bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of acetate turnover in methanogenic and sulfate-reducing sediments by radiolabeling and stable isotope labeling and by use of specific inhibitors: evidence for isotopic exchange
Authors: de Graaf W , Wellsbury P , Parkes R J , Cappenberg T E
Published: January 2020

Acetate turnover in the methanogenic freshwater anoxic sediments of Lake Vechten, The Netherlands, and in anoxic sediments from the Tamar Estuary, United Kingdom, and the Grosser Jasmunder Bodden, Germany, the latter two dominated by sulfate reduction, was determined. Stable isotopes and radioisotopes, inhibitors (chloroform and fluoroacetate), and methane flux were used to provide independent estimates of acetate turnover. Pore water acetate pool sizes were determined by gas chromatography with a flame ionization detector, and stable isotope-labeled acetate was determined by gas chromatography-mass spectrometry. The appearance of acetates with a different isotope labeling pattern from that initially added demonstrated that isotopic exchange occurred during methanogenic acetate metabolism. The predominant exchange processes were (i) D-H exchange in the methyl group and (ii) (sup13)C-(sup12)C exchange at the carboxyl carbon. These exchanges are most probably caused by the activity of the enzyme complex carbon monoxide dehydrogenase and subsequent methyl group dehydrogenation by tetrahydromethanopterine or a related enzyme. The methyl carbon was not subject to exchange during transformation to methane, and hence acetate with the methyl carbon labeled will provide the most reliable estimate of acetate turnover to methane. Acetate turnover rate estimates with these labels were consistent with independent estimates of acetate turnover (acetate accumulation after inhibition and methane flux). Turnover rates from either radioisotope- or stable isotope-labeled methyl carbon isotopes are, however, dependent on accurate determination of the acetate pool size. The additions of large amounts of stable isotope-labeled acetate elevate the acetate pool size, stimulating acetate consumption and causing deviation from steady-state kinetics. This can, however, be overcome by the application of a non-steady-state model. Isotopic exchange in sediments dominated by sulfate reduction was minimal.
Initial hydrogenation and extensive reduction of substituted 2,4-dinitrophenols
Authors: Lenke H , Knackmuss H
Published: January 2020

Rhodococcus erythropolis HL 24-1 isolated as a 2,4-dinitrophenol-degrading organism can utilize 2-chloro-4,6-dinitrophenol as the sole nitrogen, carbon, and energy source under aerobic conditions. This compound is metabolized with liberation of stoichiometric amounts of chloride and nitrite. Under anaerobic conditions, 2,4-dinitrophenol was transiently accumulated in the culture fluid, indicating a reductive elimination of chloride. During aerobic bioconversion of 2-amino-4,6-dinitrophenol by R. erythropolis HL 24-1, a reductive elimination of nitrite leading to 2-amino-6-nitrophenol was observed. Elimination of chloride or nitrite by the initial formation of a hydride Meisenheimer complex is discussed. A methyl group in the ortho position of the 2,4-dinitrophenol gives rise to an extensive reduction of the aromatic ring under aerobic conditions. Thus, 2-methyl-4,6-dinitrophenol was shown to be converted to the two diastereomers of 4,6-dinitro-2-methylhexanoate as dead-end metabolites which were identified by spectroscopic methods.
High Nitrate Concentrations in Vacuolate, Autotrophic Marine Beggiatoa spp
Authors: McHatton S C , Barry J P , Jannasch H W , Nelson D C
Published: January 2020

