Showing papers in "Archives of Microbiology in 1993"

Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals.

Abstract: The gram-negative metal-reducing microorganism, previously known as strain GS-15, was further characterized. This strict anaerobe oxidizes several short-chain fatty acids, alcohols, and monoaromatic compounds with Fe(III) as the sole electron acceptor. Furthermore, acetate is also oxidized with the reduction of Mn(IV), U(VI), and nitrate. In whole cell suspensions, the c-type cytochrome(s) of this organism was oxidized by physiological electron acceptors and also by gold, silver, mercury, and chromate. Menaquinone was recovered in concentrations comparable to those previously found in gram-negative sulfate reducers. Profiles of the phospholipid ester-linked fatty acids indicated that both the anaerobic desaturase and the branched pathways for fatty acid biosynthesis were operative. The organism contained three lipopolysaccharide hydroxy fatty acids which have not been previously reported in microorganisms, but have been observed in anaerobic freshwater sediments. The 16S rRNA sequence indicated that this organism belongs in the delta proteobacteria. Its closest known relative is Desulfuromonas acetoxidans. The name Geobacter metallireducens is proposed.

The biological role of nitric oxide in bacteria

Abstract: Bacterial N oxide metabolism is related to cellular bioenergetics and processes of nitrogen assimilation. The interest in nitric oxide (NO) centers around the dissimilatory transformation of nitrate, better known as the denitrification process. Denitrification [described as phenomenon more than 100 years ago (Gayon and Dupetit 1886)], is a distinctive mode of respiration that satisfies the bioenergetic needs of a great variety of bacteria by transforming oxyanions of nitrogen to Nz, mainly under conditions of reduced oxygen tension or strict anaerobiosis. The reaction reverses nitrogen fixation in the biogeochemical N cycle sustained by prokaryotes. Denitrification is controlled by the metalloenzymes nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide (N20) reductase and involves the corresponding enzyme substrates. The same magnitude of nitrogen fixed yearly by biological and abiological processes [combined estimates vary between 254 to 406 million tons N (Jenkinson 1990)] has to be returned to N 2 by denitrification to close the N cycle. However, because of the large anthropogenic contribution, fixation and denitrification are not balanced anymore as evident from the steady increase of nitrate in the environment. A second concern focussing around NO and the conditions of its microbial formation, is the nitrosation of secondary amines in the etiology of certain types of cancer. Since the discovery of NO in 1987 as a vasodilatory messenger (Ignarro et al. 1987; Palmer et al. 1987), the biomedical community is astounded by the diverse roles of NO in cellular communication including the central and peripheral nervous system, and in host defense mechanisms of eukaryotes (for reviews see Marletta et al. 1990; Moncada 1992; Nathan 1992; Traylor and Sharma 1992: Edelman and Gally 1992). Yet NO is not an obscure chemical and certainly no newcomer to the life sciences, as often stated in hyperbole. Early in evolution NO took its role as a central player in bacterial bioenergetics and in the global N cycle vital to all organisms. The chemistry of NO in biological systems and that of the nitroxyl anion (NO-) and nitrosonium cation (NO +) has been reviewed briefly (Stamler et al. 1992c; see also comment by Bonner and Hughes 1993). Another remarkable finding is the formation of N20 from nitrite, sometimes accompanied by NO production, by the fungus impetfectus Fusarium oxysporum and telemorphic and anamorphic relatives (Shoun et al. 1992). These fungi synthesize a special cytochrome P-450 induced only in the presence of nitrite (Shoun and Tanimoto 1991), which has been shown to have NO reductase activity (Nakahara et al. 1993). The existence of such a hemoprotein is of interest in the context that the cytokine (interferon-7 and lipopolysaccharide)-inducible form of NO synthase from macrophages is a cytochrome P-450 (White and Marletta 1992); also hepatic cytochrome P-450 monooxygenases are able to convert the Na-hydroxy-activated form of L-arginine to NO (Boucher et al. 1992). In another fungus, the slime mold Dictyostelium discoideum, NO alters the cellular aggregation behavior via ADP-ribosylation of a cytoplasmic 41-kDa protein (Tao et al. 1992). Principles of bacterial NO metabolism were spelled out nearly 40 years ago. NO was found as a product of denitrification from a clay loam microcosm with 15N nitrate as tracer (Wijler and Delwiche 1954). The study of nitrite utilization with Thiobacillus denitr~'cans established that NO was consumed and produced by a defined axenic culture (Baalsrud and Baalsrud 1954). NO was given status of an intermediate in bacterial denitrification, first as the result of studies with intact cells (Iwasaki etal. 1956; Matsubara and Mori 1968) and later from recognition that cell-free extracts of \"Pseudomonas denitr~'cans\" reduce exogenous NO (Miyata et al. 1969). Around that time Pseudomonas stutzeri ZoBell (formerly P. perfectomarina) was introduced to denitrification research and a pathway identical to that of Mori and coworkers was formulated (Payne et al. 1971):

Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

Abstract: A novel, hyperthermophilic, anaerobic, sulfurmetabolizing archaeon was isolated from a fluid sample from recently discovered hydrothermal vents in the North Fiji basin (SW Pacific), at 2000 m depth. The new organism, strain GE5, is a gram-negative, highly motile coccus. It grows between 67° and 102°C under atmospheric pressure, with an optimum at 96°C (doubling time 33 min). The upper growth temperature is extended by at least 3°C when cells are cultivated under in situ hydrostatic pressures (20 MPa). Strain GE5 is an obligate heterotroph, fermenting peptides, or mixtures of amino acids to acetate, isovalerate, isobutyrate, propionate, H2 and CO2. Hydrogen inhibits growth unless sulfur is present. In the presence of sulfur, H2S is then produced. Phylogenetic analyses of the 16 S rRNA sequence of strain GE5 places the new isolate within the Thermococcales. By its high growth temperature and physiological features the new isolate ressembles Pyrococcus sp. However it deffers by a 7% mol upper G+C-content and shows low level of DNA similarity with the two previously described species. Based on these differences the description of strain GE5 as a new species Pyrococcus abyssi (CNCM I-1302) is proposed.

Growth and flagellation of Vibrio fischeri during initiation of the sepiolid squid light organ symbiosis

Abstract: A pure culture of the luminous bacterium Vibrio fischeri is maintained in the light-emitting organ of the sepiolid squid Euprymna scolopes. When the juvenile squid emerges from its egg it is symbiont-free and, because bioluminescence is part of an anti-predatory behavior, therefore must obtain a bacterial inoculum from the surrounding environment. We document here the kinetics of the process by which newly hatched juvenile squids become infected by symbiosis-competent V. fischeri. When placed in seawater containing as few as 240 colony-forming-units (CFU) per ml, the juvenile became detectably bioluminescent within a few hours. Colonization of the nascent light organ was initiated with as few as 1 to 10 bacteria, which rapidly began to grow at an exponential rate until they reached a population size of approximately 105 cells by 12 h after the initial infection. Subsequently, the number of bacteria in the established symbiosis was maintained essentially constant by a combination of both a >20-fold reduction in bacterial growth rate, and an expulsion of excess bacteria into the surrounding seawater. While V. fischeri cells are normally flagellated and motile, these bacteria did not elaborate these appendages once the symbiosis was established; however, they quickly began to synthesize flagella when they were removed from the light organ environment. Thus, two important biological characteristics, growth rate and flagellation, were modulated during establishment of the association, perhaps as part of a coordinated series of symbiotic responses.

