bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes
Authors: Salama M S , Sandine W E , Giovannoni S J
Published: January 2020

Lactococcus lactis subsp. cremoris is widely used in the manufacture of fermented milk products. Despite numerous attempts, efforts to isolate new strains by traditional plating and identification methods have not been successful. Previously, we described oligonucleotide probes for 16S rRNAs which could be used to discriminate L. lactis subsp. cremoris from related strains. These probes were used in colony hybridization experiments to screen large numbers of colonies obtained from enrichment cultures. A total of 170 strains of L. lactis were isolated from six milk samples, two colostrum samples, and one corn sample by using oligonucleotide probe 212RLa specific for the species L. lactis. Fifty-nine of these isolates also hybridized to L. lactis subsp. cremoris-specific probe 68RCa, and 26 of the strains which hybridized to the L. lactis subsp. cremoris-specific probe had the L. lactis subsp. cremoris phenotype.
Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi
Authors: Orth A B , Royse D J , Tien M
Published: January 2020

Phanerochaete chrysosporium is rapidly becoming a model system for the study of lignin biodegradation. Numerous studies on the physiology, biochemistry, chemistry, and genetics of this system have been performed. However, P. chrysosporium is not the only fungus to have a lignin-degrading enzyme system. Many other ligninolytic species of fungi, as well as other distantly related organisms which are known to produce lignin peroxidases, are described in this paper. In this study, we demonstrated the presence of the peroxidative enzymes in nine species not previously investigated. The fungi studied produced significant manganese peroxidase activity when they were grown on an oak sawdust substrate supplemented with wheat bran, millet, and sucrose. Many of the fungi also exhibited laccase and/or glyoxal oxidase activity. Inhibitors present in the medium prevented measurement of lignin peroxidase activity. However, Western blots (immunoblots) revealed that several of the fungi produced lignin peroxidase proteins. We concluded from this work that lignin-degrading peroxidases are present in nearly all ligninolytic fungi, but may be expressed differentially in different species. Substantial variability exists in the levels and types of ligninolytic enzymes produced by different white not fungi.
Growth of
Azotobacter vinelandii
UWD in Fish Peptone Medium and Simplified Extraction of Poly-β-Hydroxybutyrate
Authors: Page William J. , Cornish Anthony
Published: January 2020

Azotobacter vinelandii
UWD was grown in a fermentor with glucose medium with and without 0.1% fish peptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-β-hydroxybutyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH
4
+
, but PHB synthesis stopped when NH
4
+
was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH
4
+
depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells that demonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present in fed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from 0.43 g/g, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growth phase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphic cells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH
3
(pH 11.4) at 45°C for 10 min. This treatment removed about 10% of the non-PHB mass from the pellet, of which 60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residual mass. The polymer molecular weight (1.7 × 10
6
to 2.0 × 10
6
) and dispersity (1.0 to 1.9) were not significantly affected (
P
= 0.05) by this treatment. In addition, the NH
3
extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bioplastics is discussed.
Analysis of Fungal Pellets by UV-Visible Spectrum Diffuse Reflectance Spectroscopy
Authors: Leštan Domen , Podgornik Helena , Perdih Anton
Published: January 2020

The application of the UV-visible spectrum diffuse reflectance spectroscopy for the determination of intracellular pH in vivo, for determination of cytochrome content, and for the noninvasive in vivo detection of the redox state of fungal mitochondrial cytochromes in filamentous fungi is introduced. The time course of the intracellular pH values, mitochondrial cytochromes, and CO-binding pigments content and the correlations between the actual redox state of cytochrome
aa
3
and saturation of growth medium with oxygen in pellets of the basidiomycete
Phanerochaete chrysosporium
were determined. As the test microorganism, the yeast
Saccharomyces cerevisiae
was used. UV-visible spectrum diffuse reflectance spectroscopy proved to be a promising method for the quick and simple analysis of light-impermeable biological structures for which the classical transmittance spectrophotometric methods are difficult to implement.
Purification and Characterization of an
l
-Aminopeptidase from
Pseudomonas putida
ATCC 12633
Authors: Hermes H. F. M. , Sonke T. , Peters P. J. H. , van Balken J. A. M. , Kamphuis J. , Dijkhuizen L. , Meijer E. M.
Published: January 2020

