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Archives of Microbiology (v.168, #4)
Behavioural responses of bacteria to light and oxygen by Judith P. Armitage (pp. 249-261).
Motile bacteria have long been known to swim towards or away from specific environmental stimuli such as nutrients, oxygen or light. Although there has been a detailed description of chemosensory responses in enteric species for several years, there has been little information on the mechanisms involved in responses to stimuli affecting electron transport as these usually also change the electrochemical proton gradient – at least transiently – and, thus, directly change flagellar rotation. There have, however, been major advances recently. Halobacterium salinarium uses a retinal-based sensory system to sense changes in specific wavelengths of light and to signal via a transmembrane sensory protein, which turns out to be homologous to the transmembrane chemoreceptors of Escherichia coli. A FAD-binding protein, also related to these receptors, signals changes in respiratory electron transport in E. coli. Rhodobacter sphaeroides cells do not respond to light or oxygen specifically, but sense a change in the rate of electron transfer, probably again using an electron-transport-chain-linked redox sensor, signalling through a common sensory pathway. These recent studies reveal that bacteria not only sense a range of environmental stimuli but also integrate the signals through common pathways to produce a balanced flagellar response.
Keywords: Key words Bacterial behaviour; Bacterial motility; Aerotaxis; Photoresponses; Photoaxis; Chemotaxis; Electron transport; Flagella
Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes by Volker F. Wendisch; Marion Spies; D. J. Reinscheid; Stephanie Schnicke; Hermann Sahm; B. J. Eikmanns (pp. 262-269).
In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.
Keywords: Key wordsCorynebacterium glutamicum; Acetate; metabolism; Acetate kinase; Phosphotransacetylase; Isocitrate lyase; Malate synthase; Acetyl-CoA; Acetyl-phosphate
New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1′,2′-dihydro-γ-carotene, 1′,2′-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains by S. Takaichi; Zheng-Yu Wang; Mitsuo Umetsu; Tsunenori Nozawa; Keizo Shimada; Michael T. Madigan (pp. 270-276).
The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1′,2′-dihydro derivatives (1′,2′-dihydro-γ-carotene and 1′,2′-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus.
Keywords: Key words Carotenoid biosynthesis; Green sulfur; bacteria; Thermophily; Chlorobium tepidum; 1′; 2′-Dihydro-γ-carotene; 1′; 2′-Dihydrochlorobactene; OH-Chlorobactene glucoside ester; Nicotine; Bacteriochlorophyll c
The major carotenoid in all known species of heliobacteria is the C30 carotenoid 4,4′-diaponeurosporene, not neurosporene by S. Takaichi; Kazuhito Inoue; Mitsuhiro Akaike; Masami Kobayashi; Hirozo Oh-oka; Michael T. Madigan (pp. 277-281).
The carotenoids of five species of heliobacteria (Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum, and Heliobacterium gestii) were examined by spectroscopic methods, and the C30 carotene 4,4′-diaponeurosporene was found to be the dominant pigment; heliobacteria were previously thought to contain the C40 carotenoid neurosporene. In addition, trace amounts of the C30 diapocarotenes diapolycopene, diapo-ζ-carotene, diapophytofluene, and diapophytoene were also found. Up to now, diapocarotenes have been found in only three species of chemoorganotrophic bacteria, but not in phototropic organisms. Furthermore, the esterifying alcohol of bacteriochlorophyll g from all known species of heliobacteria was determined to be farnesol (C15) instead of the usual phytol (C20). Heliobacteria may be unable to produce geranylgeranyol (C20).
Keywords: Key words Carotenoids; Diapocarotenes; 4; 4′-Diaponeurosporene; Bacteriochlorophyll g; Heliobacteria; Heliobacillus mobilis; Heliophilum; fasciatum; Heliobacterium chlorum; Heliobacterium; modesticaldum; Heliobacterium gestii
Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine by J. Boch; Gabriele Nau-Wagner; Susanne Kneip; E. Bremer (pp. 282-289).
Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD+, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress.
Keywords: Key words Osmoregulation; Compatible solutes; Glycine betaine synthesis; Aldehyde dehydrogenase; Bacillus subtilis
Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration by Sabine Becker; Dorina Vlad; Sylvia Schuster; Peter Pfeiffer; G. Unden (pp. 290-296).
In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O2 tension (pO2) in the medium. The pO2 values that gave rise to half-maximal synthesis of the products (pO0.5) were 0.2–0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate. The pO0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar. Thus, the pO2 for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO0.5≤ 5 mbar), which was determined earlier. An essential role for quinol oxidase bd in microaerobic growth was demonstrated. A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O2), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain. In the presence of nitrate, the amount of lactate was largely decreased. Therefore, under microoxic conditions, the pO2 appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo.
Keywords: Key wordsEscherichia coli; (Mixed acid) fermentation; Facultative anaerobic metabolism; O2; Aerobic; respiration; Regulation; FNR; ArcA
Thiosulfate as a metabolic product: the bacterial fermentation of taurine by Karin Denger; Heike Laue; A. M. Cook (pp. 297-301).
