Showing papers in "Archives of Microbiology in 1988"

The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor

Abstract: The effect of different electron acceptors on substrate degradation was studied in pure and mixed cultures of various hydrogenotrophic homoacetogenic, methanogenic, sulfate-reducing, fumarate-reducing and nitrate-ammonifying bacteria. Two different species of these bacteria which during organic substrate degradation produce and consume hydrogen, were cocultured on a substrate which was utilized only by one of them. Hydrogen, which was excreted as intermediate by the first strain (and reoxidized in pure culture), could, depending on the hydrogen acceptor present, also be used by the second organism, resulting in interspecies hydrogen transfer. The efficiency of H2 transfer was similar when methanol, lactate or fructose were used as organic substrate, although the free energy changes of fermentative H2 formation of these substrates are considerably different. In coculture experiments nitrate or fumarate>sulfate> CO2/CH4>sulfur or CO2/acetate were the preferred electron acceptors, and an increasing percentage of H2 was transferred to that bacterium which was able to utilize the preferred electron acceptor. In pure culture the threshold values for hydrogen oxidation decreased in the same order from ≤1,100 ppm for homoacetogenic bacteria to about 0.03 ppm for nitrate or fumarate reducing bacteria. The determined H2-threshold values as well as the percentage of H2 transfer in cocultures were related to the Gibbs free energy change of the respective hydrogen oxidizing reaction.

Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations

Abstract: The sequence of events following the addition of 0.5 M NaCl to cells of Escherichia coli growing in a minimal mineral medium was investigated. Immediately after upshock the cells took up a large amount of K+ and synthesized approximately half the equivalent amount of glutamate concomitantly. After 30 min the cells started to synthesize trehalose, and after 2 h they had replaced most of their initial osmoprotectants by the carbohydrate. Cell trehalose was rapidly replaced by proline, taken up from the medium when added to the osmoadapting cells. The initial rate of this proline uptake was extremely rapid, and with rates observed of up to 0.6 mmolxmin-1xg-1 of cell protein it was approximately ten times faster than that reported in the literature for non-growing cells. These results indicate that for osmoadaptation of growing cells of E. coli the uptake of proline has priority over the synthesis of trehalose, which in its turn is preferred above K+ and glutamate as osmoprotectants. We observed that two mutants with unknown lesions, but which are known to be impaired in osmoadaptation, were inhibited in replacing K+ and glutamate by trehalose, indicating that this is the basis for their defect in osmoadaptation. Further experiments revealed that neither internal pH nor the membrane potential nor the transmembrane protonmotive force are likely to be involved in osmoadaptation in E. coli. However, during osmoadaptation a high internal potassium concentration appeared to stimulate the derepression of proline-uptake systems (mainly system ProP).

Algae sequester heavy metals via synthesis of phytochelatin complexes

Abstract: A Cd-binding complex was isolated from Chlorella fusca and has been shown to be composed of phytochelating peptides, (γ-Glu-Cys) n -Gly, n=2–5. Members of six of the ten classes of Phycophyta revealed phytochelatin synthesis after exposure to cadmium ions. Phytochelatin was also induced by ions of lead, zinc, silver, copper and mercury. These experiments uneqiovocally demonstrated that algae sequester heavy metals by an identical mechanism as higher plants, namely via complexation to phytochelatins.

Chromate resistance and reduction in pseudomonas fluorescens strain LB300

Abstract: Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment Chromate resistance is conferred by the plasmid pLHB1 Strain LB300 grew in minimal salts medium with as much as 1000 μg of K2CrO4 ml−1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor P fluorescens LB303, a plasmidless, chromatesensitive variant of P fluorescens LB300, did not grow in minimal salts medium with more than 10 μg of K2CrO4 ml−1 However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction Evidently chromate resistance and chromate reduction in these organisms are unrelated Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Abstract: Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.

Thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus Thermotoga

Abstract: A second species of the extremely thermophilic, eubacterial genus Thermotoga is described as clearly distinguished from the type species Thermotoga maritima by physiological and phylogenetic criteria. It is named Thermotoga neapolitana.