Massive accumulations of very large Beggiatoa spp. are found at a Monterey Canyon cold seep and at Guaymas Basin hydrothermal vents. Both environments are characterized by high sediment concentrations of soluble sulfide and low levels of dissolved oxygen in surrounding waters. These filamentous, sulfur-oxidizing bacteria accumulate nitrate intracellularly at concentrations of 130 to 160 mM, 3,000- to 4,000-fold higher than ambient levels. Average filament widths range from 24 to 122 (mu)m, and individual cells of all widths possess a central vacuole. These findings plus recent parallel discoveries for Thioploca spp. (H. Fossing, V. A. Gallardo, B. B. Jorgensen, M. Huttel, L. P. Nielsen, H. Schulz, D. E. Canfield, S. Forster, R. N. Glud, J. K. Gundersen, J. Kuver, N. B. Ramsing, A. Teske, B. Thamdrup, and O. Ulloa, Nature (London) 374:713-715, 1995) suggest that nitrate accumulation may be a universal property of vacuolate, filamentous sulfur bacteria. Ribulose bisphosphate carboxylase-oxygenase and 2-oxoglutarate dehydrogenase activities in the Beggiatoa sp. from Monterey Canyon suggest in situ autotrophic growth of these bacteria. Nitrate reductase activity is much higher in the Monterey Beggiatoa sp. than in narrow, laboratory-grown strains of Beggiatoa spp., and the activity is found primarily in the membrane fraction, suggesting that the vacuolate Beggiatoa sp. can reduce nitrate coupled to electron flow through an electron transport system. Nitrate-concentrating and respiration potentials of these chemolithoautotrophs suggest that the Beggiatoa spp. described here are an important link between the sulfur, nitrogen, and carbon cycles at the Monterey Canyon seeps and the Guaymas Basin hydrothermal vents where they are found.
Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2
Authors: Binks P R , French C E , Nicklin S , Bruce N C
Published: January 2020

A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.
Quantitative Analysis of the Physiological Heterogeneity within Starved Cultures of Micrococcus luteus by Flow Cytometry and Cell Sorting
Authors: Kaprelyants A S , Mukamolova G V , Davey H M , Kell D B
Published: January 2020

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). We used flow cytometry and cell sorting to study populations of bacteria that had been starved for 5 months. These cells could be stained by the fluorescent lipophilic cation rhodamine 123, but such staining was almost independent of metabolically generated energy in that it was not affected by uncouplers. Two populations could be distinguished, one with a lower degree of rhodamine fluorescence (a degree of fluorescence referred to as region A and containing approximately 80% of the cells) and one with a more elevated degree of fluorescence (region B, approximately 20% of the cells). Subsequent incubation of starved cells in fresh medium in the presence of the antibiotic chloramphenicol (to which M. luteus is sensitive) resulted in the transient appearance of cells actively accumulating rhodamine 123 (and fluorescing in region B) and of larger cells exhibiting a yet-greater degree of fluorescence (region C). These more fluorescent cells accounted for as much as 50% of the total population, under conditions in which the viable and total counts were constant. Thus, metabolic resuscitation of at least one-half of the cells takes place under conditions in which cryptic growth cannot play any role. Sorting experiments revealed that the great majority of the viable cells in the starved population are concentrated in regions B and C and that the extent of rhodamine staining under conditions of starvation therefore reflects the physiological state of the cells. Physical separation of these cells from cells in region A resulted in an increase (of approximately 25-fold) in the viability of cells in regions B and C and of the population as a whole. Resuscitation of dormant cells in a most-probable-number assay in the presence of supernatant taken from growing M. luteus revealed the resuscitation of cells from regions B and C but not from region A. It is suggested that initially dormant (resuscitable) cells are concentrated in regions B and C.
Genotyping of heterotrophic bacteria from the central baltic sea by use of low-molecular-weight RNA profiles
Authors: Hofle M G , Brettar I
Published: January 2020

The Gotland Deep, an anoxic basin, was investigated for its heterotrophic microflora as a station representative of the central Baltic Sea and as an example of a brackish water environment. One hundred twenty-three bacterial strains were isolated along the water column by use of four different cultivation procedures. High-resolution electrophoresis of the low-molecular-weight (LMW) RNA (5S rRNA and tRNA) was used for analysis of the taxonomic position of the strains. The banding pattern of the LMW RNA generated by the electrophoresis allowed a taxonomic grouping at the species level of the 123 strains into 24 different genotypes. This grouping was confirmed by use of long-range gels with a substantially better resolution than that of standard gels; i.e., about 60% more tRNA bands were obtained on the long-range gels, and the distance between the bands was increased by about two-thirds. The majority of the strains (76%) could be identified to the species level by comparison with LMW RNA profiles from reference strains stored in an electronic database. Eighty-seven percent of the strains could be assigned to the families Vibrionaceae, Enterobacteriaceae, and Pseudomonadaceae (rRNA group I). The most abundant species among the isolates were Shewanella putrefaciens (48%) and a new Pseudomonas species (24%). The remaining fraction of 28% of the isolates was split into 22 other genotypes. Thirteen of these genotypes were represented by single isolates. This study demonstrates the utility of LMW RNA profiling for a rapid assessment of genotypic diversity of heterotrophic isolates from natural environments.
Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column
Authors: Ramsing N B , Fossing H , Ferdelman T G , Andersen F , Thamdrup B
Published: January 2020