An
l
-aminopeptidase of
Pseudomonas putida
, used in an industrial process for the hydrolysis of
d,l
-amino acid amide racemates, was purified to homogeneity. The highly
l
-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and α-H amino acid amides, e.g.,
l
-phenylglycine amide.
Roles of N-acetylglutaminylglutamine amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic stress
Authors: D'Souza-Ault M R , Smith L T , Smith G M
Published: January 2020

The mechanism of osmotic stress adaptation in Pseudomonas aeruginosa PAO1 was investigated. By using natural abundance 13C nuclear magnetic resonance spectroscopy, osmotically stressed cultures were found to accumulate glutamate, trehalose, and N-acetylglutaminylglutamine amide, an unusual dipeptide previously reported only in osmotically stressed Rhizobium meliloti and Pseudomonas fluorescens. The intracellular levels of these osmolytes were dependent on the chemical composition and the osmolality of the growth medium. It was also demonstrated that glycine betaine, a powerful osmotic stress protectant, participates in osmoregulation in this organism. When glycine betaine or its precursors, phosphorylcholine or choline, were added to the growth medium, growth rates of cultures in 0.7 M NaCl were increased more than threefold. Furthermore, enhancement of growth could be observed with as little as 10 microM glycine betaine or precursor added to the medium. Finally, the mechanism of osmotic stress adaptation of two clinical isolates of P. aeruginosa was found to be nearly identical to that of the laboratory strain PAO1 in all aspects studied.
Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens
Authors: Pons J L , Picard B , Niel P , Leluan G , Goullet P
Published: January 2020

Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.
Degradation of 2-chloroallylalcohol by a Pseudomonas sp
Authors: van der Waarde J J , Kok R , Janssen D B
Published: January 2020

Three Pseudomonas strains capable of utilizing 2-chloroallylalcohol (2-chloropropenol) as the sole carbon source for growth were isolated from soil. The fastest growth was observed with strain JD2, with a generation time of 3.6 h. Degradation of 2-chloroallylalcohol was accompanied by complete dehalogenation. Chloroallylalcohols that did not support growth were dechlorinated by resting cells; the dechlorination level was highest if an alpha-chlorine substituent was present. Crude extracts of strain JD2 contained inducible alcohol dehydrogenase activity that oxidized mono- and dichloroallylalcohols but not trichloroallylalcohol. The enzyme used phenazine methosulfate as an artificial electron acceptor. Further oxidation yielded 2-chloroacrylic acid. The organism also produced hydrolytic dehalogenases converting 2-chloroacetic acid and 2-chloropropionic acid.
Cyclodextrins as carriers of cholesterol and fatty acids in cultivation of mycoplasmas
Authors: Greenberg-Ofrath N , Terespolosky Y , Kahane I , Bar R
Published: January 2020

The design of fully or partly defined media for mycoplasma cultivation involves the need to provide the essential lipids, cholesterol and long-chain fatty acids, in an assimilable and nontoxic form. This study introduces cyclodextrins (CDs) as carriers of these lipids, thus suggesting alternatives to serum or bovine serum albumin (BSA). The effects of beta-CD and two forms of chemically modified beta-CD, dimethyl-beta-CD (Dimeb) and hydroxypropyl-beta-CD (Hyprob), on the growth of Mycoplasma capricolum and Acholeplasma laidlawii were investigated in a basal medium as well as in serum- and BSA-supplemented media. beta-CD was found to inhibit the growth of the sterol-requiring M. capricolum in both serum and BSA media, but it stimulated the growth of the sterol-independent A. laidlawii. Inhibition by beta-CD was explained by its capacity to form a water-insoluble CD-cholesterol complex, thus rendering it unavailable to the cells. Dimeb, despite its strong complexing ability for lipids, was found to be toxic to all mycoplasma species in both liquid cultures and agar diffusion susceptibility tests. In sharp contrast to beta-CD and Dimeb, Hyprob (with a degree of substitution of 4.2) added at 5 and 10 mM to a basal medium supplemented with lipids permitted growth of M. capricolum. Comparison of growth curves in the two conventional serum and BSA media with those in two Hyprob media revealed comparable growth and growth rates.
Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods
Authors: Koch W H , Payne W L , Wentz B A , Cebula T A
Published: January 2020