Sporomusa termitida sp. nov., an H2/CO2-utilizing acetogen isolated from termites

Abstract: H2-oxidizing CO2-reducing acetogenic bacteria were isolated from gut contents of Nasutitermes nigriceps termites. Isolates were strictly anaerobic, Gram negative, endospore-forming, straight to slightly curved rods (0.5–0.8×2–8 μm) that were motile by means of lateral flagella. Cells were oxidase negative, but catalase positive and possessed a b-type cytochrome(s) associated with the cell membrane. Cells grew anaerobically with H2+CO2 as energy source and catalyzed a total synthesis of acetate from this gas mixture. H2 uptake by a representative isolate (strain JSN-2) displayed a Km=6 μM and Vmax=380 nmol x min-1 x mg protein-1. Other substrates used as energy sources for growth and acetogenesis included CO, methanol, betaine, trimethoxybenzoate, and various other organic acids. Succinate was also fermented, but propionate was formed from this substrate instead of acetate. Of a variety of sugars and sugar alcohols tested, only mannitol supported growth. Cells grew optimally at 30° C and pH 7.2 and required yeast extract or a source of amino acids (e.g. Casamino acids) for good growth. During initial enrichment and isolation, cells appeared sensitive to various reducing agents commonly employed in media for anaerobes. The DNA base composition of strain JSN-2 was 48.6 mol% G+C. On the bases of cell morphology, substrate utilization spectrum, and DNA base composition, strain JSN-2 is here-with proposed as the type strain of the new species Sporomusa termitida.

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate: description and enzymatic studies

Abstract: An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H2-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO2. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO2, H2 and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H2, formate or e.g. valine were added as H-donors. In pure culture on alanine under N2, strain al-2 grew very poorly and produced H2 up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H2-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H2S, CH4 or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (≥0.1 μM); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO2. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO2 and H2. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.

Oxidative and reductive acetyl-CoA/carbon monoxide dehydrogenase pathway in Desulphovibrio autotrophicum. 2. Demonstration of the enzymes of the pathway and comparison of CO dehydrogenase.

Abstract: It has been proposed that in some anaerobic facultatively autotrophic bacteria the acetyl CoA/CO dehydrogenase pathway is operating both in the reductive and in the oxidative direction, depending on the growth conditions. One of these anaerobes, the Gram-negative sulfate-reducing cubacterium Desulfobacterium autotrophicum, was examined for enzymes of the proposed pathway. All the required enzyme activities were present in sufficient amounts both in autotrophically and in heterotrophically grown cells, provided that the cellular tetrahydropterin rather than tetrahydrofolate was used as cosubstrate in some of the enzyme assays. The question arises whether two sets of enzymes are operating in the reductive and oxidative direction, respectively. The key enzyme of this pathway, CO dehydrogenase, which was reasonably oxygen stable, was analysed by native polyacrylamide gel electrophoresis and anaerobic activity staining. Extracts from heterotrophically grown cells exhibited five enzyme activity bands. Extracts from autotrophically grown cells showed the same pattern but an additional activity band appeared.

Characterization of Desulfovibrio fructosovorans sp. nov.

Abstract: Desulfovibrio strain JJ isolated from estuarine sediment differed from all other described Desulfovibrio species by the ability to degrade fructose. The oxidation was incomplete, leading to acetate production. Fructose, malate and fumarate were fermented mainly to succinate and acetate in the absence of an external electron acceptor. The pH and temperature optima for growth were 7.0 and 35° C respectively. Strain JJ was motile by means of a single polar flagellum. The DNA base composition was 64.13% G+C. Cytochrome c3 and desulfoviridin were present. These characteristics established the isolate as a new species of the genus Desulfovibrio, and the name Desulfovibrio fructosovorans is proposed.

Morphology of bacterial leaching patterns by Thiobacillus ferrooxidans on synthetic pyrite

Abstract: Thiobacillus ferrooxidans has been cultivated on synthetic pyrite (FeS2) single crystals as the only energy source and the pyrite interface investigated with respect to characteristic morphological changes using scanning electron microscopy. Corrosion patterns of bacterial size were identified in different stages of development and correlated with bacterial activity. It appears that bacterial attack of the sulfide interface starts by secretion of organic substances around the contact area between the bacterial cell and the sulfide energy source. They might either be part of a “pseudo capsule” which shields the contact area or may form a sulfur absorbing and transporting organic film. Degradation of the sulfide occurs in the contact area below the bacterial cell leading to a corrosion pit which the bacterium may abandon after it has reached a depth of bacterial dimension. Electron spectroscopic (XPS) and X-ray fluorescence studies indicate a layer of organic substances covering the sulfide surface under bacterial leaching conditions, which is sufficiently thick for consideration in interfacial chemical mechanisms.