The vertical distribution of major and intermediate electron acceptors and donors was measured in a shallow stratified fjord. Peaks of zero valence sulfur, Mn(IV), and Fe(III) were observed in the chemocline separating oxic surface waters from sulfidic and anoxic bottom waters. The vertical fluxes of electron acceptors and donors (principally O2 and H2S) balanced within 5%; however, the zones of oxygen reduction and sulfide oxidation were clearly separated. The pathway of electron transfer between O2 and H2S was not apparent from the distribution of sulfur, nitrogen, or metal compounds investigated. The chemical zonation was related to bacterial populations as detected by ethidium bromide (EtBr) staining and by in situ hybridization with fluorescent oligonucleotide probes of increasing specificity. About half of all EtBr-stained cells were detectable with a general oligonucleotide probe for all eubacteria when digital image analysis algorithms were used to improve sensitivity. Both EtBr staining and hybridization indicated a surprisingly uniform distribution of bacteria throughout the water column. However, the average cell size and staining intensity as well as the abundance of different morphotypes changed markedly within the chemocline. The constant overall cell counts thus concealed pronounced population shifts within the water column. Cells stained with a delta 385 probe (presumably sulfate-reducing bacteria) were detected at the chemocline at about 5 x 10(4) cells per ml, and this concentration increased to 2 x 10(5) cells per ml beneath the chemocline. A long slim rod-shaped bacterium was found in large numbers in the oxic part of the chemocline, whereas large ellipsoid cells dominated at greater depth. Application of selective probes for known genera of sulfate-reducing bacteria gave only low cell counts, and thus it was not possible to identify the dominant morphotypes of the sulfate-reducing community.
Anaerobic, nitrate-dependent microbial oxidation of ferrous iron
Authors: Straub K L , Benz M , Schink B , Widdel F
Published: January 2020

Enrichment and pure cultures of nitrate-reducing bacteria were shown to grow anaerobically with ferrous iron as the only electron donor or as the additional electron donor in the presence of acetate. The newly observed bacterial process may significantly contribute to ferric iron formation in the suboxic zone of aquatic sediments.
Homology between genes for aromatic hydrocarbon degradation in surface and deep-subsurface Sphingomonas strains
Authors: Kim E , Aversano P J , Romine M F , Schneider R P , Zylstra G J
Published: January 2020

The cloned genes for aromatic hydrocarbon degradation from Sphingomonas yanoikuyae B1 were utilized in Southern hybridization experiments with Sphingomonas strains from the surface and deep-subsurface environments. One hybridization pattern was obtained with BamHI-digested genomic DNAs for two surface strains, while a differing pattern was seen for five deep-subsurface strains. The cross-hybridizing genes were located in the chromosomes of the surface strains and on plasmids in the deep-subsurface strains.
Purification and characterization of microbial gellan lyase
Authors: Hashimoto W , Inose T , Nakajima H , Sato N , Kimura S , Murata K
Published: January 2020

Gellan lyase was purified from the culture fluid of soil samples incubated in a medium containing gellan as a sole carbon source. The enzyme was a monomer with a molecular mass of 140 kDa and was most active at pH 7.5 and 45 degrees C. The enzyme was highly specific to gellan and lowered the viscosity of the polymer.
Development of an oligonucleotide probe for Aureobasidium pullulans based on the small-subunit rRNA gene
Authors: Li S , Cullen D , Hjort M , Spear R , Andrews J H
Published: January 2020

Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surfaces and has potential as a biocontrol agent of pathogens. To assess the feasibility of rRNA as a target for A. pullulans-specific oligonucleotide probes, we compared the nucleotide sequences of the small-subunit rRNA (18S) genes of 12 geographically diverse A. pullulans strains. Extreme sequence conservation was observed. The consensus A. pullulans sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 A. pullulans strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum was also identified. These probes have potential in monitoring and quantifying fungi in leaf surface and other microbial communities.
Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes
Authors: Bogan B W , Lamar R T
Published: January 2020