The polymerase chain reaction was used to selectively amplify sequences within the cholera toxin operon from Vibrio cholerae O1. Oysters, crabmeat, shrimp, and lettuce were seeded with V. cholerae and then homogenized or washed with alkaline peptone water, followed by short-term (6- to 8-h) enrichment. A detection limit of as few as 1 V. cholerae CFU per 10 g of food was obtained with amplification reactions from crude bacterial lysates. The method is extremely rapid and obviates the need for DNA isolation from a variety of complex food matrices.
Survival differences among freeze-dried genetically engineered and wild-type bacteria
Authors: Israeli E , Shaffer B T , Hoyt J A , Lighthart B , Ganio L M
Published: January 2020

Because the death mechanisms of freeze-dried and air-dried bacteria are thought to be similar, freeze-drying was used to investigate the survival differences between potentially airborne genetically engineered microorganisms and their wild types. To this end, engineered strains of Escherichia coli and Pseudomonas syringae were freeze-dried and exposed to air, visible light, or both. The death rates of all engineered strains were significantly higher than those of their parental strains. Light and air exposure were found to increase the death rates of all strains. Application of death rate models to freeze-dried engineered bacteria to be released into the environment is discussed.
Genetic basis of tetracycline resistance in food-borne isolates of Listeria innocua
Authors: Facinelli B , Roberts M C , Giovanetti E , Casolari C , Fabio U , Varaldo P E
Published: January 2020

Eleven of 12 tetracycline-resistant Listeria innocua strains, isolated from chicken or turkey frankfurters and mozzarella cheese, were shown to carry DNA sequences which hybridized with the Tet M probe; of these, two strains also hybridized with Tet K. The remaining strain hybridized with the Tet K probe only. The Tet M determinant appeared to be located on the chromosome; in one case, it was transferable by conjugation to recipients Listeria monocytogenes, Listeria ivanovii, and Enterococcus faecalis.
Growth and survival of Shigella flexneri in common Bangladeshi foods under various conditions of time and temperature
Authors: Islam M S , Hasan M K , Khan S I
Published: January 2020

Survival and growth of Shigella flexneri were assessed in various foods, including boiled rice, lentil soup, milk, cooked beef, cooked fish, mashed potato, mashed brinjal, and raw cucumber. Growth at 25 and 37 degrees C and survival at 5 degrees C were observed by viable counts on MacConkey agar. The organism grew well in all tested foods and growth increased from 10(5) to 10(8) to 10(10) cells per ml or g within 6 to 18 h after inoculation at 25 and 37 degrees C.
Distribution of viral abundance in the reef environment of Key Largo, Florida
Authors: Paul J H , Rose J B , Jiang S C , Kellogg C A , Dickson L
Published: January 2020