Competition for limiting amounts of oxygen between Nitrosomonas europaea and Nitrobacter winogradskyi grown in mixed continuous cultures

Abstract: Chemolithotrophic nitrifying bacteria are dependent on the presence of oxygen for the oxidation of ammonium via nitrite to nitrate. The success of nitrification in oxygen-limited environments such as waterlogged soils, will largely depend on the oxygen sequestering abilities of both ammonium- and nitrite-oxidizing bacteria. In this paper the oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter winogradskyi serotype agilis were determined with cells grown in mixed culture in chemostats at different growth rates and oxygen tensions. Reduction of oxygen tension in the culture repressed the oxidation of nitrite before the oxidation of ammonium was affected and hence nitrite accumulated. K m values found were within the range of 1–15 and 22–166 μM O2 for the ammonium- and nitrite-oxidizing cells, respectively, always with the lowest values for the N. europaea cells. Reduction of the oxygen tension in the culture lowered the half saturation constant K m for oxygen of both species. On the other hand, the maximal oxygen consumption rates were reduced at lower oxygen levels especially at 0 kPa. The specific affinity for oxygen indicated by the V max/K m ratio, was higher for cells of N. europaea than for N. winogradskyi under all conditions studied. Possible consequences of the observed differences in specific affinities for oxygen of ammonium-and nitrite-oxidizing bacteria are discussed with respect to the behaviour of these organisms in oxygen-limited environments.

Insoluble hydrophobin complexes in the walls of schizophyllum-commune and other filamentous fungi

Abstract: Two closely related cysteine-rich hydrophobic proteins, Sc3p and Sc4p, of the basidiomycete Schizophyllum commune are developmentally regulated and associated with the walls of aerial hyphae and fruit-body hyphae. They are present in the walls as hot-SDS-insoluble complexes which can be extracted with formic acid. The hydrophobins can then be dissociated by oxidation with performic acid. However, extraction of the walls with trifluoroacetic acid results in both solubilization and dissociation of the hydrophobin complexes into monomers. This suggests that non-covalent interactions are responsible for formation of these insoluble complexes. Carboxymethylation with iodoacetic acid only occurred after reduction with DTT indicating all cysteines in the monomeric hydrophobins involved in intramolecular disulfide bridges. Abundant proteins with similar properties were found in walls from all other filamentous fungi tested, including the basidiomycetes Pleurotus ostreatus, Coprinus cinereus, Agaricus bisporus, and Phanerochaete chrysosporium, the ascomycetes Aspergillus nidulans, Neurospora crassa, and Penicillium chrysogenum, and the zygomycete Mucor mucedo.

Anaerobic transformation of 2,4,6-trinitrotoluene (TNT).

Abstract: A sulfate-reducing bacterium using trinitrotoluene (TNT) as the sole nitrogen source was isolated with pyruvate and sulfate as the energy sources. The organism was able to reduce TNT to triaminotoluene (TAT) in growing cultures and cell suspensions and to further transform TAT to still unknown products. Pyruvate, H2, or carbon monoxide served as the electron donors for the reduction of TNT. The limiting step in TNT conversion to TAT was the reduction of 2,4-diamino-6-nitrotoluene (2,4-DANT) to triaminotoluene. The reduction proceeded via 2,4-diamino-6-hydroxylaminotoluene (DAHAT) as an intermediate. The intermediary formation of DAHAT was only observed in the presence of carbon monoxide or hydroxylamine, respectively. The reduction of DAHAT to triaminotoluene was inhibited by both CO and NH2OH. The inhibitors as well as DANT and DAHAT significantly inhibited sulfide formation from sulfite. The data were taken as evidence for the involvement of dissimilatory sulfite reductase in the reduction of DANT and/or DAHAT to triaminotoluene. Hydrogenase purified from Clostridium pasteurianum and carbon monoxide dehydrogenase partially purified from Clostridium thermoaceticum also catalyzed the reduction of DANT in the presence of methyl viologen or ferredoxin, however, as the main reduction product DAHAT rather than triaminotoluene was formed. The findings could explain the function of CO as an electron donor for the DANT reduction (to DAHAT) and the concomitant inhibitory effect of CO on triaminotoluene formation (from DAHAT) by the inhibition of sulfite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

Abstract: The ambivalent relations of sulfate-reducing bacteria to molecular O2 have been studied with ten freshwater and marine strains. Generally, O2 was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O2 concentrations of below 15 μM (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O2 as electron acceptor (up to one doubling of protein). However, O2 was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O2 tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O2 concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 μM. The specific rates of O2 uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O2 concentrations in the gas phase.