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.
Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Batch and Fed-Batch Cultures
Authors: Wang P , Krawiec S
Published: January 2020

The DbtS(sup+) phenotype (which confers the ability to oxidize selectively the sulfur atom of dibenzothiophene [DBT] or dibenzothiophene sulfone [DBTO(inf2)]) of Rhodococcus erythropolis N1-36 was quantitatively characterized in batch and fed-batch cultures. In flask cultures, production of the desulfurization product, monohydroxybiphenyl (OH-BP), was maximal at pH 6.0, while specific productivity (OH-BP cell(sup-1)) was maximal at pH 5.5. Quantitative measurements in fermentors (in both batch and fed-batch modes) demonstrated that DBTO(inf2) as the sole sulfur source yielded a greater amount of product than did DBT. Specifically, 100 (mu)M DBT maximally yielded (apprx=)40 (mu)M OH-BP, while 100 (mu)M DBTO(inf2) yielded (apprx=)60 (mu)M OH-BP. Neither maintaining the pH at 6.0 nor adding an additional carbon source increased the yield of OH-BP. The presence of SO(inf4)(sup2-) in growth media repressed expression of desulfurization activity, but SO(inf4)(sup2-) added to suspensions of cells grown in DBT or DBTO(inf2) did not inhibit desulfurization activity.
Comparison of Quasielastic Light Scattering and Laser Diffractometry as Nondestructive Probes into the Structure of Bacillus sphaericus Spores Produced at Different Temperatures
Authors: Molina-Garcia A D , de Pieri L A , Ludlow I , Waites W M , de la Torre J G , Harding S E
Published: January 2020

Quasielastic light scattering (QLS) and laser diffractometry (LD) are relatively novel nondestructive procedures for estimating the sizes of bacterial spores in suspension. This study for the first time directly compared the two with a destructive procedure, namely, scanning electron microscopy (SEM), for quasispherical spores of Bacillus sphaericus. Because of the different physical aspect measured, the sizes derived by QLS and LD are, as could be expected for spores with an exosporium, significantly different. The larger estimates obtained by QLS (1.70, 1.58, and 1.14 (mu)m for spores produced at 15(deg)C [BS15], 20(deg)C [BS20], and 30(deg)C [BS30], respectively) than by LD (0.56 [BS15], 0.58 [BS20], and 0.52 [BS30] (mu)m) and SEM (0.64 [BS15], 0.58 [BS20], and 0.70 [BS30] (mu)m) are explained in terms of the detection by QLS, LD, and SEM of different spore layers and the degree of nonsphericity of the latter.
Evaluation of three portable samplers for monitoring airborne fungi
Authors: Mehta S K , Mishra S K , Pierson D L
Published: January 2020

Airborne fungi were monitored at five sample sites with the Burkard portable, the RCS Plus, and the SAS Super 90 air samplers; the Andersen 2-stage impactor was used for comparison. All samplers were calibrated before being used simultaneously to collect 100-liter samples at each site. The Andersen and Burkard samplers retrieved equivalent volumes of airborne fungi; the SAS Super 90 and RCS Plus measurements did not differ from each other but were significantly lower than those obtained with the Andersen or Burkard samplers. Total fungal counts correlated linearly with Cladosporium and Penicillium counts. Alternaria species, although present at all sites, did not correlate with total count or with amounts of any other fungal genera. Sampler and location significantly influenced fungal counts, but no interactions between samplers and locations were found.
Evaluation of strains isolated by growth on naphthalene and biphenyl for hybridization of genes to dioxygenase probes and polychlorinated biphenyl-degrading ability
Authors: Pellizari V H , Bezborodnikov S , Quensen J F , Tiedje J M
Published: January 2020