The distribution of viral and microbial abundance in the Key Largo, Fla., reef environment was measured. Viral abundance was measured by transmission electron microscope direct counts and plaque titer on specific bacterial hosts in water and sediment samples from Florida Bay (Blackwater Sound) and along a transect from Key Largo to the outer edge of the reef tract in Key Largo Sanctuary. Water column viral direct counts were highest in Blackwater Sound of Florida Bay (1.2 x 10(7) viruses per ml), decreased to the shelf break (1.7 x 10(6) viruses per ml), and were inversely correlated with salinity (r = -0.97). Viral direct counts in sediment samples ranged from 1.35 x 10(8) to 5.3 x 10(8)/cm(3) of sediment and averaged nearly 2 orders of magnitude greater than counts in the water column. Viral direct counts (both sediment and water column measurements) exceeded plaque titers on marine bacterial hosts (Vibrio natriegens and others) by 7 to 8 orders of magnitude. Water column viral abundance did not correlate with bacterial direct counts or chlorophyll a measurements, and sediment viral parameters did not correlate with water column microbial, viral, or salinity data. Coliphage, which are indicators of fecal pollution, were detected in two water column samples and most sediment samples, yet their concentrations were relatively low (<2 to 15/liter for water column samples, and <2 to 108/cm(3) of sediment). Our findings indicate that viruses are abundant in the Key Largo environment, particularly on the Florida Bay side of Key Largo, and that processes governing their distribution in the water column (i.e., salinity and freshwater input) are independent of those governing their distribution in the sediment environment.
Plasmid Transfer between Marine Bacteria in the Aqueous Phase and Biofilms in Reactor Microcosms
Authors: Angles M. L. , Marshall K. C. , Goodman A. E.
Published: January 2020

Plasmid transfer of broad-host-range plasmid RP1 from marine
Vibrio
sp. strain S14 to marine strain SW5 under optimum conditions on the surface of nutrient plates was improved 2 orders of magnitude by using the plasmid transfer process to select an SW5 recipient more efficient than the wild type in receiving and/or maintaining the plasmid. This recipient strain, SW5H, was used to form biofilms under flow conditions on the surfaces of glass beads in reactors. The S142(RP1) donor strain was introduced to the reactors after either 48 or 170 h of biofilm formation, and production of transconjugants in the aqueous phases and biofilms without selection pressure was assessed. Plasmid transfer to the recipient cells in the biofilm was detected for biofilms formed for 170 h but not in those formed for 48 h. The plasmid transfer frequency was significantly higher (
P
< 0.05) among cells attached to the bead surfaces in the biofilm than among cells in the aqueous phase.
Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae
Authors: Southgate V J , Steyn A J , Pretorius I S , Van Vuuren H J
Published: January 2020

Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.
Correlative Association between Resident Plasmids and the Host Chromosome in a Diverse
Agrobacterium
Soil Population
Authors: Bouzar Hacène , Ouadah Djaouida , Krimi Zoulikha , Jones Jeffrey B. , Trovato Maurizio , Petit Annik , Dessaux Yves
Published: January 2020

Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a population of
Agrobacterium
spp. estimated at 3 × 10
7
CFU/g. Characterization of 72 strains selected from four different isolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on five different test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. All tumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilization patterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed of five distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B). chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism, pathogenicity, and agrocin sensitivity, chr A strains carried a nopaline Ti plasmid (pTi), whereas chr C strains had an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chr F) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic and catabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin 84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains were agrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in this
Agrobacterium
soil population, there is a preferential association between the resident plasmids and their bacterial host.
Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium
Authors: Rabus R , Nordhaus R , Ludwig W , Widdel F
Published: January 2020

