bluuebunny/crossref_metadata_2025 huggingface dataset:
Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat
Authors: Ferris M J , Ruff-Roberts A L , Kopczynski E D , Bateson M M , Ward D M
Published: January 2020

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.
Use of an ipb-lux Fusion To Study Regulation of the Isopropylbenzene Catabolism Operon of Pseudomonas putida RE204 and To Detect Hydrophobic Pollutants in the Environment
Authors: Selifonova O V , Eaton R W
Published: January 2020

A DNA segment involved in the regulation of the isopropylbenzene (cumene) catabolism operon (ipb) of plasmid pRE4 from Pseudomonas putida RE204 and the Vibrio fischeri luciferase genes, luxCDABE, were used to create an ipbRo/pA(prm1)-luxCDABE reporter fusion plasmid, pOS25. Escherichia coli HMS174(pOS25) produces light in the presence of inducers of the ipb operon. These inducers were shown to be hydrophobic compounds and to include monoalkylbenzenes, substituted benzenes and toluenes, some alkanes and cycloalkanes, chlorinated solvents, and naphthalenes. Complex hydrocarbon mixtures, such as gasoline, diesel fuel, jet fuels (JP-4 and JP-5), and creosote, were also inducers of ipb-lux. Bacteria carrying the ipb-lux reporter may be useful as bioindicators of hydrocarbon pollution in the environment and may be particularly valuable for examining the bioavailability of inducing pollutants.
Development and characterization of lactose-positive pediococcus species for milk fermentation
Authors: Caldwell S L , McMahon D J , Oberg C J , Broadbent J R
Published: January 2020

Bacteriophages against Streptococcus thermophilus are a growing problem in the Italian cheese industry. One possible control method involves replacing S. thermophilus in mozzarella starter blends with lactic acid bacteria from a different genus or species. In this study, we evaluated lactose-positive pediococci for this application. Because we could not identify any commercially available pediococci with fast acid-producing ability in milk, we transformed Pediococcus pentosaceus ATCC 25744, P. pentosaceus ATCC 25745, and Pediococcus acidilactici ATCC 12697 by electroporation with pPN-1, a 35-kb Lactococcus lactis lactose plasmid. Transformants of P. pentosaceus ATCC 25745 and P. acidilactici ATCC 12697 were then used to examine lactose-positive pediococci for properties related to milk fermentation. Both transformants rapidly produced acid and efficiently retained pPN-1 in lactose broth, and neither bacterium was attacked by bacteriophages in whey collected from commercial cheese facilities. Paired starter combinations of Pediococcus spp. and Lactobacillus helveticus LH100 exhibited synergistic pH reduction in milk, and small-scale cheese trials showed that these cultures could be used to manufacture part-skim mozzarella cheese. Results demonstrate that lactose-positive pediococci have potential as replacement cocci for S. thermophilus in Italian cheese starter blends and may facilitate development of new strain rotation schemes to combat S. thermophilus bacteriophage problems in mozzarella cheese plants.
Prevention of histamine formation in cheese by bacteriocin-producing lactic Acid bacteria
Authors: Joosten H , Nunez M
Published: January 2020

The susceptibility of 13 amine-forming lactobacilli to several bacteriocins was investigated by an agar diffusion assay. All strains were susceptible to nisin and to five bacteriocins of enterococcal origin. Pediocin PA-1, bavaricin A, lactococcin A, and a bacteriocin from Enterococcus faecalis 1061 did not show inhibitory activity. Two bacteriocin-producing enterococci and a nisin-producing Lactococcus lactis strain were employed as starters in separate cheese-making experiments. Outgrowth of histamine producer Lactobacillus buchneri St2A, which was added to the milk at levels of up to 190 CFU/ml, was almost completely inhibited. No histamine formation was detected in the cheeses made with bacteriocin-producing starters. In the control cheese without bacteriocin, St2A reached levels of 1.1 x 10(sup8) CFU/g, and 200 mg of histamine per kg was found after 4 months of ripening. To our knowledge, this is the first report of bacteriocin-mediated inhibition of histamine formation in foods.
A microsensor for nitrate based on immobilized denitrifying bacteria
Authors: Larsen L H , Revsbech N P , Binnerup S J
Published: January 2020