Approximately equal numbers of bacteria were isolated from primarily tropical soils by growth on biphenyl and naphthalene to compare their competence in polychlorinated biphenyl (PCB) degradation. The strains isolated by growth on biphenyl catalyzed more extensive PCB degradation than the strains isolated by growth on naphthalene, suggesting that naphthalene cocontamination may be only partially effective in stimulating the cometabolism of lower chlorinated PCBs. Probes were made from the bph, nah, and tod genes encoding the large iron iron sulfur protein of the dioxygenase complex and hybridized to 19 different strains. The hybridization patterns did not correlate well with the substrates of isolation, suggesting that there is considerable diversity in these genes in nature and that probe hybridization is not a reliable indication of catabolic capacity. The strains with the most extensive PCB degradation capacity did strongly hybridize to the bph probe, but a few strains that exhibited strong hybridization had poor PCB-degrading ability. Of the 19 strains studied, 5 hybridized to more than one probe and 2, including one strong PCB degrader, hybridized to all three probes. Southern blots showed that the bph and nah probes hybridized to separate bands, suggesting that multiple dioxygenases were present. Multiple dioxygenases may be an important feature of competitive decomposers in nature and hence may not be rare. Most of the isolates identified were members of the beta subgroup of the Proteobacteria, a few were gram positive, and none were true Pseudomonas species.
A virion concentration method for detection of human enteric viruses in oysters by PCR and oligoprobe hybridization
Authors: Jaykus L A , De Leon R , Sobsey M D
Published: January 2020

This article reports the development of a method to purify and concentrate intact virions from oyster extracts to volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase PCR (RT-PCR) and confirmation by oligonucleotide probe hybridization. Fifty-gram oyster samples were processed by an adsorption-elution -precipitation method and then seeded with 10(1) to 10(5) PFU of poliovirus type 1 (PV1) or hepatitis A virus (HAV). Seeded viruses in oyster extracts were purified by fluorocarbon extraction and concentrated by polyethylene glycol (PEG) precipitation and elution. Virus recovery after elution of PEG precipitates was dependent upon PEG concentration and averaged 60% for PV1 and 40% for HAV. The next processing step used the protein-precipitating agent Pro-Cipitate (Affinity Technology, Inc., Brunswick, N.J.) in an adsorption-elution -precipitation scheme to further concentrate viruses and reduce sample volumes to 100 microliter. Oyster extracts processed by Pro-Cipitate adsorption-elution-precipitation were directly compatible with RT-PCR and yielded virus recoveries of > 80% for both PV1 and HAV. When extracts from 50-g oyster samples were seeded and processed by the combined concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 10 PFU for both PV1 and HAV and with low levels of Norwalk virus. Virus recoveries based on cell culture infectivity were 25 to 35% for PV1 and 5 to 10% for HAV. When tested on artificially contaminated raw oysters, the combined method successfully detected > or = 10(3) PFU of PV1 and HAV and 10(5) RT-PCR-amplifiable units of Norwalk virus. Virus detection by RT-PCR and cell culture infectivity was consistent and well correlated among replicate samples and at different virus titers. The procedure developed in this study is rapid, sensitive, and effective for the direct detection of enteric viruses in oysters by RT-PCR.
Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR
Authors: Schwab K J , De Leon R , Sobsey M D
Published: January 2020

To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with IMDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, Pro-Cipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 mu l. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by AbCap method 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results of enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.
A Role for Fructose 1,6-Diphosphate in the ATPase-Mediated Energy-Spilling Reaction of Streptococcus bovis
Authors: Bond D R , Russell J B
Published: January 2020

The amount of ATP produced by Streptococcus bovis was larger than the amount that could be attributed to growth and maintenance, and even glucose-limited continuous cultures used ATP inefficiently (spilled ATP). Rapid-dilution-rate cultures always spilled more ATP than those growing at slow dilution rates, but rates of ATP spilling could also be enhanced by amino acid deprivation (with only ammonia as a nitrogen source). Energy spilling and intracellular ATP were not correlated, but energy spilling was always greatest when the rate of lactate production was high. The relationship between lactate production and energy spilling was supported by the observation that amino acid deprivation increased lactate production and ATP spilling. The lactate production rate of nongrowing (energy-spilling) S. bovis cells was fructose 1,6-diphosphate (FDP) dependent, and previous work showed that the lactate dehydrogenase of S. bovis was activated by FDP (M. J. Wolin, Science 146:775-777, 1964). The role of FDP in energy spilling was supported by the observation that the membrane-bound ATPase of S. bovis could be stimulated by FDP. FDP decreased the K(infm) for ATP by as much as fivefold. Other glycolytic intermediates could not stimulate the ATPase of washed membrane preparations, and FDP had no effect on soluble ATPase activity.
Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus
Authors: Yamagishi A , Tanimoto T , Suzuki T , Oshima T
Published: January 2020