A toluene-degrading sulfate-reducing bacterium, strain Tol2, was isolated from marine sediment under strictly anoxic conditions. Toluene was toxic if applied directly to the medium at concentrations higher than 0.5 mM. To provide toluene continuously at a nontoxic concentration, it was supplied in an inert hydrophobic carrier phase. The isolate had oval, sometimes motile cells (1.2 to 1.4 by 1.2 to 2.0 microns). The doubling time was 27 h. Toluene was completely oxidized to CO2, as demonstrated by measurement of the degradation balance. The presence of carbon monoxide dehydrogenase and formate dehydrogenase indicated a terminal oxidation of acetyl coenzyme A via the CO dehydrogenase pathway. The use of hypothetical intermediates of toluene degradation was tested in growth experiments and adaptation studies with dense cell suspensions. Results do not support a degradation of toluene via one of the cresols or methylbenzoates, benzyl alcohol, or phenylacetate as free intermediate. Benzyl alcohol did not serve as growth substrate; moreover, it was a strong, specific inhibitor of toluene degradation, whereas benzoate utilization was not affected by benzyl alcohol. Sequencing of 16S rRNA revealed a relationship to the metabolically dissimilar genus Desulfobacter and on a deeper level to the genus Desulfobacterium. The new genus and species Desulfobacula toluolica is proposed.
Purification, characterization, and lytic activity against Naegleria fowleri of two amoebicins produced by Bacillus licheniformis A12
Authors: Gálvez A , Valdivia E , González-Segura A , Lebbadi M , Martínez-Bueno M , Maqueda M
Published: January 2020

Bacillus licheniformis A12 produces two amoebolytic substances (amoebicins A12-A and A12-B) in liquid media during sporulation. Both substances have been purified and characterized. They are heat- and protease-resistant peptides containing aspartic acid, glutamic acid, serine, proline, and tyrosine in a molar ratio of 5:2:2:2:2. No fatty acids or carbohydrates have been detected. Their molecular weight is 1,430. Purified amoebicins A12-A and A12-B exhibit amoebolytic action against Naegleria fowleri. They also exhibit antibiotic action against yeasts (Saccharomyces heterogenicus and Cryptococcus neoformans) and several fungal species (Aspergillus niger, Microsporum canis, Mucor plumbeus, and Trychophyton mentagrophytes). Their antibacterial spectrum appears to be restricted to Bacillus megaterium, Corynebacterium glutamicum, and Sarcina sp.
Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure
Authors: Wagner M , Amann R , Lemmer H , Schleifer K H
Published: January 2020

Bacterial community structures in activated sludge samples from aeration tanks of a two-stage system with a high-load first stage and a low-load second stage were analyzed with oligonucleotide probes. The probes were complementary to conserved regions of the rRNA of the alpha, beta, and gamma subclasses of proteobacteria and of all bacteria. Group-specific cell counts were determined by in situ hybridization with fluorescent probe derivatives. Contributions of the proteobacterial subclasses to total bacterial rRNA were quantified by dot blot hybridization with digoxigenin-labeled oligonucleotides. The activated sludge samples were dominated by proteobacteria from the alpha, beta, or gamma subclass. These proteobacteria account for about 80% of all active bacteria found in the activated sludge. For both samples the community structures determined with molecular techniques were compared with the composition of the heterotrophic saprophyte flora isolated on nutrient-rich medium. Probes were used to rapidly classify the isolates and to directly monitor population shifts in nutrient-amended, activated sludge samples. The rich medium favored growth of gamma-subclass proteobacteria (e.g., enterobacteria) and selected against beta-subclass proteobacteria. The culture-dependent community structure analysis of activated sludge produced partial and heavily biased results. A more realistic view will be obtained by using in situ techniques.
Development of a Rapid Assimilable Organic Carbon Method for Water
Authors: LeChevallier Mark W. , Shaw Nancy E. , Kaplan Louis A. , Bott Thomas L.
Published: January 2020

A rapid method for measurement of assimilable organic carbon (AOC) is proposed. The time needed to perform the assay is reduced by increasing the incubation temperature and increasing the inoculum density. The ATP luciferin-luciferase method quickly enumerates the test organisms without the need for plate count media or dilution bottles. There was no significant difference between AOC values determined with strain P17 for the ATP and plate count procedures. For strain NOX, the plate count procedure underestimated bacterial levels in some samples. Comparison of AOC values obtained by the Belleville laboratory (by the ATP technique) and the Stroud Water Research Center (by plate counts) showed that values were significantly correlated and not significantly different. The study concludes that the rapid AOC method can quickly determine the bacterial growth potential of water within 2 to 4 days.
Application of rRNA-based probes for observing marine nanoplanktonic protists
Authors: Lim E L , Amaral L A , Caron D A , DeLong E F
Published: January 2020