The Production and Utilization of Intermediary Elemental Sulfur During the Oxidation of Reduced Sulfur-Compounds by Thiobacillus-Ferrooxidans

Abstract: The intermediary production of elemental sulfur during the microbial oxidation of reduced sulfur compounds has frequently been reported. Thiobacillus ferrooxidans, an acidophilic chemolithoautotroph, was found to produce an insoluble sulfur compound, primarily elemental sulfur, during the oxidation of thiosulfate, trithionate, tetrathionate and sulfide. This was confirmed by light and electron microscopy. Sulfur was produced from sulfide by an oxidative step, while the production from tetrathionate was initiated by a hydrolytic step, probably followed by a series of chemical reactions. The oxidation of intermediary sulfur was severely inhibited by sulfhydryl-binding reagents such as N-ethylmaleimide, by the addition of uncouplers or after freezing and thawing of the cells, which probably damaged the cell membrane. The mechanisms behind these inhibitions have not yet been clarified. Finally, it was observed that elemental sulfur oxidation by whole cells depended on the medium composition. The absence of sulfate or selenate reduced the sulfur oxidation rate.

The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

Abstract: Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO2 or an auxiliary oxidant. NO - 3 , N2O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO - 3 was reduced extensively to NO - 3 , TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO2 or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

Abstract: The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.

Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli.

Abstract: The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate. The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1–2 min and restore a higher initiation frequency. High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process. It is inferred that turgor could control DNA replication and cell division in two separate ways. Glycine betaine action is not mediated by K+ ions as the internal level of K+ ions is not modified significantly following glycine betaine accumulation.

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Abstract: Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO2 via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K m; 1 U·mg-1=1 μmol·min-1·mg protein-1 at 37°C): Acetate kinase (6.3 U·mg-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg-1, 0.06 mM CH3−FH4); methylenetetrahydrofolate dehydrogenase (3.6 U·mg-1, 0.9 mM NAD, 0.1 mM CH2=FH4); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg-1); formyltetrahydrofolate synthetase (3 U·mg-1, 1.4 mM FH4, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg-1.

Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhizium anisopliae

Abstract: Synthesis of chymoelastase and trypsin by the entomopathogenic fungus Metarhizium anisopliae occurs rapidly (<2 h) during carbon and nitrogen derepression in minimal media. Enzyme levels were enhanced when minimal media were supplemented with insect cuticle or other insoluble polymetic nutrients (e.g. cellulose) that were insufficient to produce catabolite repression. Addition of more readily utilized metabolites (e.g. glucose or alanine) repressed protease production confirming that production is constitutive but repressible. Operational control of protease release involves synthesis rather than secretion because catabolite repression reduced endocellular activity (associated with a sedimentable vacuole containing fraction) as well as extracellular enzyme levels. Studies with metabolic inhibitors indicated that production of Pr1 and Pr2 does not require DNA synthesis. However, synthesis is substantially reduced by inhibitors of transcription (actinomycin D and 8-azoguanine) and translation (cyclohexamide and puromycin). Inhibition by 8-azoguanine is relieved by guanine. These results imply that the operative steps in protease regulation involve de novo synthesis of mRNA. Inhibition of enzyme production by an AMP analogue adenosine 5′-0-thiophosphate implies an involvement for AMP-dependent enzyme systems in derepression. However, neither exogenous cAMP nor an inhibitor of cAMP phosphodiesterase relieved catabolite repression by glucose or NH4Cl. Use of o-vanadate to inhibit plasmalemma ATPase confirmed that secretion of chymoelastase-like protease and trypsin-like protease via the cell membrane is an active process.

Pseudo vitamin B12 or 5-hydroxybenzimidazolyl-cobamide are the corrinoids found in methanogenic bacteria

Abstract: Thirteen species of methanogenic bacteria were analyzed for corrinoids. Pseudo vitamin B12 (Coα-[α-(7-adenyl)]-cobamide) was the predominant cobamide of methanococcales and Methanoplanus. All other methanogens contained factor III (Coα-[α-(5-hydroxybenzimidazolyl)]-cobamide). Vitamin B12 (Coα-[α-(5,6-dimethylbenzimidazolyl)]-cobamide) was not detected in any of these archaebacteria. Their cobamide content was 100 to 1400 nmol per gram cell dry weight, indicating that abundant cobamides are essential for methanogens.

Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum. I: Characterization and metabolic function of the cellular tetrahydropterin

Abstract: In Desulfobacterium autotrophicum, a facultatively autotrophic, sulfate reducing eubacterium, CO2-fixing enzymes of the acetyl CoA/CO dehydrogenase pathway but neither key enzymes of the Calvin cycle nor those of the reductive citric acid cycle were detectable. This finding substantiates the proposal that the former pathway is operating in the reductive direction for CO2 fixation into acetyl CoA, and in the oxidative direction for acetyl CoA oxidation to CO2. However, the specific activity of an essential one-carbon metabolizing enzyme of this pathway, formyl-tetrahydrofolate synthetase, was unsufficient (0.01 μmol·min-1 ·mg-1), and its apparent Km-value for l-tetrahydrofolate (10 mM) was inadequate. It is shown that the physiological cosubstrate in this bacterium is not tetrahydrofolic acid, but a tetrahydropterin containing 4 mol of l-glutamate per mol of pterin. With this coenzyme the specific formyltetrahydropterin synthetase activity (1.7 μmol·min-1·mg-1) was 170-fold higher and the apparent Km for the tetrahydropterin (0.07 mM) was 140-fold lower. This indicates that it may be essential to reexamine the enzymes of acetyl CoA/CO dehydrogenase pathway in this organism and others with the proper cellular cosubstrate.

Inhibition of motility and phototaxis in the green flagellate, Euglena gracilis , by UV-B radiation

Abstract: The effects of solar UV-B radiation on the green flagellate, Euglena gracilis, are measured under controlled conditions. Both photoorientation and motility are drastically impaired even after short exposure times of a few hours to sunlight not filtered by an ozone cuvette. Phototactic orientation starts to deteriorate after about 90 min and is completely lost after about 5 h. The percentage of motile cells in a population decreases likewise after an exposure of about 2 h and the velocity distributions shows a reduced speed of movement after an initial photokinetic increase. The damage is irreversible: in populations exposed for >2 h no living cell was found 24 h later. The UV-B sensitivity seems to be independent of the culture age at least over three weeks: While the percentage of motile cells changes with a peak at about 8 d, the relative UV-B induced inhibition is constant and depends only on the UV dose. DNA seems not to be the primary UV-B target since UV-B inhibition could not be repaired during subsequent dark or moderate light conditions even after low doses.

Differential roles for menaquinone and demethylmenaquinone in anaerobic electron transport of E. coli and their fnr-independent expression

Abstract: Escherichia coli grown with glucose in the absence of added electron acceptors contained 3–4 times more naphthoquinones (menaquinone plus demethylmenaquinone) than in the presence of O2. Presence of electron acceptors resulted in a slight additional increase of the naphthoquinone content. A strain defective in the fnr gene, which encodes the transcriptional activator of anaerobic respiration, showed the same response. With fumarate or dimethyl sulfoxide present, 94% of the naphthoquinones consisted of menaquinone, while with nitrate up to 78% was demethylmenaquinone. With trimethylamine N-oxid as the acceptor the proportion was intermediate. From the donor substrates of anaerobic respiration only glycerol had a significant influence on the ratio of the contents of the 2 quinones. It is concluded that FNR, the gene product of the fnr gene, is not required for anaerobic derepression of naphthoquinone viosynthesis. Menaquinone appears to be involved specifically in the respiration with fumarate or dimethyl sulfoxide, and demethylmenaquinone in nitrate respiration. Both naphthoquinones appear to serve in trimethylamine N-oxide respiration.

Sulphated exopolysaccharides produced by two unicellular strains of cyanobacteria, Synechocystis PCC 6803 and 6714

Abstract: The exopolysaccharides (EPS) of two unicellular strains of cyanobacteria Synechocystis PCC 6803 and 6714, formed labile, radial structures, uniformly distributed on the cell surface, and stainable by specific dyes for acidic polysaccharides. The two strains produced EPS at similar rates, which depended, along with the duration of the producing phase, on the incubation conditions. The exopolysaccharides from both strains were constituted of at least 11–12 mono-oses, probably forming several types of polymers. They contained about 15–20% (w/w) uronic derivatives and 10–15% (w/w) osamines. Proteins represented 20–40% of total weight. A most interesting feature was the presence of 7–8% (molar ratio) sulphate residues, a characteristic that is otherwise limited to exopolysaccharides produced by eukaryotic algae.