We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus. A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced. The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile.
Coupling of Metabolism and Bioconversion: Microbial Esterification of Citronellol with Acetyl Coenzyme A Produced via Metabolism of Glucose in an Interface Bioreactor
Authors: Oda S , Inada Y , Kobayashi A , Kato A , Matsudomi N , Ohta H
Published: January 2020

Microbial esterification of citronellol with acetyl coenzyme A (acetyl-CoA) produced via metabolism of glucose was performed with Hansenula saturnus IFO 0809 in an interface bioreactor by using a hydrophilic carrier (an agar plate or an agar-coated filter pad) and a hydrophobic organic solvent (decane). An increase in the glucose concentration on an agar plate led to a decrease in oxidation of citronellol and an increase in the amount of citronellyl acetate. Fed-batch addition of citronellol efficiently alleviated substrate toxicity and resulted in the accumulation of high levels of citronellyl acetate. Furthermore, triple coupling of acetyl-CoA formation, microbial reduction of citronellal to citronellol, and esterification of the resulting citronellol with acetyl-CoA was also an efficient way to prepare citronellyl acetate. By using coupling systems, citronellyl acetate could be efficiently produced without any acetyl donor.
Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation
Authors: Top E M , Maltseva O V , Forney L J
Published: January 2020

The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway. The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF. Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB. To capture such plasmids from soil populations, we used a recipient strain of A. eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted. Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained. Among the transconjugants obtained were clones that contained a ca. 75-kb plasmid, pEMT8. Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not. Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4. Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp. strain RASC. Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF. Moreover, cell extracts from A. eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A. eutrophus(pEMT8)(pBH501aE) cultures did. These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE.
Bacteria Are Omnipresent on Phanerochaete chrysosporium Burdsall
Authors: Seigle-Murandi F , Guiraud P , Croize J , Falsen E , Eriksson K L
Published: January 2020

Bacteria have been isolated from 10 different strains of Phanerochaete chrysosporium, a white rot fungus which degrades lignocellulosic materials. The investigations showed that one or more bacterial species were always associated with the fungus. Various attempts to eliminate the bacteria on the fungus were unsuccessful. Three different bacterial species were isolated and identified. One of these was Agrobacterium radiobacter, while another may represent a new taxon close to the genus Burkholderia. A third strain remains unidentified but is most probably a member of rRNA superfamily IV or the Woese (alpha) group. Besides P. chrysosporium, 23 other white rot fungi and 9 brown rot fungi were also investigated. None of these was associated with bacteria. The physiological significance of the association between the fungus and the bacteria remains to be elucidated.
Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes
Authors: Kuhner C H , Hartman P A , Allison M J
Published: January 2020

The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.
Microbial transformation of 2,4,6-trinitrotoluene in aerobic soil columns
Authors: Bruns-Nagel D , Breitung J , von Low E , Steinbach K , Gorontzy T , Kahl M , Blotevogel K , Gemsa D
Published: January 2020

2,4,6-Trinitrotoluene (TNT)-contaminated soil material of a former TNT production plant was percolated aerobically in soil columns. Nineteen days of percolation with a potassium phosphate buffer supplemented with glucose or glucose plus ammonium sulfate caused an over 90% decline in the amount of extractable nitroaromatics in soils containing 70 to 2,100 mg of TNT per kg (dry weight). In the percolation solution, a complete elimination of TNT was achieved. Mutagenicity and soil toxicity were significantly reduced by the percolation process. 4-N-Acetylamino-2-amino-6-nitrotoluene was generated in soil and percolation fluid as a labile TNT metabolite.
Purification and characterization of a novel antibacterial protein from the marine bacterium D2
Authors: James S G , Holmström C , Kjelleberg S
Published: January 2020

