Tryptophan metabolism in Klebsiella aerogenes: regulation of the utilization of aromatic amino acids as sources of nitrogen.

Klebsiella aerogenes utilized aromatic amino acids as sole sources of nitrogen but not as sole sources of carbon. K. aerogenes abstracted the alpha-amino group of these compounds by transamination and excreted the arylpyruvate portions into the medium. When tryptophan was utilized as the sole source of nitrogen by K. aerogenes, indolepyruvate was excreted into the medium, where it polymerized non-enzymatically to form a brick red pigment. At least four separate aromatic aminotransferase activities were found in K. aerogenes. One activity (aromatic aminotransferase I) appeared to be solely responsible for the aminotransferase reaction necessary for the growth of K. aerogenes when tryptophan was the source of nitrogen; the loss of this activity by mutation (tut) prevented the growth of cells on media containing this and other aromatic amino acids. None of the other aminotransferase activities in the cells could substitute for aromatic aminotransferase in this regard. Tryptophan-dependent pigment formation in K. aerogenes was positively controlled by the intracellular level of glutamine synthetase. Nevertheless, the aromatic aminotransferase activity in cells varied less than 2-fold in response to 10-fold or greater changes in the levels of glutamine synthetase. Glutamine synthetase affected the ability of the cells to take up tryptophan from the medium.     

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species.

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.     

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.     

Hot water systems as sources of Legionella pneumophila in hospital and nonhospital plumbing fixtures.

Samples obtained from plumbing systems of hospitals, nonhospital institutions and homes were cultured for Legionella spp. by plating the samples directly on a selective medium. Swab samples were taken from the inner surfaces of faucet assemblies (aerators, spouts, and valve seats), showerheads, and shower pipes. Water and sediment were collected from the bottom of hot-water tanks. Legionella pneumophila serogroups 1, 5, and 6 were recovered from plumbing fixtures of the hospitals and nonhospital institutions and one of five homes. The legionellae (7 to 13,850 colony-forming units per ml) were also present in water and sediment from hot-water tanks maintained at 30 to 54 degrees C, but not in those maintained at 71 and 77 degrees C. Legionella micdadei was isolated from one tank. Thus legionellae are present in hot-water tanks which are maintained at warm temperatures or whose design results in warm temperatures at the bottom of the tanks. We hypothesize that hot-water tanks are a breeding site and a major source of L. pneumophila for the contamination of plumbing systems. The existence of these bacteria in the plumbing systems and tanks was not necessarily associated with disease. The extent of the hazard of this contamination needs to be delineated.     

Dihydroxy and monohydroxy fatty acids in Legionella pneumophila

Five strains of Legionella pneumophila were examined for the presence of hydroxy fatty acid. The cellular distribution of the fatty acids was also determined, as was the variation of hydroxy acid production on five growth media. The strains tested all produced approximately 5 mol% of hydroxy fatty acid, most of which was found in the nonextractable, alkali-stable, acid-labile (wall-associated, amide-linked) fraction. Three major hydroxy acids were found, along with several minor components. The major hydroxy acids were analyzed by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and infrared spectrophotometry. These compounds were tentatively identified as 3-hydroxy-12-methyltridecanoate, 3-hydroxy-n-eicosanoate, and a novel dihydroxy acid, 2,3-dihydroxy-12-methyltridecanoate. The total amount of hydroxy acid produced, as well as the profile of the hydroxy acids, remained relatively unchanged with respect to strain and growth medium.     

Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

ATP and other nucleotide interaction with model compounds of amino acids.