Thiosulfate (S2O3 2–) is a natural product that is widely utilized in natural ecosystems as an electron sink or as an electron donor. However, the major biological source(s) of this thiosulfate is unknown. We present the first report that taurine (2-aminoethanesulfonate), the major mammalian solute, is subject to fermentation. This bacterial fermentation was found to be catalyzed by a new isolate, strain GKNTAU, a strictly anaerobic, gram-positive, motile rod that formed subterminal spores. Thiosulfate was a quantitative fermentation product. The other fermentation products were ammonia and acetate, and all could be formed by cell-free extracts.
Keywords: Key words Taurine; Fermentation; Anaerobic; desulfonation; Thiosulfate
The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells by Giovanni Moschettini; Alejandro Hochkoeppler; Barbara Monti; Bruna Benelli; D. Zannoni (pp. 302-309).
Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with E m,7 of +180, +72 and –5 mV (561–575 nm), and +244 and +27 mV (551–540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with E m,7 of +60 and –45 mV and +290, +250, +135, –20 and –105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (E m,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c 2, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85–90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa 3 type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c 2 or a branched respiratory chain.
Keywords: Key words Cytochromes; Halophilic purple nonsulfur bacteria; High-potential iron-sulfur proteins; Respiratory chain; Rhodospirillum salinarum
Anaerobic metabolism of l-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica by Sabine Schneider; M. E.-S. Mohamed; G. Fuchs (pp. 310-320).
The anaerobic metabolism of phenylalanine was studied in the denitrifying bacterium Thauera aromatica, a member of the β-subclass of the Proteobacteria. Phenylalanine was completely oxidized and served as the sole source of cell carbon. Evidence is presented that degradation proceeds via benzoyl-CoA as the central aromatic intermediate; the aromatic ring-reducing enzyme benzoyl-CoA reductase was present in cells grown on phenylalanine. Intermediates in phenylalanine oxidation to benzoyl-CoA were phenylpyruvate, phenylacetaldehyde, phenylacetate, phenylacetyl-CoA, and phenylglyoxylate. The required enzymes were detected in extracts of cells grown with phenylalanine and nitrate. Oxidation of phenylalanine to benzoyl-CoA was catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase (NAD+), phenylacetate-CoA ligase (AMP-forming), enzyme(s) oxidizing phenylacetyl-CoA to phenylglyoxylate with nitrate, and phenylglyoxylate:acceptor oxidoreductase. The capacity for phenylalanine oxidation to phenylacetate was induced during growth with phenylalanine. Evidence is provided that α-oxidation of phenylacetyl-CoA is catalyzed by a membrane-bound enzyme. This is the first report on the complete anaerobic degradation of an aromatic amino acid and the regulation of this process.
Keywords: Key wordsThauera aromatica; l-phenylalanine; metabolism; Phenylalanine transaminase; Phenylpyruvate decarboxylase; Phenylacetaldehyde; dehydrogenase; Phenylacetate-CoA ligase; α-Oxidation; of phenylacetyl-CoA; Phenylglyoxylate:acceptor; oxidoreductase
The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases by M. Pedraza-Reyes; Félix Gutiérrez-Corona (pp. 321-327).
The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-β-d-cellobioside and 4-methylumbelliferyl-β-d-cellotrioside. These enzymatic preparations also showed a greater capacity at 70° C than at 42° C to degrade chitosan oligomers of a minimum size of six units. Conversely, the β-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42° C than at 70° C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.
Keywords: Key wordsβ-1; 4 Glucanase; Endocellulase; Chitosanase; Myxobacter
Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale by Sylke Kreimer; Brigitte Söhling; J. R. Andreesen (pp. 328-337).
The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale.
Keywords: Key words Thioredoxin; Thioredoxin reductase; Glycine reductase; Disulfide exchange reaction; Clostridium litorale
Desulfovibrio inopinatus, sp. nov., a new sulfate-reducing bacterium that degrades hydroxyhydroquinone (1,2,4-trihydroxybenzene) by Wolfram Reichenbecher; B. Schink (pp. 338-344).
A new sulfate-reducing bacterium was isolated from marine sediment with hydroxyhydroquinone (1,2,4-trihydroxybenzene) as the sole electron and carbon source. Strain HHQ 20 grew slowly with doubling times of > 20 h and oxidized hydroxyhydroquinone, lactate, pyruvate, ethanol, fructose, and ribose incompletely to acetate and carbon dioxide, with concomitant reduction of sulfate to sulfide. Cells were large, vibrio-shaped, and gram-negative with a G+C content of 49.7 mol%, and contained desulfoviridin. Based on analysis of the 16S rRNA sequence, strain HHQ 20 was found to be related to the genus Desulfovibrio but formed a separate line, thus justifying the establishment of a new species within this genus. Hydroxyhydroquinone was the only aromatic compound utilized among numerous hydroxybenzoates, hydroxybenzenes, methoxybenzoates, and methoxybenzenes tested, suggesting that phloroglucinol and resorcinol are not degradation intermediates. Cell-free extracts of strain HHQ 20 did not contain pyrogallol-phloroglucinol transhydroxylase activity. First experiments indicated that this strain uses a new reductive pathway for anaerobic hydroxyhydroquinone degradation.
Keywords: Key words Anaerobic degradation; Trihydroxybenzenes; Hydroxyhydroquinone; Pelobacter massiliensis; Desulfovibrio sp.