An extremely thermophilic Methanococcus from a deep sea hydrothermal vent and its plasmid

Abstract: An extremely thermophilic methanogen was isolated from a hydrothermal vent core sample from Guaymas Basin, Gulf of California, at a depth of 2003 m. The isolate, designated strain AG86, was a coccoid autotroph using H2-CO2 as energy and carbon source with a growth temperature range of 48 to 92°C, optimum, 85°C. AG86 required NaCl and Mg2+ and trace amounts of selenite and tungstate. Vitamins were not required. However, yeast extract, Casamino acids and Trypticase stimulated growth significantly. When grown in the presence of these stimulants and at the optimal growth temperature and pH 6.5, the minimum doubling time was 20 min. Cells were fragile and readily lysed by detergents. The mol% G+C was 33%. These results and partial 16S rRNA sequencing indicated that AG86 belonged to the genus Methanococcus and closely resembled Methanococcus jannaschii. Tests for extrachromosomal DNA revealed a plasmid in AG86 and two plasmids in M. jannaschii. Different patterns were obtained from restriction endonuclease digestion of the three plasmids, and no homology was observed with DNA-DNA hybridization.

Defects in cytochrome cd1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Abstract: Mutants with defective respiratory nitrite utilization (Nir- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd 1 content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c 552 about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd 1. Mutant strain MK202 lacked cytochrome cd 1 and, simultaneously, had low amounts of cytochrome c 552 and the split α-peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d 1 prosthetic group. Irrespective of these biochemically distinct Nir- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd 1 is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd 1. They also indicate the functional or regulatory interdependence of c-type cytochromes.

Lactate conversion to acetate, CO2 and H2 in cell suspensions of Desulfovibrio vulgaris (Marburg): indications for the involvement of an energy driven reaction

Abstract: Cell suspensions of Desulfovibrio vulgaris were found to catalyze, in the absence of sulfate, the complete conversion of 1 lactate to 1 acetate, 1 CO2, and 2 H2 (ΔG′0=-8.8 kJ/mol) and of 1 pyruvate to 1 acetate, 1 CO2, and 1 H2 (ΔG′0=-52 kJ/mol). Protonophores, the proton translocating ATPase inhibitor N,N′-dicyclohexylcarbodiimide, and arsenate specifically inhibited H2 formation from lactate but not from pyruvate. The results suggest that lactate oxidation to pyruvate and H2 (ΔG′0=+43.2 kJ/mol) is energy driven.

A new purple sulfur bacterium from stratified freshwater lakes, Amoebobacter purpureus sp. nov.

Abstract: Six strains of a new purple sulfur bacterium were isolated from the chemocline of four different freshwater lakes. Single cells were spherical to oval, nonmotile and contained gas vacuoles in the central part of the cytoplasm. All strains contained bacteriochlorophyll a and okenone as the major carotenoid. The intracytoplasmic membrane system was of vesicular type. All strains resembled each other in growth conditions and utilization of simple organic carbon sources. The strains were able to grow microaerophilic in the dark, used hydrogen sulfide, elemental sulfur or thiosulfate as electron donor, and lacked assimilatory sulfate reduction. On the basis of all characteristics the new bacterium represents a new species of the genus Amoebobacter, A. purpureus sp. nov.

Optimum culture conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1

Abstract: We sought the optimum conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1. The use of isovaleronitrile or isobutyronitrile as an inducer greatly enhanced benzonitrilase formation. When Rhodococcus rhodochrous J1 was cultivated at 28°C for 96 h in a medium consisting of 0.1 ml of isovaleronitrile, 0.5 g of polypeptone, 0.3 g of malt extract, 0.3 g of yeast extract and 1 g of glycerol per 100 ml of tap water (pH 7.2), and isovaleronitrile was fed twice at the concentrations of 0.1% (v/v) and 0.2% (v/v) at 55 h and 77 h, respectively, during the course of cultivation, the enzyme activity in the culture broth reached approximately 3,100-times higher than the initially obtained level.

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

Abstract: A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO2. H2, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros.

Abstract: The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.

Characterization of a new mesophilic, secondary alcohol-utilizing methanogen, Methanobacterium palustre spec. nov. from a peat bog

Abstract: The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H2/CO2 and formate in complex medium. It also grew autotrophically on H2/CO2. Furthermore, growth on 2-propanol/CO2 was observed. Methane was formed from CO2 by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO2 apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable. On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum. An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP+ was the only electron carrier that was utilized. No reaction was found with NAD+, F420, FMN and FAD.