With the use of a new liquid scintillation technique, the partitioning and interfacial interaction of ³H-ATP and other adenine nucleotides to various amphiphiles has been studied. The amphiphiles consisted of lipophilic groups attached to amino acid residues or certain functional groups and were as follows: trimethylindole, octadecylamine, octadecyl alcohol, dodecylguanidine, stearylamide, acetododecyl amide, p-nonyl phenol, stearyl mercaputan, stearic acid, and imidazole. A toluene solution containing the lipophilic substance and fluor molecules was layered onto aqueous solutions containing a ³H-nucleotide, and the interaction followed by the liquid scintillation technique. A strong interaction with the nucleotides occurred with dodecylguanidine, octadecylamine, and trimethylindole, while the interaction with the other amphiphiles was slight or absent. The time course of the interaction followed first order kinetics irrespective of the number of reactive species; the rate of diffusion of the nucleotide being the rate-determining step. The technique permitted a simple analysis of formation constants; which for ATP were 1–2 orders of magnitude greater than for ADP and AMP. In the presence of Ca²⁺, but not Mg²⁺, an interaction occurred between ATP and either stearic acid, stearyl mercaputan, and p-nonyl phenol. On the other hand, in the presence of Mg²⁺, but not Ca²⁺, an interaction occurred between stearic acid and polyadenylic acid. The results are discussed in relation to ATP interactions with proteins and lipids.     

Cysteine metabolism in Legionella pneumophila: characterization of an L-cystine-utilizing mutant

Growth of Legionella pneumophila on buffered charcoal-yeast extract (BCYE) medium is dependent on L-cysteine (but not L-cystine), which is added in excess over what is required for nutrition. We investigated the biochemical and genetic bases for this unusual requirement and determined that much of the L-cysteine in BCYE medium is rapidly oxidized to L-cystine and is unavailable to the bacteria. Analysis of cysteine consumption during bacterial growth indicated that of the 11% consumed, 3.85% (approximately 0.1 mM) was incorporated into biomass. The activities of two key cysteine biosynthetic enzymes (serine acetyltransferase and cysteine synthase) were not detected in cell extracts of L. pneumophila, and the respective genes were not present in the genome sequences, confirming cysteine auxotrophy. Kinetic studies identified two energy-dependent cysteine transporters, one with high affinity (apparent Km, 3.29 microM) and the other with low affinity (apparent Km, 93 microM), each of which was inhibited by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Cystine was not transported by L. pneumophila; however, a mutant strain capable of growth on L-cystine (CYS1 mutant) transported L-cystine with similar kinetics (Km, 4.4 microM and 90 microM). Based on the bipartite kinetics, requirement for proton motive force, and inhibitor studies, we suggest that a high-affinity periplasmic binding protein and a major facilitator/symporter (low affinity) mediate uptake. The latter most likely is functional at high cysteine concentrations and most likely displays altered substrate specificity in the CYS-1 mutant. Our studies provide biochemical evidence to support a general view that L. pneumophila is restricted to an intracellular lifestyle in natural environments by an inability to utilize cystine, which most likely ensures that the dormant cyst-like transmissible forms do not germinate outside suitable protozoan hosts.     

Intermediary metabolism and life-history trade-offs: differential metabolism of amino acids underlies the dispersal-reproduction trade-off in a wing-polymorphic cricket

Although the differential flow of metabolites through alternate pathways of intermediary metabolism is thought to be an important functional cause of life-history trade-offs, this phenomenon remains understudied. Using a radiolabeled amino acid, we quantified genetic differences in in vivo amino acid metabolism between morphs of the wing-polymorphic cricket Gryllus firmus that trade off early-age reproduction and dispersal capability. Lines selected for the flight-capable morph, which delays reproduction, oxidized a greater proportion of radiolabeled glycine and converted a greater amount into somatic lipid, mainly triglyceride (flight fuel). By contrast, lines selected for the flightless, reproductive morph converted a substantially greater proportion of glycine into ovarian protein. Compensatory interactions between amino acid and lipid metabolism make up a key aspect of specialization for dispersal versus reproduction in G. firmus: increased oxidation of amino acids by the flight-capable morph spares fatty acid for enhanced conversion into triglyceride flight fuel. By contrast, increased oxidation of fatty acid by the flightless morph spares amino acids for enhanced biosynthesis of ovarian protein. Studies of amino acid and lipid metabolism in G. firmus currently represent the most detailed analyses of genetic modifications of intermediary metabolism that underlie a functionally important life-history trade-off found in natural populations.     