A biofilm-forming marine bacterium, D2, isolated from the surface of the tunicate Ciona intestinalis, was found to produce a novel, 190-kDa protein with antibacterial activity. The protein contained at least two subunits of 60 and 80 kDa, joined together by noncovalent bonds, and was shown to be released by D2 cells into the surrounding medium during stationary phase. N-terminal sequence analysis revealed no close similarity of this protein to any other proteins within the Swiss Prot database. Bacteriocidal activity against a wide variety of marine and medical bacterial isolates was observed, 77% of the strains tested being sensitive to the protein. Bacterial strains varied in their resistance to the D2 protein, with D2 itself being among the most sensitive with an MBC in liquid suspension of 4 micrograms/ml. An apparent increased resistance of D2 to the protein as the cells progressed further into stationary phase was observed and seen as a possible explanation for its survival despite the production of an autoinhibitory factor. The ability of the D2 bacterium to produce an antibacterial factor in addition to its inhibitory effects on marine invertebrates and algae (S. Egan et al., unpublished data) indicates that D2 has the potential to greatly affect the survival of a range of colonizers of the marine surface environment.
Development of metal tolerance in soil bacterial communities exposed to experimentally increased metal levels
Authors: Diaz-Ravina M , Baath E
Published: January 2020

The development of metal tolerance in soil bacterial communities exposed to different heavy metals was examined under laboratory conditions. An agricultural soil amended with different Zn concentrations was studied most intensively, and measurements were made over a 28-month incubation period by means of the thymidine incorporation technique. Tolerance levels were not affected by metal concentrations lower than 2 mmol of Zn kg (dry weight) of soil(sup-1), but above this value, the level of Zn tolerance increased exponentially with the logarithm of the soil Zn concentration. An increased metal tolerance was detected after only 2 days of Zn exposure. Thereafter, stable tolerance values were observed at different sampling times for bacterial communities exposed to up to 8 mmol of Zn kg (dry weight)(sup-1), indicating no changes in tolerance with time. The tolerance of bacterial communities exposed to 32 mmol of Zn kg (dry weight)(sup-1) increased rapidly within the second week of incubation, but then the values remained unchanged until the end of the experiment. Bacterial communities from soil contaminated with 16 mmol of Zn kg (dry weight)(sup-1) showed an increase of the same magnitude, but the increase started later, after 4 months of incubation, and took place for a much longer period (more than 1 year). Cd, Cu, and Ni addition also resulted in metal-tolerant communities, and the level of tolerance increased with prolonged incubations of the soils. The bacterial community at the end of the incubation period also exhibited a lower pH optimum and an increased tolerance to low osmotic potential. The results suggest that the increase in metal tolerance of the community after adding metals can be attributed to an immediate effect due to the death of sensitive species and a later effect due to different competitive abilities and adaptation of surviving bacteria.
Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum
Authors: Hermann T , Kramer R
Published: January 2020

Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.
Atropine Metabolism by Pseudomonas sp. Strain AT3: Evidence for Nortropine as an Intermediate in Tropine Breakdown and Reactions Leading to Succinate
Authors: Bartholomew B A , Smith M J , Trudgill P W , Hopper D J
Published: January 2020

Pseudomonas strain AT3, isolated by elective culture with atropine, hydrolyzed atropine and grew diauxically, first on the tropic acid and then on the tropine. Tropine was also used as a sole carbon and energy source. The methyl group of tropine was eliminated as formaldehyde, and the nortropine thus formed was a precursor of 6-hydroxycyclohepta-1,4-dione. Ammonia was detected as a product of nitrogen elimination. 6-Hydroxycyclohepta-1,4-dione was oxidized to cyclohepta-1,3,5-trione by an induced NAD(sup+)-specific dehydrogenase. Although cyclohepta-1,3,5-trione is a (beta)-diketone with two potential hydrolytic cleavage sites, an induced hydrolase was specific for one of these sites, with 4,6-dioxoheptanoate as the only hydrolysis product. Unlike the alternative cleavage product (3,6-dioxoheptanoate), this compound is also a (beta)-diketone, and a second hydrolytic cleavage formed succinate and acetone. Although Pseudomonas strain AT3 was not capable of growth with acetone, the compound was not detected in the culture medium and may have been lost to the atmosphere. Exhaustive experimentation with a wide range of conditions did not result in detection of the enzymes required for cleavage of the carbon-nitrogen bonds leading to the formation of nortropine and 6-hydroxycyclohepta-1,4-dione.
Inactivation of Aeromonas hydrophila by Fe(II)-related-radical generation in oxidizing groundwaters
Authors: Kersters I , Verstraete W
Published: January 2020