A biosensor for NO(inf3)(sup-) was constructed by attaching a 30- to 70-(mu)m-wide capillary with immobilized denitrifying bacteria in front of an N(inf2)O microsensor. These bacteria reduced O(inf2) so that only bacteria in the very tip of the sensor were exposed to O(inf2) whereas bacteria at a greater depth could carry out the anaerobic process of denitrification. In the presence of acetylene, which inhibits nitrous oxide reductase, bacteria reduced NO(inf3)(sup-) (or NO(inf2)(sup-)) from the surrounding medium to N(inf2)O and the concentration sensed by the N(inf2)O microsensor was directly proportional to the concentration of NO(inf3)(sup-) in the medium. By applying a 250-(mu)m-long capillary in front of the N(inf2)O microsensor, the 90% response time of the biosensor was 50 s. Biosensors may also be made with nitrous oxide-deficient strains so that acetylene inhibition can be omitted.
Characterization of a Novel Strain of Bacillus thuringiensis
Authors: Lopez-Meza J E , Ibarra J E
Published: January 2020

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.
Effects of low temperatures on viability of Cryptosporidium parvum oocysts
Authors: Fayer R , Nerad T
Published: January 2020

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.
Characterization of Listeria monocytogenes isolates by esterase electrophoresis
Authors: Harvey J , Gilmour A
Published: January 2020

Multiple bands of alpha-naphthyl-propionyl esterase (alpha NPE) activity observed following starch gel electrophoresis of cell extracts allowed 219 Listeria monocytogenes isolates from milk, nondairy foods, and clinical and veterinary sources to be assigned to 17 different alpha NPE types. Multilocus enzyme electrophoresis (MEE) analysis was used to obtain electrophoretic mobility data for alpha NPEs and nine other metabolic enzymes; on the basis of these data, the 219 strains were assigned to 59 electrophoretic types. Each of the methods separated the strains into two main groups, and there was extensive agreement on assignment of the strains to the groups. Although the method is less discriminatory than MEE, the usefulness of alpha NPE electrophoresis alone as a rapid, simple typing method for L. monocytogenes for certain purposes is indicated by agreement among alpha NPE, MEE, and restriction fragment length polymorphism types as molecular markers for persistent and transient L. monocytogenes strains found in sequential raw-milk and nondairy food samples obtained from different processors.
Straw compost and bioremediated soil as inocula for the bioremediation of chlorophenol-contaminated soil
Authors: Laine M M , Jorgensen K S
Published: January 2020

We evaluated the use of straw compost and remediated soil as inocula for bioremediation of chlorophenol-contaminated soil. The in situ biotransformation of pentachlorophenol (PCP) and mineralization of radiolabeled [U-(sup14)C]PCP by straw compost and remediated soil were studied under field-simulating conditions before and after 3 months of adaptation with PCP in a percolator. After PCP adaptation, the straw compost mineralized up to 56% of the [(sup14)C]PCP. No partial dechlorination of PCP was found. The native straw compost did not mineralize PCP, but partial dechlorination of PCP occurred (i) at pH 8 under near-thermophilic conditions (45(deg)C) and (ii) at pH 7 under aerobic and mesophilic conditions. No biotransformation reactions occurred at room temperature (25(deg)C) at pH 8. Enrichment in the percolator enhanced the mineralization rate of remediated soil to 56% compared with that of the native remediated soil, which mineralized 24% of [(sup14)C]PCP added. Trace amounts of chloroanisoles as the only biotransformation products were detected in PCP-adapted remediated soil. Both inoculants studied here showed effective mineralization of PCP when they were adapted to PCP in the percolator. No harmful side reactions, such as extensive methylation, were observed.
Photic Volume in Photobioreactors Supporting Ultrahigh Population Densities of the Photoautotroph Spirulina platensis
Authors: Gitelson A , Qiuang H , Richmond A
Published: January 2020