The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Abstract: Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C551 may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.

Enzymes of the reductive citric acid cycle in the autotrophic eubacterium Aquifex pyrophilus and in the archaebacterium Thermoproteus neutrophilus

Abstract: The autotrophic carbon fixation pathway was studied in the thermophilic hydrogen oxidizing eubacterium Aquifex pyrophilus and in the thermophilic sulfur reducing archaebacterium Thermoproteus neutrophilus. Neither organism contained ribulose-1,5-bisphosphate carboxylase activity suggesting that the Calvin cycle is not operating. Rather, all enzymes of the reductive citric acid cycle were found in A. pyrophilus. In T. neutrophilus ATP citrate lyase activity was detected which has not been achieved so far; this finding corroborates earlier work suggesting the presence of the reductive citric acid cycle in this archaebacterium. The reductive citric acid cycle for autotrophic CO2 fixation now has been documented in the eubacterial branches of the proteobacteria, in green sulfur bacteria, and in the thermophilic Knallgas bacteria as well as in the branch of the sulfur dependent archaebacteria.

Effect of redox potential on methanogenesis by Methanosarcina barkeri

Abstract: Concentrations of 0.5% O2 immediately inhibited CH4 production from methanol by Methanosarcina barkeri. Simultaneously, the redox potential of the medium increased to about +100 mV. However, the rates of CH4 production were not significantly affected, when the redox potential of an anoxic medium was adjusted to values between -420 mV and +100 mV by addition of titanium (III) citrate, sodium dithionite, flavin adenine dinucleotide, or sodium ascorbate. When the redox potential was adjusted to values between -80 mV and +550 mV by means of mixtures of ferrocyanide and ferricyanide, CH4 production was not inhibited until a redox potential of about +420 mV was reached. M. barkeri was able to reduce 0.5 mM ferricyanide solution at +430 mV within 0.005%.

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS-) of Pseudomonas stutzeri.

Abstract: The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export The sequence showed 638% positional identity with the amino acid sequence of "Achromobacter cycloclastes" nitrite reductase Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified The nirK gene was transferred to the mutant MK202 of P stutzeri which lacks cytochrome cd1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro Expression of the nirS gene from P stutzeri in P aureofaciens and E coli led to nonfunctional gene products Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d1, indicating that both heterologous hosts synthesized a cytochrome cd1 without the d1-group

Transient, specific and extremely rapid release of osmolytes from growing cells of Escherichia coli K-12 exposed to hypoosmotic shock.

Abstract: The influence of hypoosmotic shock on the solute content of growing Escherichia coli K-12 cells was investigated at 37°C. Within 20 s after the shock the cells had released most of their osmolytes K+, glutamate and trehalose. This release was specific and not due to rupture of the cell membrane, since under these conditions i) the cells neither lost protein nor ATP, ii)[14C]-labeled sucrose did not enter the cytoplasm from the periplasm, and iii) except for their glutamate and aspartate level, which decreased, the amino acid pool of alanine, lysine and arginine of the cells remained approximately constant. Within a minute after the shock the cells started to reaccumulate parts of their previously released glutamate, aspartate and K+, but not trehalose and resumed growth within 10 min after the shock. Experiments with K+-transport mutants showed that none of the genetically-identified K+ transport systems is involved in the K+-release process. Reaccumulation of K+ took place via the uptake systems TrkG and TrkH. The possibility is discussed that the exit of solutes after hypoosmotic shock occurs via several stretch-activated channels, which each allow the release of a specific osmolyte.