The survival of Aeromonas hydrophila AWWX1 in filter-sterilized phreatic groundwaters was studied by using viable counts. Aeromonas counts rapidly decreased 2 to 3 log units in oxidizing raw groundwaters from Snellegem and Beernem, Belgium (Snellegem-raw and Beernem-raw, respectively), containing high concentrations of Fe2+ (460 to 1,070 microM). The rapid decline in viable counts of Aeromonas cells in the oxidizing raw groundwater of Snellegem was prevented by the addition of an Fe2+ chelator (2,2'-dipyridyl) or compounds (i.e., ascorbic acid and catalase) that act on toxic oxygen species. The results suggest that free radicals, generated spontaneously in oxidizing Fe2+-containing groundwaters, caused the inactivation of A. hydrophila AWWX1. Evidence that free radicals are generated under the given conditions was provided by the observation that propylphosphonic acid, a compound which is very susceptible to radicals, was degraded upon addition to these waters. A. hydrophila PWBS, Pseudomonas fluorescens P17, Spirillum strain NOX, and heterotrophs showed decreases in culturability in filter-sterilized Snellegem-raw water similar to that shown by A. hydrophila AWWX1. These findings indicate that free radicals generated in Fe2+-containing groundwaters upon aeration are capable of inactivating various bacterial species.
New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity
Authors: Fimland G , Blingsmo O R , Sletten K , Jung G , Nes I F , Nissen-Meyer J
Published: January 2020

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.
Elution of viruses by ionic and nonionic surfactants
Authors: Fujito B T , Lytle C D
Published: January 2020

The ionic and nonionic surfactants sodium dodecyl sulfate and Triton X-100, respectively, eluted two viruses, phi X174 and PRD1, which were adsorbed to the ionic and nonionic binding membranes cationic polysulfone and nitrocellulose, respectively. Results indicated that complete elution was readily achieved only when combinations of surfactants and binding membranes were matched (i.e., ionic-ionic or nonionic-nonionic).
Immunomagnetic-Electrochemiluminescent Detection of Bacillus anthracis Spores in Soil Matrices
Authors: Bruno J G , Yu H
Published: January 2020

Rapid (<=1.5-h) detection of anthrax spores in soil suspensions was accomplished by an immunomagnetic electrochemiluminescence method. Strain-dependent detection limits in the range of 10(sup2) to 10(sup5) spores were achieved in buffer. The rank order of sensitivity for the assay in buffer was Sterne > Ames > Vollum 1B. Detection was up to 3 orders of magnitude less sensitive in soil suspensions, and the rank order of sensitivity was altered.
Fungal biotransformation of the antihistamine azatadine by Cunninghamella elegans
Authors: Zhang D , Hansen E B , Deck J , Heinze T M , Sutherland J B , Cerniglia C E
Published: January 2020

The metabolism of the antihistamine azatadine by the zygomycete fungus Cunninghamella elegans ATCC 9245 was investigated. Within 72 h from the addition of the drug to 48-h-old cultures grown in Sabouraud dextrose broth, 95% of azatadine was biotransformed. Two major metabolites, 7-hydroxyazatadine (25%) and 8-hydroxyazatadine (50%), and two minor metabolites, N-desmethylazatadine and 9-hydroxyazatadine, were isolated by high-performance liquid chromatography and characterized by mass spectrometric and proton nuclear magnetic resonance spectroscopic analyses.
Methanogenic transformation of methylfurfural compounds to furfural
Authors: Boopathy R
Published: January 2020

The metabolic conversion of 5-methylfurfural and 2-methylfurfural to furfural by a methanogenic bacterium, Methanococcus sp. strain B, was studied. This bacterium was found to use methylfurfural compounds as a growth substrate and to convert them stoichiometrically to furfural. For every mole of methylfurfurals metabolized, almost 1 mol of furfural and 0.7 mol of methane were produced. Several methanogenic bacteria did not carry out this conversion. The metabolic conversion of methylfurfurals is likely to be of value in the anaerobic treatment of methylfurfural-containing wastewaters such as those produced by the paper and pulp industries and oatmeal processing industries. This study adds to the list of the limited number of compounds that are known to serve as electron donors for methanogenesis.