Characterization of the photic zone and light penetration depth in cultures with ultrahigh cell densities represents a major issue in mass cultures of phytoautotrophic microorganisms grown in enclosed photobioreactors. In a study of the effect of underwater optical properties on the penetration depth of photosynthetically active radiation, the inherent optical properties of algal suspensions, i.e., absorption and scattering coefficients, as well as their apparent optical properties, i.e., the reflectance and the vertical attenuation coefficient of downwelling irradiance, were determined by using high-spectral-resolution radiometric measurements. The vertical attenuation coefficient was used to estimate quantitatively the depth of light penetration into a reactor containing an ultrahigh cell density (chlorophyll concentration, up to 300,000 mg m(sup-3)). For such a high cell density, the photic volume in the reactor was found to be extremely small; nevertheless, it differed between the blue and red light (less than 0.06 mm) and the green light (about 0.5 mm). This suggests a singular role for green light under the unique circumstances existing in ultrahigh-cell-density cultures of photoautotrophs.
Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici
Authors: Lairini K , Perez-Espinosa A , Pineda M , Ruiz-Rubio M
Published: January 2020

The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.
The genes for secretion and maturation of lactococcins are located on the chromosome of Lactococcus lactis IL1403
Authors: Venema K , Dost M H , Beun P A , Haandrikman A J , Venema G , Kok J
Published: January 2020

Southern hybridization and PCR analysis were used to show that Lactococcus lactis IL1403, a plasmid-free strain that does not produce bacteriocin, contains genes on its chromosome that are highly homologous to lcnC and lcnD and encode the lactococcin secretion and maturation system. The lcnC and lcnD homologs on the chromosome of IL1403 were interrupted independently by Campbell-type integrations. Both insertion mutants were unable to secrete active lactococcin. Part of the chromosomal lcnC gene was cloned and sequenced. Only a few nucleotide substitutions occurred, compared with the plasmid-encoded lcnC gene, and these did not lead to changes in the deduced amino acid sequence. No genes homologous to those for lactococcin A, B, or M could be detected in IL1403, and the strain does not produce bacteriocin activity.
Fermentation and Sulfur Reduction in the Mat-Building Cyanobacterium Microcoleus chthonoplastes
Authors: Moezelaar R , Bijvank S M , Stal L J
Published: January 2020

The mat-building cyanobacterium Microcoleus chthonoplastes carried out a mixed-acid fermentation when incubated under anoxic conditions in the dark. Endogenous storage carbohydrate was fermented to acetate, ethanol, formate, lactate, H(inf2), and CO(inf2). Cells with a low glycogen content (about 0.3 (mu)mol of glucose per mg of protein) produced acetate and ethanol in equimolar amounts. In addition to glycogen, part of the osmoprotectant, glucosyl-glycerol, was degraded. The glucose component of glucosyl-glycerol was fermented, whereas glycerol was released into the medium. Cells with a high content of glycogen (about 2 (mu)mol of glucose per mg of protein) did not utilize glucosyl-glycerol. These cells produced more acetate than ethanol. M. chthonoplastes was also capable of using elemental sulfur as the electron acceptor during fermentation, resulting in the production of sulfide. With sulfur present, acetate production increased whereas ethanol production decreased. Also, less formate was produced and the evolution of hydrogen ceased completely. In general, the carbon recoveries were satisfactory but the oxidation-reduction balances were too high. The latter could be explained by assuming the reduction of ferric iron, which is associated with the cells, mediated by the oxidation of formate. The switch from photoautotrophic to fermentative metabolism did not require de novo protein synthesis, and fermentation started immediately upon transfer to dark anoxic conditions. From the molar ratios of the fermentation products and from measurement of enzyme activities in cell extracts, we concluded that glucose derived from glycogen and glucosyl-glycerol is degraded via the Embden-Meyerhof-Parnas pathway.
Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli
Authors: Millard C S , Chao Y P , Liao J C , Donnelly M I
Published: January 2020

Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight.
Transformation of Azo Dye Isomers by Streptomyces chromofuscus A11
Authors: Pasti-Grigsby M B , Burke N S , Goszczynski S , Crawford D L
Published: January 2020

Fourteen mono-azo dyes were used to study the effects of substitution patterns on the biodegradability of dimethyl-hydroxy-azobenzene 4(prm1)-sulfonic acids by Streptomyces chromofuscus A11. Two substitution patterns were analyzed: (i) all possible substitution patterns of the two methyl and hydroxy substitution groups, 2-hydroxy (3,5; 4,5; 5,6) dimethyl and 4-hydroxy (2,3; 2,5; 2,6; 3,5) dimethyl isomers of azobenzene 4(prm1)-sulfonic acid; and (ii) replacement of the sulfonic group with a carboxylic group in these sulfonated azo dyes. The structural pattern of the hydroxy group in para position relative to the azo linkage and of two methyl substitution groups in ortho position relative to the hydroxy group was the most susceptible to degradation. Replacement of the sulfonic group with a carboxylic group enhanced overall dye degradability by S. chromofuscus A11.
Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential
Authors: Rodríguez M , Núñez F , Córdoba J J , Bermúdez E , Asensio M A
Published: January 2020

Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.
Detection of Novel Proline 3-Hydroxylase Activities in Streptomyces and Bacillus spp. by Regio- and Stereospecific Hydroxylation of l-Proline
Authors: Mori H , Shibasaki T , Uozaki Y , Ochiai K , Ozaki A
Published: January 2020

During the screening of microbial proline hydroxylases, novel proline 3-hydroxylase activities, which hydroxylate free l-proline to free cis-3-hydroxy-l-proline, were detected in whole cells of Streptomyces sp. strain TH1 and Bacillus sp. strains TH2 and TH3 from 3,000 strains isolated from soil. The reaction product was purified from a reaction mixture of Streptomyces sp. strain TH1, and its chemical structure was identified as cis-3-hydroxy-l-proline by instrumental analyses. Proline 3-hydroxylase activity was also detected in Streptomyces canus ATCC 12647 which produces the 3-hydroxyproline-containing peptide antibiotic telomycin. Bacillus sp. strains TH2 and TH3 were found to accumulate cis-3-hydroxy-l-proline in culture media at 426 and 352 (mu)M, respectively. It was suggested that hydroxylation occurred in a highly regio- and stereospecific manner at position 3 of l-proline because no hydroxylation product other than cis-3-hydroxy-l-proline was observed. Proline 3-hydroxylases of these strains were first characterized on crude enzyme preparations. Since 2-oxoglutarate and ferrous ion were required for hydroxylation of l-proline, these 3-hydroxylases were thought to belong to a family of 2-oxoglutarate-related dioxygenases. The reaction was inhibited by Co(sup2+), Zn(sup2+), and Cu(sup2+). l-Ascorbic acid accelerated the reaction. The optimum pH and temperature were 7.5 and 35(deg)C, respectively.
Effects of viruses on nutrient turnover and growth efficiency of noninfected marine bacterioplankton
Authors: Middelboe M , Jorgensen N , Kroer N
Published: January 2020

The effects of virus infection and lysis of a marine Vibrio sp. on C, N, and P turnover and the growth efficiency of noninfected bacterioplankton were studied in a series of dilution cultures. The cultures were enriched with various sources of organic matter and N and P. The growth of the Vibrio host and the growth of the natural bacterioplankton were measured by immunofluorescence and 4(prm1),6-diamidino-2-phenylindole staining methods, respectively. Lysis products resulting from infection of the Vibrio sp. caused an increase in metabolic activity and cell production by the noninfected bacterioplankton. In P-limited cultures, the addition of viruses increased the uptake of dissolved organic carbon by 72% and the potential alkaline phosphatase activity by 89% compared with control cultures without viruses. Our data suggest that input of available phosphorus through virus-induced Vibrio lysates occurred, which caused an increase in the bacterial nutrient uptake. The growth efficiency of noninfected bacteria was reduced in the presence of viruses compared with the control without viruses (growth efficiencies, 0.08 (plusmn) 0.03 and 0.24 (plusmn) 0.02, respectively). We suggest that the decrease in growth efficiency may be explained by an increase in bacterial energy demand associated with extracellular degradation of polymeric organic nitrogen and phosphorus in cell lysates.
The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum
Authors: Abu Kwaik Y
Published: January 2020

Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.
Development of fungal inocula for bioaugmentation of contaminated soils
Authors: Lestan D , Lamar R T
Published: January 2020

This report describes novel fungal inocula for bioaugmentation of soils contaminated with hazardous organic compounds. The inocula are in the form of pelleted solid substrates coated with a sodium alginate suspension of fungal spores or mycelial fragments and incubated until overgrown with the mycelium of selected lignin-degrading fungi. The organisms evaluated were Phanerochaete chrysosporium (BKM F-1767, ATCC 42725), P. sordida (HHB-8922-Sp), Irpex lacteus (Mad-517, ATCC 11245), Bjerkandera adusta (FP-135160-Sp, ATCC 62023), and Trametes versicolor (MD-277). The pelleted fungal inocula resisted competition and proliferation from indigenous soil microbes, were lower in moisture content than current fungal inocula, and had sufficient mechanical strength to allow handling and introduction into the soil without a change in the mechanical consistency of the pellets. Inoculated at a rate of 3% in artificially contaminated nonsterile soil, I. lacteus, B. adusta, and T. versicolor removed 86, 82, and 90%, respectively, of the pentachlorophenol in 4 weeks. A mathematical model was developed to explain moisture distribution in a hydrogel-coated pelleted substrate.
A marine oligobacterium harboring genes known to be part of aromatic hydrocarbon degradation pathways of soil pseudomonads
Authors: Wang Y , Lau P C , Button D K
Published: January 2020

The far-ranging distribution of genes for aromatic hydrocarbon catabolism, predominantly studied in soil pseudomonads, is extended to a marine oligobacterium by finding five homologous sequences in a 5.7-kb chromosomal DNA from a new isolate, Cycloclasticus oligotrophus RB1. RB1 is capable of growth in unamended seawater or mineral salts media supplemented with a variety of aromatic compounds, including toluene, o-, m-, or p-xylenes, as sole carbon sources. The five open reading frames, designated xylM, K, G, C1, and C2, are 57% A+T-rich. XylM is predicted to be an integral membrane protein; XylK and XylG possess glutathione S-transferase (GST) and 2-hydroxy-5methyl-6-oxohexa2,4-dienoate dehydrogenase activities, respectively; XylC1C2 are homologs of the large and small subunits of the iron sulfur protein component of the biphenyl dioxygenase (e.g., BphA1A2).
Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes
Authors: Pérez-Gonzalez J A , De Graaff L H , Visser J , Ramón D
Published: January 2020

Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains.
Inhibition of Growth of a Graphium sp. on Gaseous n-Alkanes by Gaseous n-Alkynes and n-Alkenes
Authors: Curry S , Ciuffetti L , Hyman M
Published: January 2020

The growth of a filamentous fungus, a Graphium sp., on n-alkanes (C(inf2) to C(inf4)) was inhibited by low concentrations of acetylene, propyne, 1-butyne, ethylene, and propylene. Acetylene and other unsaturated hydrocarbons had no effect on the growth of the Graphium sp. on potato dextrose broth, ethanol, or acetate. Our results suggest that n-alkynes and n-alkenes are selective inhibitors of a nonspecific monooxygenase enzyme responsible for the initial oxidation of n-alkanes.
Solvent-Augmented Mineralization of Pyrene by a Mycobacterium sp
Authors: Jimenez I Y , Bartha R
Published: January 2020