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.
Comparison of the azoreductase and nitroreductase from Clostridium perfringens
Authors: Rafii F , Cerniglia C E
Published: January 2020

The purified azoreductase and nitroreductase of Clostridium perfringens, which have similar electrophoretic properties, both reacted in a Western blot (immunoblot) with a polyclonal antibody raised against the azoreductase. The activity of both enzymes was enhanced by flavin adenine dinucleotide and was inhibited by menadione, o-iodosobenzoic acid, and the antibody against azoreductase. Reduction of the azo dye Direct Blue 15 by the azoreductase was inhibited by nitroaromatic compounds. The apparent Km of the enzyme for reduction of Direct Blue 15 in the presence of 1-nitropyrene was higher than the Km with the azo dye alone, demonstrating competitive inhibition. The data show that the same protein is involved in the reduction of both azo dyes and nitroaromatic compounds.
Glutamate Dehydrogenase Is Not Essential for Glutamate Formation by
Corynebacterium glutamicum
Authors: Kholy Elke R. Börmann-El , Eikmanns Bernhard J. , Gutmann Marcella , Sahm Hermann
Published: January 2020

Two
Corynebacterium glutamicum
strains, one being glutamate dehydrogenase (GDH) negative and the other possessing 11-fold-higher specific GDH activity than the parental wild type, were constructed and used to analyze the role of GDH in
C. glutamicum
. The results indicate (i) that GDH is dispensable for glutamate synthesis required for growth and (ii) that although a high level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.
Development of Steady-State Diffusion Gradients for the Cultivation of Degradative Microbial Consortia
Authors: Wolfaardt G. M. , Lawrence J. R. , Hendry M. J. , Robarts R. D. , Caldwell D. E.
Published: January 2020

A diffusion gradient plate was constructed and evaluated for its potential use in the isolation of degradative microbial consortia from natural habitats. In this model, a steady-state concentration gradient of diclofop methyl, established by diffusion through an agarose gel, provided the carbon for microbial growth. Colonization of the gel surface was observed with epifluorescence and scanning confocal laser microscopy to determine microbial responses to the diclofop gradient. A detectable gradient developed over a narrow band (<10 mm). Consequently, quantitative analyses of the microbial response to the gradient were difficult to obtain. A two-dimensional, finite-element numerical transport model for advective-diffusive transport was used to simulate concentration and flux profiles in the physical model. The simulated profiles were correlated with the measured concentration gradient (
R
2
= 0.89) and the cell numbers on the gel surface (
R
2
= 0.85). The numerical model was subsequently used to redesign the physical model. The detectable concentration gradient in the modified physical model extended over the length of the gel (38 mm). The simulated profile again showed a good correlation with the measured profile (
R
2
= 0.96) and the microbial responses to the concentration gradient (
R
2
= 0.99). It was concluded that these gradients provide the steady-state environments needed to sustain steady-state consortia. They also provide a physical pathway for the development of degradative biofilms from low to high concentrations of toxicants and simulate conditions under which low concentrations of toxicant are supplied at a constant flux over long periods of time, such as the conditions that could occur in natural environments.
Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in
trans
, on Incoming-Phage Proliferation
Authors: O'Sullivan Daniel J. , Hill Colin , Klaenhammer Todd R.
Published: January 2020

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in
trans
in
Lactococcus lactis
NCK203, Per50, the cloned origin of replication from phage φ50, interferes with φ50 replication. The
per50
fragment was found to afford negligible protection to NCK203 against φ50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected φ50 infection, reducing the efficiency of plaquing (EOP) to 2.5 × 10
-4
and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage φ31 and demonstrated to function as an origin of replication by enabling replication of
per31
-containing plasmids, in NCK203, on φ31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of φ31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 × 10
-7
but the plaques were large and contained Per31-resistant phages. Characterization of these “new” phages revealed at least two different types that were similar to φ31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.
Stable Tagging of
Rhizobium meliloti
with the Firefly Luciferase Gene for Environmental Monitoring
Authors: Cebolla Angel , Ruiz-Berraquero Francisco , Palomares Antonio Jose
Published: January 2020