Acetyl-CoA synthetase (ADP forming) in archaea, a novel enzyme involved in acetate formation and ATP synthesis

Abstract: The anaerobic hyperthermophilic archaea Desulfurococcus amylolyticus, Hyperthermus butylicus, Thermococcus celer, Pyrococcus woesei, the hyperthermophilic bacteria Thermotoga maritima and Clostridium thermohydrosulfuricum and the aerobic mesophilic archaeon Halobacterium saccharovorum were grown either on complex media, on sugars or on pyruvate as carbon and energy sources. During growth acetate was formed as fermentation product by all organisms. The enzymes involved in acetyl-CoA formation from pyruvate and in acetate formation from acetyl-CoA were investigated: These data indicate that acetyl-CoA synthetase (ADP forming) represents a typical archaeal property rather than an enzyme specific for hyperthermophiles. It is proposed that in all acetate forming archaea the formation of acetate and of ATP from acetyl-CoA, ADP and Pi are catalyzed by acetyl-CoA synthetase (ADP forming), whereas in all acetate forming (eu)bacteria these reactions are catalyzed by two enzymes, phosphate acetyltransferase and acetate kinase.

Effect of dissolved inorganic carbon on the expression of carboxysomes, localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

Abstract: In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO2+HCO inf3 sup- ) in the growth medium. In cells grown under 5% CO2 and in those bubbled with air, carboxysomes were present in low numbers ( 8 · longitudinal section-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO inf3 sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.

Syntrophic interactions during degradation of 4-aminobenzenesulfonic acid by a two species bacterial culture.

Abstract: During synthrophic growth of Hydrogenophaga palleronii (strain S1) and Agrobacterium radiobacter (strain S2) with 4-aminobenzene sulfonate (4ABS) only strain S1 desaminates 4ABS by regioselective 3,4-dioxygenation. The major part of the metabolite catechol-4-sulfonate (4CS) is excreted and further metabolized by strain S2. Although both organisms harbour activities of protocatechuate pathways assimilation of the structural analog 4CS requires first of all enzyme activities with broader substrate specificity: protocatechuate 3,4-dioxygenase and carboxymuconate cycloisomerase activities were identified which in addition to the natural substrates also convert 4CS requires first of all enzyme activities with Carboxymethyl-4-sulfobut-2-en-4-olide (4SL) was identifed as a metabolite. Its further metabolism requires a desulfonating enzyme which eliminates sulfite from (4SL) and generates maleylacetate. Convergence with the 3-oxoadipate pathway is catalyzed by a maleyl acetate reductase, which was identified in cell-free extracts of both organisms S1 and S2. Characteristically, only strain S1 can oxidize sulfite and thus contributes to the interdependence of the two bacteria during growth with 4ABS.

Bacterial sulphur respiration

Abstract: The reduction of elemental sulphur to HSis coupled to the phosphorylation of ADP with inorganic phosphate in the catabolism of certain anaerobic bacteria. This process is called sulphur respiration. Since the discovery of sulphur respiration with the isolation of Desulfuromonas acetoxidans (Pfennig and Biebl 1976), many other bacteria have been found that catalyze the reduction of elemental sulphur (for review see Widdel 1988; Stetter et al. 1990: Adams 1990; Fauque et al. 1991). Most of these organisms belong to the group of extremely thermophilic archaebacteria. Also some species of Thermotoga, the earliest branch of the eubacterial domain, as well as the methanogens catalyze sulphur reduction. These organisms are thought to be more closely related to the common ancestor than any other living organism. Therefore, sulphur respiration may represent one of the first means of biological energy conservation in evolution (Stetter and Gaag 1983). For bacteria that grow with H 2 or formate as electron donors and sulphur as acceptor it is evident from growth that sulphur reduction is coupled to phosphorylation, since the growth reactions do not allow substrate level phosphorylation. In contrast, the metabolic significance of sulphur reduction is uncertain in other bacteria. After a brief discussion of the biology of sulphur reducers, this review will focus on the chemistry, enzymology and bioenergetics of catabolic sulphur reduction. Sulphur reduction serving anabolic purposes will not be discussed here (for review see Le Faou et al. 1990).

Cloning and sequencing of curA encoding curvacin A, the bacteriocin produced by Lactobacillus curvatus LTH1174.