The biodegradation of polycyclic aromatic hydrocarbon pollutants is constrained, in part, by their solid physical state and very low water solubility. Searching for ways to overcome these limitations, we isolated from soil a bacterium capable of growing on pyrene as a sole source of carbon and energy. Acid-fast stain, morphology, and fatty acid profile identified it as a Mycobacterium sp. In a mineral salts solution, the isolate mineralized 50% of a 250-(mu)g/ml concentration of [(sup14)C]pyrene in 2 to 3 days. Detergent below the critical micelle concentration increased the pyrene mineralization rate to 154%, but above the critical micelle concentration, the detergent severely inhibited pyrene mineralization. The water-miscible solvent polyethylene glycol was inhibitory. The hydrophobic solvents heptamethylnonane, decalin, phenyldecane, and diphenylmethane were also inhibitory at several concentrations tested, but the addition of paraffin oil, squalene, squalane, tridecylcyclohexane, and cis-9-tricosene at 0.8% (vol/vol) doubled pyrene mineralization rates by the Mycobacterium sp. without being utilized themselves. The Mycobacterium sp. was found to have high cell surface hydrophobicity and adhered to the emulsified solvent droplets that also contained the dissolved pyrene, facilitating its mass transfer to the degrading bacteria. Cells physically adhering to solvent droplets metabolized pyrene 8.5 times as fast as cells suspended in the aqueous medium. An enhanced mass transfer of polycyclic aromatic hydrocarbon compounds to microorganisms by suitable hydrophobic solvents might allow the development of solvent-augmented biodegradation techniques for use in aqueous or slurry-type bioreactors.
The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection
Authors: Steidler L , Yu W , Fiers W , Remaut E
Published: January 2020

We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.
Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions
Authors: Otte S , Grobben N G , Robertson L A , Jetten M S , Kuenen J G
Published: January 2020

Nitrous oxide can be a harmful by-product in nitrogen removal from wastewater. Since wastewater treatment systems operate under different aeration regimens, the influence of different oxygen concentrations and oxygen fluctuations on denitrification was studied. Continuous cultures of Alcaligenes faecalis TUD produced N2O under anaerobic as well as aerobic conditions. Below a dissolved oxygen concentration of 5% air saturation, the relatively highest N2O production was observed. Under these conditions, significant activities of nitrite reductase could be measured. After transition from aerobic to anaerobic conditions, there was insufficient nitrite reductase present to sustain growth and the culture began to wash out. After 20 h, nitrite reductase became detectable and the culture started to recover. Nitrous oxide reductase became measurable only after 27 h, suggesting sequential induction of the denitrification reductases, causing the transient accumulation of N2O. After transition from anaerobic conditions to aerobic conditions, nitrite reduction continued (at a lower rate) for several hours. N2O reduction appeared to stop immediately after the switch, indicating inhibition of nitrous oxide reductase, resulting in high N2O emissions (maximum, 1.4 mmol liter-1 h-1). The nitrite reductase was not inactivated by oxygen, but its synthesis was repressed. A half-life of 16 to 22 h for nitrite reductase under these conditions was calculated. In a dynamic aerobic-anaerobic culture of A. faecalis, a semisteady state in which most of the N2O production took place after the transition from anaerobic to aerobic conditions was obtained. The nitrite consumption rate in this culture was equal to that in an anaerobic culture (0.95 and 0.92 mmol liter-1 h-1, respectively), but the production of N2O was higher in the dynamic culture (28 and 26% of nitrite consumption, respectively).
The composition of fluorescent pseudomonad populations associated with roots is influenced by plant and soil type
Authors: Latour X , Corberand T , Laguerre G , Allard F , Lemanceau P
Published: January 2020

