate catalyses the association of inactive subunits of mol.wt. 45000 to form the polymeric 12-unit active enzyme of mol.wt. 560000. The time required for complete association is highly dependent on the concentration of the enzyme, temperature and pH. 6. The associated enzyme has a pH optimum of 8.0 and K(m) with benzonitrile as substrate of 4mm. The activation energy of the reaction as deduced from the Arrhenius plot is 51.8kJ/mol. 7. Enzyme activity is inhibited by thiol-specific reagents and several metal ions. 8. Studies with different substrates indicate that the nitrilase is specific for nitrile groups directly attached to the benzene ring. Various substituents in the ring are compatible with activity, though ortho-substitution, except by fluorine, renders the nitrile invulnerable to attack. 9. The environmental implications of these findings and the possible significance of the enzyme in the regulation of metabolism are discussed.

Aminohydrolases↗
PubMed1985

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.

Aniline Compounds↗
PubMed1993

Microbial metabolism of quinoline and related compounds. XVII. Degradation of 3-methylquinoline by Comamonas testosteroni 63.

A bacterial strain which utilizes 3-methylquinoline as sole source of carbon, nitrogen and energy was isolated from activated sludge. On the basis of its morphological and physiological characteristics, this isolate was classified as Comamonas testosteroni. Four metabolites of 3-methylquinoline degradation were isolated from the culture supernatant and identified as 3-methyl-2-oxo-1,2-dihydroquinoline, 6-hydroxy-3-methyl-2-oxo-1,2-dihydroquinoline, 5,6-dihydroxy-3-methyl-2-oxo-1,2-dihydroquinoline and 2,5,6-trihydroxy-3-methylpyridine. Based on these results, a degradation pathway for 3-methylquinoline is proposed.

Culture Media↗
PubMed1981

Isolation and preliminary characterization of mutants of the cyanobacterium Nostoc muscorum resistant to growth inhibition by methylamine.

The wild-type heterocystous and nitrogen-fixing (Het+Nif+) N. muscorum and its non-heterocystous non-nitrogen-fixing (Het-Nif-) mutant strain both fail to grow in different inorganic nitrogen media containing 1 mM methylamine hydrochloride (MA). Mutants of the Het+Nif+ and Het-Nif- parents resistant to growth inhibition by 5 mM MA and thus designated as MAR strains were isolated with a frequency of 2.5(+/- 2.4) x 10(6). A MAR strain of the Het+Nif+ and a MAR strain of the Het-Nif- parent were characterized for growth, heterocyst formation and acetylene reducing activity in the presence and absence of methylamine in N2 medium. The Het+Nif+ MAR strain grows better in MA containing than in MA-free N2 medium, and all cultures grown with MA are found to lack both acetylene reducing activity and heterocyst. The Het-Nif-MAR strain shows good growth in MA-containing N2 medium but no growth in MA-free N2 medium. Furthermore, both the Het+Nif+MAR and Het-Nif-MAR strains show better growth in the presence than in the absence of MA in NO3- and HN4+ media. These results appear to suggest that the MAR phenotype in N. muscorum is due to the metabolic utilization of the ammonium analog as a nitrogen source.

Cyanobacteria↗
PubMed1975

Relationship between ruminal ammonia and nonprotein nitrogen utilization by ruminants. II. Application of published evidence to the development of theoretical model for predicting nonprotein nitrogen utilization.

Results from published experiments dealing with several aspects of nitrogen utilization by ruminants were used to test the concept of zero utilization of nonprotein nitrogen under conditions where more ammonia (greater than 5 mg ammonia nitrogen/100 ml) is in the rumen than can be converted to microbial protein. Results from experiments where the flow of non-ammonia nitrogen to the abomasum of sheep was measured indicate that when urea was the source of supplemental nitrogen, a constant quantity of amino acids reached the abomasum for all rations ranging from 10 to 23% crude protein. From growth studies, addition of nonprotein nitrogen to low protein, high energy rations caused an improved rate of gain. Additions of nonprotein nitrogen to rations resulting in predicted ruminal ammonia concentrations greater than 5 mg ammonia nitrogen/100 ml rumen fluid were without benefit. From lactation studies, nonprotein nitrogen supplementation did not improve milk production if the ration contained more than 12.5% crude protein prior to supplementation or if the predicted ruminal ammonia concentration was greater than 4 mg ammonia nitrogen/100 ml rumen fluid. The importance of the amino acid requirement of the animal as well as the composition of the ration in designing and evaluating nitrogen supplementation studies is discussed.

Ammonia↗