The metabolism of amino acids in the bovine lens. Their oxidation as a source of energy

1. The metabolism by the bovine lens of nine (14)C-labelled l-amino acids was studied. These were: alanine, aspartate, glutamate, leucine, lysine, proline, serine, tyrosine and tryptophan. 2. All were taken up by the tissue and incorporated into protein. 3. Aspartate and glutamate, although poorly taken up, were readily metabolized to CO(2). Radioactivity from glutamate was also found in glutathione, glutamine, proline and ophthalmic acid. Aspartate was converted into glutamate, glutathione, proline, alanine and lactate. 4. Alanine was largely converted into lactate, which was released into the medium, but incorporation of radioactivity into CO(2), glutamate, glutathione, aspartate and lipids also occurred. 5. Radioactivity from leucine was detected in CO(2), lipids, glutamate, glutathione, proline and glutamine. 6. Lysine was only slightly broken down by the bovine lens; radioactivity was observed in CO(2), glutamate, glutathione, proline and two unidentified compounds. 7. Proline was metabolized to glutamate from which CO(2), glutathione and glutamine were formed. Hydroxyproline in the capsule collagen was labelled. 8. Radioactivity from serine was found in CO(2), lipids, glutathione, glycine, cystine, ATP, lactate and three unidentified compounds, one of which was probably taurine. 9. Neither tyrosine nor tryptophan were metabolized by the bovine lens. 10. The ability of the lens to metabolize amino acids was also shown by measurement of NH(3) production: more NH(3) was formed when glucose was absent from the incubation medium. 11. These experiments suggest that oxidation of amino acids is a source of energy for the lens.     

Transformation of some hydroxy amino acids to other amino acids.

It has been observed that beta-hydroxy-alpha-amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give beta-hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of beta-hydroxy-alpha-amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

Proteinases of Legionella: phenylalanineaminopeptidase of L. pneumophila

Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-L-threonyl-L-phenylalanyl-L-prolylglycyl-aminosilo chrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7-9 and had an activity optimum in the range of pH 8-9.5 with L-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards L-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.     

Microalgae as sources of pharmaceuticals and other biologically active compounds

In the last decade the screening of microalgae, especially the cyanobacteria (blue-green algae), for antibiotics and pharmacologically active compounds has received ever increasing interest. A large number of antibiotic compounds, many with novel structures, have been isolated and characterised. Similarly many cyanobacteria have been shown to produce antiviral and antineoplastic compounds. A range of pharmacological activities have also been observed with extracts of microalgae, however the active principles are as yet unknown in most cases. Several of the bioactive compounds may find application in human or veterinary medicine or in agriculture. Others should find application as research tools or as structural models for the development of new drugs. The microalgae are particularly attractive as natural sources of bioactive molecules since these algae have the potential to produce these compounds in culture which enables the production of structurally complex molecules which are difficult or impossible to produce by chemical synthesis.     

Hibernation hypothermia and metabolism in hedgehogs--changes in free amino acids and related compounds

1. Hypothermia in midwinter revealed a marked increase in GABA and glutamine due to active decarboxylation and amidation of glutamic acid. This influenced the glutamate-aspartate pathway and resulted in a significant drop in levels of both acids. 2. Elevated levels of GABA and taurine during hibernation pointed to their role as inhibitory neurotransmitters. 3. Amidation of glutamate induced a noticeable drop in ammonia concurring with increased urea and low uric acid levels. 4. Hypothermia in summer revealed a significant role of temperature as a determining factor in the hibernation cycle. Arousal was a repeated, though reversed, phenomenon in this cycle.     

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.     

Growth of Legionella pneumophila in continuous culture.

A method was developed to grow Legionella pneumophila in continuous culture. A chemostat was used to simulate nutrient-limited, submaximal growth in the natural environmental and to provide a precisely controlled growth regimen. Cultures grew under forced aeration under conditions yielding up to 38% saturation of dissolved oxygen; supplemental CO2 (5%) at the same gas flow rates as ambient air had no effect on culture growth. Pleomorphism was observed during growth under all conditions. Pigment was produced only at D less than 0.03 h-1. Catalase was produced at higher growth rates but not at higher temperatures. The pathogenicity was unaffected by altering either the growth rate or the growth temperature.     

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.