Populations of fluorescent pseudomonads isolated from an uncultivated soil and from the roots of two plant species were previously shown to differ (P. Lemanceau, T. Corberand, L. Gardan, X. Latour, G. Laguerre, J.-M. Boeufgras, and C. Alabouvette, Appl. Environ. Microbiol. 61:1004-1012, 1995). The diversities of fluorescent pseudomonads, from two uncultivated soils and from the roots of two plant species cultivated in these two soils, were compared. The phenotypic diversity of the bacterial isolates was characterized on the basis of biochemical and physiological tests and on the basis of their ability to utilize 147 different organic compounds. The genotypic diversity of the isolates was characterized on the basis of the types of 16S genes coding for rRNA (rDNA), their repetitive extragenic palindromic patterns by PCR, and plasmid profiles. Taxonomic identification of the isolates was achieved with both biochemical and physiological tests and by comparing their 16S rDNA types to those of reference and type strains of fluorescent Pseudomonas spp. Numerical analysis of phenotypic characteristics allowed the clustering of isolates that showed high levels of similarity. This analysis indicated that both soil type and host plant had an effect on the diversity of fluorescent pseudomonads. However, of the two factors studied, the soil was clearly the dominating one. Indeed, the populations associated with the roots of each plant species varied from one soil to the other. This variation could possibly be ascribed to the differences recorded between the phenotypically diverse populations of fluorescent pseudomonads from the two uncultivated soils. The plant selection was, at least partly, plant specific. It was not related to bacterial species and biovars or to the presence of plasmid DNA. The phenotypic clustering of isolates was well correlated with genotypic characterization by repetitive extragenic palindrome-PCR fingerprinting.
Effect of Phosphorus on the Synechococcus Cell Cycle in Surface Mediterranean Waters during Summer
Authors: Vaulot D , Lebot N , Marie D , Fukai E
Published: January 2020

The effect of phosphorus (P) and nitrogen (N) additions on the Synechococcus cell cycle was tested with natural populations from the Mediterranean Sea in summer. In the absence of stimulation, the Synechococcus cell cycle was synchronized to the light-dark cycle. DNA synthesis began around 1600, a maximum of S-phase cells was observed at around dusk (2100), and a maximum of G(inf2)-phase cells was observed at around 2400. Addition of P (as PO(inf4)(sup3-)) caused, in all cases, a decrease in the fraction of cells in G(inf2) at around 1800, no change at around 2400, and an increase at around 1200 the next day, while addition of N (as NO(inf3)(sup-)) had no effect. We hypothesize that P addition induced a shortening of the G(inf1) phase, resulting in cells entering and leaving the S and G(inf2) phases earlier. These data suggest very strongly that the Synechococcus cells were P limited rather than N limited during this period of the year. In most cases, additions as low as 20 nM P induced a cell cycle response. From dose-response curves, we established that the P concentration inducing a 50% change in the percentage of cells in G(inf2) was low, close to 10 nM, at the beginning of the sampling period (30 June) and increased to about 50 nM by the end (9 July), suggesting a decrease in the severity of P limitation. This study extends recent observations that oligotrophic systems may be P rather than N limited at certain times of the year.
Metabolism of phenanthrene by the white rot fungus Pleurotus ostreatus
Authors: Bezalel L , Hadar Y , Fu P P , Freeman J P , Cerniglia C E
Published: January 2020

The white rot fungus Pleurotus ostreatus, grown for 11 days in basidiomycetes rich medium containing [14C] phenanthrene, metabolized 94% of the phenanthrene added. Of the total radioactivity, 3% was oxidized to CO2. Approximately 52% of phenanthrene was metabolized to trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) (28%), 2,2'-diphenic acid (17%), and unidentified metabolites (7%). Nonextractable metabolites accounted for 35% of the total radioactivity. The metabolites were extracted with ethyl acetate, separated by reversed-phase high-performance liquid chromatography, and characterized by 1H nuclear magnetic resonance, mass spectrometry, and UV spectroscopy analyses. 18O2-labeling experiments indicated that one atom of oxygen was incorporated into the phenanthrene trans-9,10-dihydrodiol. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was 9R,10R, which is different from that of the principal enantiomer produced by Phanerochaete chrysosporium. Significantly less phenanthrene trans-9,10-dihydrodiol was observed in incubations with the cytochrome P-450 inhibitor SKF 525-A (77% decrease), 1-aminobenzotriazole (83% decrease), or fluoxetine (63% decrease). These experiments with cytochrome P-450 inhibitors and 18O2 labeling and the formation of phenanthrene trans-9R,10R-dihydrodiol as the predominant metabolite suggest that P. ostreatus initially oxidizes phenanthrene stereoselectively by a cytochrome P-450 monoxygenase and that this is followed by epoxide hydrolase-catalyzed hydration reactions.
Stereospecific Biohydroxylations of Protected Carboxylic Acids with Cunninghamella blakesleeana
Authors: Kreiner M , Braunegg G , de Raadt A , Griengl H , Kopper I , Petsch M , Plachota P , Schoo N , Weber H , Zeiser A
Published: January 2020