Abstract: Curvacin A is a bacteriocin produced by Lactobacillus curvatus LTH1174 which is a potential starter organism for the production of fermented dry sausages. This peptide inhibits the growth of the opportunistic food pathogens Listeria monocytogenes and Enterococcus faecalis and thus, curvacin A may enable better performance of a starter and improvement of the hygienic status of meat products. Oligonucleotides were constructed deduced from the peptide sequence and used for the identification of the curvacin A structural gene cur A on a 60 kb plasmid of L. curvatus LTH1174. Plasmidcured derivatives of this strain were unable to produce curvacin A but were still resistant to the bacteriocin. Cur A was cloned into Escherichia coli NM554 and its nucleotide sequence was determined. Sequencing revealed the presence of an additional open reading frame of 51 amino acids with unknown function. A promoter was detected upstream of cur A by primer extension. Both reading frames form a single transcript. Curvacin A is synthesised as a prepeptide of 59 amino acids which is proteolytically processed to the mature bacteriocin of 41 amino acids.

Formation of Dimethylsulfide and Methanethiol from Methoxylated Aromatic Compounds and Inorganic Sulfide by Newly Isolated Anaerobic Bacteria

Abstract: Anaerobic enrichment cultures supplemented with methoxylated aromatic acids (3,4,5-trimethoxybenzoic acid or syringic acid) showed intense gas production and smell of methylated sulfur compounds. Via direct dilution of mud samples, two strictly anaerobic rod-shaped bacteria were isolated which produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. The volatile organic sulfur compounds were derived from the methoxyl carbon and the sulfide added as a reducing agent to the medium. Both isolates also grew with gallate, phloroglucinol, or pyrogallol without producing dimethylsulfide or methanethiol. The aromatic substrates were degraded to acetate. Determination of the stoichiometries of substrate degradation and sulfur compound formation indicated a stepwise methylation of sulfide to dimethylsulfide with methanethiol as an intermediate. The principle biochemical pathways leading to formation of methylated sulfur compounds by the new isolates have not been elucidated so far.

Carbon source control on β-glucanases, chitobiase and chitinase from Trichoderma harzianum

Abstract: The cell-wall degrading enzymes β-glucanase and chitinase have been suggested to be essential for the mycoparasitic action of Trichoderma species against plant fungal pathogens. For this reason, the production in different carbon sources of extracellular β-1,3-glucanase, β-1,6-glucanase, chitobiase and chitinase was studied in a mycoparasitic strain of Trichoderma harzianum. Maximal β-glucanase specific activities were detected in media supplemented with either pustulan (β-1,6-glucan), nigeran (α-1,3-glucan alternating with α-1,4-glucan), chitin or Saccharomyces cerevisiae or Botrytis cinerea purified cell walls, whereas the highest chitinase specific activity was obtained in medium supplemented with chitin. Furthermore, β-glucanase, chitobiase and chitinase activities showed an increase parallel to increasing concentrations of either pustulan or chitin added to the cultures, although the extent of this increase varied with the different enzymes. The culture filtrates of T. harzianum grown in these carbon sources also showed lytic activity on purified cell walls of S. cerevisiae and B. cinerea. The enzyme synthesis seemed to be repressed by glucose, 8-hydroxyquinoline, which inhibits transcription, or cycloheximide, an inhibitor of protein synthesis.

Characterization of a DMSP-degrading bacterial isolate from the Sargasso Sea

Abstract: A bacterium which cleaves dimethylsulfoniopropionate (DMSP) to form dimethylsulfide (DMS) was isolated from surface Sargasso Sea water by a DMSP enrichment technique. The isolate, here designated LFR, is a Gram-negative, obligately aerobic, rod-shaped, carotenoid-containing bacterium with a DNA G+C content of 70%. Sequencing and comparison of its 16S ribosomal ribonucleic acid (rRNA) with that of known eubacteria revealed highest similarity (91% unrestricted sequence similarity) to Roseobacter denitrificans (formerly Erythrobacter species strain OCh114), an aerobic, bacteriochlorophyll-containing marine representative of the α-Proteobacteria. However, physiological differences between the two bacteria, and the current lack of other characterized close relatives, preclude assignment of strain LFR to the Roseobacter genus. Screening of fifteen characterized marine bacteria revealed only one, Pseudomonas doudoroffii, capable of degrading DMSP to DMS. Strain LFR is deposited with the American Type Culture Collection (ATCC 51258) and 16S rRNA sequence data are available under GenBank accession number 15345.