A system for stable tagging of gram-negative bacteria with the firefly luciferase gene,
luc
, is described. A previously constructed fusion constitutively expressing
luc
from the λ
p
R
promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn
5
delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such as
Escherichia coli, Rhizobium meliloti, Pseudomonas putida
, and
Agrobacterium tumefaciens
. The system permitted the detection of tagged
R. meliloti
in the presence of more than 10
5
CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescent
R. meliloti
also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment.
Influence of Metronidazole, CO, CO
2
, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus
Neocallimastix
sp. Strain L2
Authors: Marvin-Sikkema Femke D. , Rees Elizabeth , Kraak Marjan N. , Gottschal Jan C. , Prins Rudolf A.
Published: January 2020

The effects of metronidazole, CO, methanogens, and CO
2
on the fermentation of glucose by the anaerobic fungus
Neocallimastix
sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H
2
, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When
Neocallimastix
sp. strain L2 was cocultured with the methanogenic bacterium
Methanobrevibacter smithii
, the fermentation pattern changed in the opposite direction: H
2
and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO
2
in the gas phase resulted in increased H
2
and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in
Neocallimastix
sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.
Composition of Non-Microbially Reducible Fe(III) in Aquatic Sediments
Authors: Phillips Elizabeth J. P. , Lovley Derek R. , Roden Eric E.
Published: January 2020

The production of small quantities of Fe(II) during the initial phase of microbial Fe(III) reduction greatly increased the amount of Fe(III) that could be extracted from freshwater sediments with oxalate. This finding and other evidence suggest that the oxalate-extractable Fe(III) that is unavailable for microbial reduction in anoxic sediments is not in the form of mixed Fe(III)-Fe(II) forms, as was previously suggested, but rather is in the form of highly crystalline Fe(III) oxides.
Use of vaginal tampons in sewer surveys for non-O1. Vibrio cholerae
Authors: Pazzaglia G , Lesmana M , Tjaniadi P , Subekti D , Kay B
Published: January 2020

Vaginal tampons were shown to be a practical alternative to conventional Moore swabs for isolating Vibrio cholerae from sewage. Associated laboratory investigations demonstrated improved isolation of V. cholerae by using 12- or 18-h enrichments in alkaline peptone water, in comparison with 6-h enrichments, when cultures were incubated at ambient temperatures.
Starved cells of the fatty acid auxotroph Escherichia coli AK7 develop abnormal sensitivity to media with low osmolarity
Authors: Scaravaglio O R , Farías R N , Massa E M
Published: January 2020

The unsaturated fatty acid auxotroph Escherichia coli AK7 supplied with linolenic acid, while appearing normal during logarithmic growth, showed a fast decline in CFU during starvation as a result of an osmotic downshift when transferred to standard agar plates unsupplemented with an osmolyte such as 300 mosM sucrose or salt (NaCl or KCl). The starved cells could recover their osmoresistance when an energy source was added to the starvation medium.
Detection of two smooth colony phenotypes in a Salmonella enteritidis isolate which vary in their ability to contaminate eggs
Authors: Petter J G
Published: January 2020