Cunninghamella blakesleeana DSM 1906 was found to be an efficient biocatalyst for the biotransformation of cycloalkylcarboxylic acids into hydroxy and oxo derivatives. When cultivated in submerged culture, the fungus grew in pellets. In comparison with malt extract-glucose-peptone-yeast extract medium (medium E), Czapek-Dox medium was found to reduce pellet size. Cycloalkylcarboxylic acids were protected against microbial degradation by chemical transformation into 2-cycloalkyl-1,3-benzoxazoles. The transformations of protected cyclopentyl-, cyclohexyl-, cycloheptyl-, and cyclooctylcarboxylic acids by C. blakesleeana were investigated. The biotransformations were performed in medium E by using an aerated, stirred-tank bioreactor. The transformation of 2-cyclopentyl-1,3-benzoxazole yielded (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol as the main product. The main by-product was (1R)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-one, and 2-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol was also obtained in small amounts. During the experiment, the enantiomeric excess of the main product increased up to 64%. 2-Cyclohexyl-1,3-benzoxazole was hydroxylated to 4-(benz-1,3-oxazol-2-yl)cyclohexan-1-ol. 2-Cycloheptyl-1,3-benzoxazole and 2-cyclooctyl-1,3-benzoxazole were transformed into several alcohols and ketones, all in low yields (2 to 19%).
Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor
Authors: van Rijn J , Tal Y , Barak Y
Published: January 2020

Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate.
Isolation and characterization of a copper-resistant methanogen from a copper-mining soil sample
Authors: Kim B K , Conway de Macario E , Nölling J , Daniels L
Published: January 2020

A copper-resistant methanogen for which the CuSO4 MICs were approximately 2- to 36-fold higher than those for other methanogens tested was isolated from a copper-mining area in the upper peninsula of Michigan. The rod-shaped methanogen used H2-CO2 or formate, but not acetate or methanol, as a growth substrate. Standing incubation with H2-CO2 medium resulted in a mat-like surface growth, dependent on the presence of hydrogen. The presence of 1 mM cupric salt resulted in longer filamentous and intertwined cells. Antigenic fingerprinting, 16S rRNA gene analysis, morphology, and substrate use suggest that the new isolate is a novel strain of Methanobacterium bryantii that is able to use formate.
Effects of atrazine on Ochrobactrum anthropi membrane fatty acids
Authors: Laura D , De Socio G , Frassanito R , Rotilio D
Published: January 2020

Ochrobactrum anthropi is a gram-negative bacillus recognized as a human opportunist pathogen isolated in clinical specimens and not of clinical significance. We report a new aspect of this bacterium, that it has been isolated from activated sludge. In fact, it is able to grow on atrazine (2-chloro-4-ethylamino-6-isopropyl-amine-s-triazine) by utilizing it as the only source of carbon. Our results show that atrazine (0.03 g/liter) causes a dramatical increase in the degree of saturation of membrane fatty acids. Analysis and identification of bacterial fatty acids were performed by gas chromatography and gas chromatography-mass spectrometry techniques.
Lip-like genes in Phanerochaete sordida and Ceriporiopsis subvermispora, white rot fungi with no detectable lignin peroxidase activity
Authors: Rajakumar S , Gaskell J , Cullen D , Lobos S , Karahanian E , Vicuna R
Published: January 2020

Lignin peroxidase-like genes were PCR amplified from Phanerochaete sordida and Ceriporiopsis subvermispora, fungi lacking lignin peroxidase (LiP) activity. Amplification products were highly similar to previously described LiP genes. Using reverse transcription-coupled PCR a LiP-like cDNA clone was amplified from P. sordida RNA. In contrast, no evidence was obtained for transcription of C. subvermispora LiP genes.