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2

Abstract: In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes ‘malic enzyme’, NAD(P)H: ferredoxin oxidoreductase, pyruvate: ferredoxin oxidoreductase, hydrogenase, acetate: succinate CoA transferase and succinate thiokinase leading to the formation of H2, CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role-especially in zoospores — as H2-evolving, ATP-generating and O2-scavenging organelles.

Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene

Abstract: We have determined the nucleotide sequences of two structural genes of the Escherichia coli gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway: gabD, coding for succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16) and gabP, coding for the 4-aminobutyrate (GABA) transport carrier (GABA permease). We have previously reported the nucleotide sequence of the third structural gene of the cluster, gabT, coding for glutamate: succinic semialdehyde transaminase (EC 2.6.1.19). All three gab genes are transribed unidirectionally and their orientation within the cluster is 5′-gabD-gabT-gabP-3′. gabT and gabP are separated by an intergenic region of 234-bp, which contains three repetetive extragenic palindromic (REP) sequences. The gabD gene consists of 1,449 nucleotides specifying a protein of 482 amino acids with a molecular mass of 51.7 kDa. The protein shows significant homologies to the NAD+-dependent aldehyde dehydrogenase (EC 1.2.1.3) from Aspergillus nidulans and several mammals, and to the tumor associated NADP+-dependent aldehyde dehydrogenase (EC 1.2.1.4) from rat. The permease gene gabP comprises 1,401 nucleotides coding a highly hydrophobic protein of 466 amino acids with a molecular mass of 51.1 kDa. The GABA permease shows features typical for an integral membrane protein and is highly homologous to the aromatic acid carrier from E. coli, the proline, arginine and histidine permeases from Saccharomyces cerevisiae and the proline transport protein from A. nidulans. Uptake of GABA was increased ca. 5-fold in transformants of E. coli containing gabP plasmids. Strong overexpression of the gabP gene under control of the isopropyl-2-d-thiogalactoside (IPTG) inducible tac promoter, however, resulted in a severe growth inhibition of the transformed strains. The GABA carrier was characterized using moderately overexpressing transformants. The K m of GABA uptake was found to be 11.8 μM and the Vmax 0.33 nmol/min · mg cells. Uptake of GABA was stimulated by ammonium sulfate and abolished by 2,4-dinitrophenol. Aspartate competed with GABA for uptake.

Zymobacter palmae gen. nov., sp. nov., a new ethanol-fermenting peritrichous bacterium isolated from palm sap

Abstract: Zymobacter palmae gen. nov., sp. nov. was proposed for a new ethanol-fermenting bacterium that was isolated from palm sap in Okinawa Prefecture, Japan. The bacterium is gram-negative, facultatively anaerobic, catalase-positive, oxidase-negative, nonspore-forming and peritrichously flagellated. It requires nicotinic acid for growth. It ferments hexoses, α-linked di- and tri-saccarides, and sugar alcohols (fructose, galactose, glucose, mannose, maltose, melibiose, saccharose, raffinose, mannitol and sorbitol). Fifteen percent of maltose in broth medium is effectively fermented, whereas glucose with a concentration higher than 10% delayed growth initiation and decreased growth rates. Maltose is fermented to produce ethanol and CO2 with a trace amount of acids. Approximately 2 mol of ethanol are produced from 1 mol moiety of hexose of maltose. The organism possesses ubiquinone-9. The G+C content of the DNA is 55.8+-0.4 mol%. Major cellular fatty acids were palmitic and oleic acids and cyclopropanic acid of C19:0. Characteristic hydroxylated acid was 3-hydroxy dodecanoic acid. The bacterium is distinct from other ethanol-fermenting bacteria belonging to the genera Zymomonas Kluyver and van Niel 1936 and Saccharobacter Yaping et al. 1990 with respect to chemotaxonomic and other phenotypic characters to warrant to compose a new genus and a new species. The type strain is strain T109 (= IAM 14233).

Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate

Abstract: Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).

Polysulfide as a possible substrate for sulfur-reducing bacteria

Abstract: Because of its low solubility it is unlikely that elemental sulfur serves as the direct substrate for sulfur-reducing bacteria. To test the hypothesis that polysulfide may represent a soluble intermediate of sulfur reduction, the maximal polysulfide concentrations formed from elemental sulfur in aqueous sulfide solutions were measured at near neutral pH and at temperatures up to 90°C. The saturation concentrations decreased by two orders of magnitude when the pH was lowered from 7 to 6 at a given temperature, and increased about tenfold when the temperature was raised from 37°C to 90°C at a given pH. The dissolution of 0.1 mM zerovalent sulfur in 1 mM sulfide (H2S+HS−) required a pH of 7.5 at 20°C and of only 6.1 at 100°C. A comparison with the growth optima of sulfur-reducers suggests that polysulfide is present at sufficient concentration at the growth conditions of the Bacteria and the moderately acidophilic Archaea. Polysulfide is apparently not available at the growth conditions of the extremely acidophilic Archaea. Alternative mechanisms for the sulfur utilization under these conditions are discussed.

Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases.

Abstract: The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR β-subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis of DNA fragments revealed three adjacent open reading frames in the order norA, norX, norB. The DNA sequences of norX and norB represented complete genes while the open reading frame of norA was truncated by the cloning site. The deduced amino acid sequence of protein NorB contained four cysteine clusters with striking homology to those of iron-sulfur centers of bacterial ferredoxins. NorB shares significant sequence similarity to the β-subunits (NarH, NarY) of the two dissimilatory nitrate reductases (NRA, NRZ) of Escherichia coli. Additionally, the derived amino acid sequence of the truncated open reading frame of norA showed striking resemblance to the α-subunits (NarG, NarZ) of the E. coli nitrate reductases.

Impact of light dark regimen on growth-rate, biomass formation and bacteriochlorophyll synthesis in erythromicrobium-hydrolyticum

Abstract: The impact of illumination on specific growth rate, biomass formation, and synthesis of photopigment was studied in Erythromicrobium hydrolyticum, an obligately aerobic heterotrophic bacterium having the ability to synthesize bacteriochlorophyll a. In dark-grown continuous cultures the concentration of protein increased with increasing dilution rate, the concentration of bacteriochlorophyll a showed the opposite effect. At a dilution rate of 0.08 h-1 (68% of μmax in the dark) and SR-acetate of 11.8 mM, the concentration of BChla of illuminated cultures in steady-state was 11–22 nM, compared to 230–241 nM in cultures incubated in darkness. No significant differences were observed in the concentration of protein. A shift from darkness to light conditions resulted in increased specific growth rates resulting in increased biomass formation, thus showing that light enhances growth by serving as an additional energy source. This phenomenon, however, was temporary because bacteriochlorophyll synthesis is inhibited by light. In contrast to incubation in continuous light or dark, incubation under light/dark regimen resulted in permanently enhanced biomass formation. In the dark periods, bacteriochlorophyll was synthesized at elevated rates (compared to constant darkness), thus compensating the inhibitory effect of light in the preceding period. It thus appears that the organism is well-adpated to life in environments with alternating light/dark conditions. The ecological relevance of the observations is discussed.

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus - involvement of reactions of the Embden-Meyerhof pathway

Abstract: The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture.

Abstract: The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014-0.076 h-1) and extracellular pH (6.11-6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentration to response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07-0.11 h-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04-0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11-0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determine that maintenance coefficient (0.04-0.06 g cellulose/g cells.h) and true growth yield (0.23-0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower mu max on microcrystalline cellulose.