Salmonella enteritidis isolates were obtained from eggs after infection of Leghorn hens with a parent isolate (SE6) known to only infrequently contaminate eggs. Isolates from eggs exhibited two phenotypes that were subtly different. One phenotype was typically smooth, while the other was transiently rough. Both sets of isolates were phage type 13A and positive for D1 epitopes. Immunoblot analysis of entire colonies and gas chromatographic analysis of purified lipopolysaccharide revealed that the phenotypic difference between isolates was due to a quantitative difference in O antigen and possibly a qualitative difference in the lipid A core region. In addition, the two isolates had different opacity properties when examined at 600 nm. When the two isolates were used to inject egg-laying hens, a significant difference in the ability to contaminate eggs was detected.
DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca
Authors: Jung E D , Lao G , Irwin D , Barr B K , Benjamin A , Wilson D B
Published: January 2020

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.
Molecular cloning and sequence analysis of the crtB gene of Thermus thermophilus HB27, an extreme thermophile producing carotenoid pigments
Authors: Hoshino T , Fujii R , Nakahara T
Published: January 2020

We have cloned and sequenced a 1.5-kb chromosomal fragment of Thermus thermophilus which promoted the overproduction of carotenoids in T. thermophilus. An open reading frame (ORF-A) coding for a polypeptide with 289 amino acids was responsible for carotenoid overproduction. The putative ORF-A protein showed significant homology with the amino acid sequences of crtB gene products (phytoene syntheses) of other microorganisms. The clone containing the ORF-A on a multicopy plasmid produced about three times as much carotenoid as that produced by the host strain, suggesting that the crtB gene product is a rate-limiting enzyme for carotenoid biosynthesis in T. thermophilus.
Cytochrome aa3 gene regulation in members of the family Rhizobiaceae: comparison of copper and oxygen effects in Bradyrhizobium japonicum and Rhizobium tropici
Authors: Gabel C , Bittinger M A , Maier R J
Published: January 2020

Dithionite-reduced minus ferricyanide-oxidized difference spectra on membranes from Rhizobium tropici (formerly Rhizobium leguminosarum bv. phaseoli) incubated at progressively lower O2 concentrations showed only a slight concomitant decrease in A603, the alpha-peak of cytochrome aa3. In contrast to previous results on Bradyrhizobium japonicum, R. tropici showed no significant O2-mediated reduction in the level of either coxA transcription or cytochrome aa3 activity (as measured by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine [TMPD] oxidase) even in the cells incubated at 12.5 microM O2. Bean nodule R. tropici bacteroids contained 65% of the fully aerobic free-living levels of the coxA transcript. Primer extension analyses established the transcription initiation site of the R. tropici coxA genes. Sequence analyses of the regions upstream of the transcription initiation site revealed no homology with previously reported Rhizobiaceae family promoters, including the coxA promoter of B. japonicum. The R. tropici deduced CoxA sequence itself is highly homologous to the B. japonicum and Paracoccus denitrificans CoxA sequences. In both B. japonicum and R. tropici, coxA transcript levels were the same for cells grown with copper (0.02 microM) in the medium or in medium completely devoid of copper. However, a posttranscriptional effect of copper deprivation was observed for both bacteria; difference absorption spectra on membranes from cells grown without copper showed that B. japonicum lacked spectroscopically detectable cytochrome aa3, whereas R. tropici retained approximately 50% of normal cytochrome aa3 levels.
Electrophoretic Transfer from Polyacrylamide Gel to Nitrocellulose Sheets, a New Method To Characterize Multilocus Enzyme Genotypes of
Klebsiella
Strains
Authors: Combe Marie-Laure , Pons Jean-Louis , Sesboue Richard , Martin Jean-Pierre
Published: January 2020

A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-μl samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of
Klebsiella
strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of
Klebsiella pneumoniae
and 24 strains of
Klebsiella oxytoca
demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.
Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes
Authors: Laguerre Gisèle , Allard Marie-Reine , Revoy Françoise , Amarger Noelle
Published: January 2020

Forty-eight strains representing the eight recognized
Rhizobium
species, two new
Phaseolus
bean
Rhizobium
genomic species,
Bradyrhizobium
spp.,
Agrobacterium
spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa.
Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR
Authors: Khan A A , Cerniglia C E
Published: January 2020

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.